an empirical framework for combining surgery with …...an empirical framework for combining surgery...

An empirical framework for combining

surgery with immune therapy

Matthew David Brown MBBS (Hons)

This thesis is presented to The University of Western Australia

for the degree of Doctor of Philosophy

School of Surgery & Pathology

2007

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To my parents, who have always inspired me. To my friends and

mentors, who spurred me on when things seemed hopeless. And to

my darling Sarah, who shares the journey with me.

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Table of Contents

DECLARATION .................................................................................... - 9 -

PROFILE .............................................................................................. - 10 -

ABSTRACT .......................................................................................... - 15 -

INDEX OF FIGURES ......................................................................... - 18 -

1. INTRODUCTION ......................................................................... - 21 -

1.1. RATIONALE FOR SURGERY/IMMUNE THERAPY ......................................... - 25 -

1.1.1. Effects of surgery on inflammation........................................................ - 25 -

1.1.2. Effects of surgery on innate immunity ................................................... - 25 -

1.1.3. Effects of surgery on adaptive immunity ............................................... - 26 -

1.1.4. Surgery and overall immune function .................................................... - 26 -

1.1.5. Surgery and immunity: conflicting paradigms ....................................... - 27 -

1.1.6. Surgery improves anti-tumour immunity ............................................... - 27 -

1.1.7. Other benefits of surgery/immune therapy ............................................ - 28 -

1.1.8. Against surgery/immune therapy ........................................................... - 29 -

1.2. IMMUNE EFFECTS OF SENTINEL NODE BIOPSY ......................................... - 29 -

1.2.1. The evolution of lymph node surgery .................................................... - 29 -

1.2.2. Sentinel lymph nodes ............................................................................. - 30 -

1.2.3. Immune function of sentinel lymph nodes ............................................. - 30 -

1.2.4. Immune consequences of sentinel node surgery .................................... - 31 -

1.2.5. Nodal invasion and tumour proximity ................................................... - 32 -

1.2.6. Lymphadenectomy and immune therapy ............................................... - 33 -

1.3. SURGERY AND TUMOUR-SPECIFIC IMMUNITY .......................................... - 33 -

1.3.1. Surgery and antigen presentation ........................................................... - 34 -

1.3.2. Surgery and immune suppression networks ........................................... - 38 -

1.3.2.1. Alleviate tumour-derived suppressive factors............................................... - 38 -

1.3.2.2. Reduce effector requirements ....................................................................... - 38 -

1.3.2.3. Reduce MSC ................................................................................................. - 39 -

1.3.2.4. Provide “antigen holiday” ............................................................................. - 39 -

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1.3.2.5. Change memory phenotype .......................................................................... - 41 -

1.3.2.6. Release effectors ........................................................................................... - 42 -

1.3.2.7. Improve overall cell mediated immunity ...................................................... - 42 -

1.4. AIMS AND HYPOTHESES ............................................................................. - 42 -

2. METHODS .................................................................................... - 45 -

2.1. CELL LINES AND CULTURE TECHNIQUES ................................................... - 45 -

2.1.1. Cell harvest............................................................................................. - 45 -

2.1.2. Mycoplasma screening ........................................................................... - 45 -

2.1.3. AB1 ........................................................................................................ - 46 -

2.1.4. AB1HA .................................................................................................. - 46 -

2.1.5. Renca ...................................................................................................... - 47 -

2.1.6. RencaHA ................................................................................................ - 47 -

2.2. MURINE SPECIES ........................................................................................ - 48 -

2.2.1. BALB/c and BALB/c nu-/-

mice ............................................................. - 48 -

2.2.2. CL4 TCR transgenic mice ...................................................................... - 49 -

2.2.3. HNT TCR transgenic mice .................................................................... - 49 -

2.3. IN VIVO PROCEDURES ................................................................................ - 49 -

2.3.1. Anaesthesia ............................................................................................ - 49 -

2.3.2. Analgaesia .............................................................................................. - 50 -

2.3.3. Subcutaneous inoculation....................................................................... - 50 -

2.3.4. Axillary inoculation ............................................................................... - 51 -

2.3.5. Intranodal inoculation ............................................................................ - 51 -

2.3.6. Intravenous inoculation .......................................................................... - 51 -

2.3.7. Intracardiac inoculation .......................................................................... - 52 -

2.3.8. Intrarenal inoculation ............................................................................. - 52 -

2.3.9. Resection studies .................................................................................... - 53 -

2.3.10. Lymphadenectomy ................................................................................. - 53 -

2.3.11. Monitoring ............................................................................................. - 54 -

2.3.12. Tumour size assessments ....................................................................... - 54 -

2.3.13. Survival analysis .................................................................................... - 54 -

2.3.14. Adoptive cell transfers ........................................................................... - 55 -

2.4. IN VIVO CFSE PROLIFERATION ASSAY ...................................................... - 55 -

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2.4.1. Preparation of CL4 cells for adoptive transfer ....................................... - 55 -

2.4.2. Adoptive transfer for antigen presentation ............................................. - 56 -

2.4.3. Analysis for in vivo antigen presentation............................................... - 56 -

2.4.4. Statistical analysis for antigen presentation ........................................... - 57 -

2.5. IN VIVO CTL ASSAY .................................................................................... - 57 -

2.5.1. Pulsed and target reference peaks .......................................................... - 57 -

2.5.2. Adoptive transfer for in vivo CTL lysis assay ....................................... - 58 -

2.5.3. Analysis for in vivo CTL lysis ............................................................... - 58 -

2.5.4. Statistical analysis for in vivo CTL Assay ............................................. - 59 -

2.6. DC PHENOTYPING ....................................................................................... - 59 -

2.6.1. Isolation of DCs from lymph nodes ....................................................... - 59 -

2.6.2. Staining of DCs ...................................................................................... - 60 -

2.6.3. DC flow cytometry ................................................................................. - 60 -

2.6.4. Analysis of DC phenotypes .................................................................... - 60 -

2.7. TREG ASSAYS ................................................................................................ - 60 -

2.7.1. Cell surface and intracellular staining for Treg ....................................... - 61 -

2.7.2. Flow cytometry for Treg .......................................................................... - 61 -

2.7.3. Statistical analysis of Treg ....................................................................... - 62 -

2.8. MSC STUDIES ............................................................................................. - 62 -

2.9. HA-SPECIFIC CD8+: DETECTION/PHENOTYPE .......................................... - 63 -

2.9.1. Pentamer calibration............................................................................... - 63 -

2.9.2. Assessment of tumour-specific memory cells ....................................... - 63 -

2.10. THERAPIES .................................................................................................. - 65 -

2.10.1. Toll Like Receptor (TLR) ligand therapy .............................................. - 65 -

2.10.2. poly I:C .................................................................................................. - 65 -

2.10.3. CpG-ODN 1668 ..................................................................................... - 65 -

2.10.4. 3M019TM

................................................................................................ - 66 -

2.10.5. Activating anti-CD40 antibody therapy ................................................. - 66 -

2.11. IN VIVO DEPLETION STUDIES ...................................................................... - 66 -

2.11.1. CD4+/CD8

+T cell depletions .................................................................. - 67 -

2.11.2. Treg depletion .......................................................................................... - 67 -

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2.12. IDENTIFICATION OF SENTINEL NODES ........................................................ - 68 -

2.12.1. Methylene blue ....................................................................................... - 68 -

2.13. DC TRACKING ............................................................................................. - 68 -

2.14. HISTOLOGY ................................................................................................. - 69 -

2.14.1. H&E staining .......................................................................................... - 69 -

2.14.2. Resection specimens .............................................................................. - 70 -

2.14.3. Kidneys .................................................................................................. - 70 -

2.14.4. Lymph nodes .......................................................................................... - 70 -

2.14.5. Lungs ...................................................................................................... - 70 -

2.15. CULTURE OF NECROPSY SPECIMENS .......................................................... - 71 -

2.15.1. Lung ....................................................................................................... - 71 -

2.15.2. Lymph nodes .......................................................................................... - 71 -

2.16. HA-SPECIFIC REAL TIME PCR ................................................................... - 71 -

2.16.1. Extraction of DNA ................................................................................. - 72 -

2.16.2. PCR of DNA templates .......................................................................... - 72 -

3. SURGERY AND CROSS PRESENTATION ............................ - 74 -

3.1. INTRODUCTION ........................................................................................... - 74 -

3.2. RESULTS ...................................................................................................... - 75 -

3.2.1. AB1HA in wild type and immunodeficient mice .................................. - 75 -

3.2.2. HA-specific presentation during AB1HA growth.................................. - 76 -

3.2.3. Specificity of CL4 proliferation ............................................................. - 76 -

3.2.4. HA presentation after surgery ................................................................ - 77 -

3.2.5. Completeness of resection...................................................................... - 80 -

3.2.6. HA presentation from recurrent AB1HA ............................................... - 84 -

3.2.7. Antigen presentation to CD4+ T cells post-op ....................................... - 84 -

3.2.8. Post-operative DC phenotype ................................................................ - 86 -

3.2.9. Cross presentation and in vivo CTL function ........................................ - 86 -

3.2.10. Recurrent tumour and systemic CTL responses..................................... - 91 -

3.3. DISCUSSION ................................................................................................. - 91 -

3.4. SUMMARY ................................................................................................... - 94 -

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4. SINECOMITANT IMMUNITY .................................................. - 97 -

4.1. INTRODUCTION ........................................................................................... - 97 -

4.2. RESULTS ...................................................................................................... - 98 -

4.2.1. Concomitant immunity in the AB1HA model ....................................... - 98 -

4.2.2. Sinecomitant immunity in the AB1HA model ....................................... - 98 -

4.2.3. Sinecomitant immunity in the wounded flank ....................................... - 99 -

4.2.4. Sinecomitant immunity and re-challenge dose .................................... - 100 -

4.2.5. Surgical trauma and sinecomitant immunity........................................ - 101 -

4.2.6. HA in sinecomitant immunity to AB1HA ........................................... - 101 -

4.2.7. T cell dependence of sinecomitant immunity ...................................... - 103 -

4.2.8. Persistent tumour and sinecomitant immunity ..................................... - 104 -

4.2.9. Persistent antigen and sinecomitant immunity ..................................... - 107 -

4.2.10. Distribution of HA specific effectors post-op ...................................... - 108 -

4.2.11. Suppression and sinecomitant immunity ............................................. - 110 -

4.2.12. Sinecomitant immunity and immune therapy ...................................... - 120 -

4.3. DISCUSSION ............................................................................................... - 122 -

4.4. SUMMARY ................................................................................................. - 132 -

5. TUMOUR IMMUNITY & SENTINEL NODES ..................... - 135 -

5.1. INTRODUCTION ......................................................................................... - 135 -

5.2. RESULTS .................................................................................................... - 136 -

5.2.1. Identification of sentinel nodes ............................................................ - 136 -

5.2.2. Dendritic tracking and the sentinel nodes ............................................ - 137 -

5.2.3. Tumour proximity and node function .................................................. - 138 -

5.2.4. Tumour invasion and nodal function ................................................... - 141 -

5.2.5. Surgical dissection of the sentinel nodes ............................................. - 141 -

5.2.6. Antigen ablation and sentinel node excision ........................................ - 141 -

5.2.7. Tumour antigen presentation after node removal ................................ - 146 -

5.2.8. Sentinel node removal and re-challenge .............................................. - 147 -

5.2.9. Sentinel sampling and staged lymphadenectomy ................................ - 149 -

5.3. DISCUSSION ............................................................................................... - 149 -

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5.4. SUMMARY ................................................................................................. - 161 -

6. RENCAHA ................................................................................... - 163 -

6.1. INTRODUCTION ......................................................................................... - 163 -

6.2. RESULTS .................................................................................................... - 164 -

6.2.1. Initial experience with RencaHA ......................................................... - 164 -

6.2.2. Subcutaneous RencaHAM ................................................................... - 165 -

6.2.3. Intravenous RencaHAM....................................................................... - 167 -

6.2.4. Orthotopic (intra-renal) RencaHAM .................................................... - 169 -

6.2.5. Lymph node metastases from RencaHAM .......................................... - 170 -

6.3. DISCUSSION ............................................................................................... - 176 -

6.4. SUMMARY ................................................................................................. - 180 -

7. THESIS SUMMARY .................................................................. - 183 -

7.1. PRINCIPAL FINDINGS ................................................................................ - 184 -

7.1.1. Effects of surgery on antigen presentation ........................................... - 184 -

7.1.2. Surgery & tumour-specific CTLs......................................................... - 184 -

7.1.3. Sentinel lymph nodes & anti-tumour immunity................................... - 185 -

7.1.4. Properties of sinecomitant immunity ................................................... - 185 -

7.2. CONCLUSIONS ........................................................................................... - 186 -

7.3. FUTURE DIRECTIONS ................................................................................ - 188 -

APPENDIX A: REFERENCES ........................................................ - 190 -

APPENDIX B: ABBREVIATIONS ................................................. - 216 -

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Declarat ion

The experiments in this thesis constitute work carried out by the candidate unless

otherwise stated. The thesis is less than 100,000 words in length (inclusive of tables,

figures, bibliography and appendices) and complies with the stipulations set out for the

degree of Doctor of Philosophy of The University of Western Australia.

Dr Andrew Currie, Dr Robert van der Most, and Dr Kathy Heel have aided with the

calibration of flow cytometry instruments when four colour flow cytometry was

performed. Immune therapy protocols for experimentation in mice were provided by

Professor Bruce Robinson and Dr Delia Nelson. Finally, Irma Larma (the Urological

Research Centre Research Assistant) worked under the supervision of the candidate for

the final twelve months of experimentation. She prepared histological sections and

performed the RT-PCR experiments in their entirety.

The following organisations are gratefully acknowledged for their financial support:

Sporting Chance Cancer Foundation, Royal Australasian College of Surgeons, Abbott

Australasia, Australasian Urological Foundation, Sir Charles Gairdner Hospital Clinical

Staff Association and The University of Western Australia (Athelstan & Amy Saw

Fellowship). These organisations did not influence the research direction or

experimental design of this thesis.

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Prof i le

Grants, Scholarships & Awards

Villus Marshall Prize, USANZ Annual Scientific Meeting, 2007

Finalist (Villus Marshall Prize), USANZ Annual Scientific Meeting, 2006

Finalist (ASI Young Scientist of 2006), Australasian Society of Immunology, 2006

Best Registrar Scientific Paper, Royal Australasian College of Surgeons (WA), 2006

SCGH Young Investigator Award, Sir Charles Gairdner Hospital, 2006

Inaugural SCGH Clinical Staff Association PhD Scholarship, SCGH, 2005

Best Surgical Oncology Paper, Royal Australasian College of Surgeons ASC, 2005

AUF/Abbott Registrar Scholarship, Australasian Urological Foundation, 2005

Raelene Boyle Scholarship, Royal Australasian College of Surgeons, 2004

Athelstan and Amy Saw Fellowship, University of Western Australia, 2004

Best Registrar Research Paper, Urological Society of Australasia State Meeting, 2004

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Associated Papers and Published Abstracts

Brown M, Vivian J, Currie A, Robinson B, Hall J. Post operative tumour antigen

presentation. ANZ Journal of Surgery 2005;75(Suppl):A106-A107

Broomfield S, Currie A, van der Most R, Brown M, van Bruggen I, Robinson BWS,

Lake RA. Partial, but not complete, tumour debulking promotes protective anti-tumour

immunity when combined with chemotherapy and adjuvant immunotherapy. Cancer

Research 2005;65:7580-7584

Brown MD, Vivian JB, Currie AJ, Robinson BWS. Surgery re-sets the anti-tumour

immune response. Tissue Antigens 2005;66(5):367

Brown MD, Vivian JB, Currie AJ, Robinson BWS. Post-operative tumour antigen

presentation in murine models of malignancy. BJU International 97(Supp 1):9

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Conference Presentations

Are Tumour Vaccines Logical? Urological Society of Australasia State Meeting (WA),

Bunker Bay Western Australia, November 5-7 2005

Postoperative Tumour Neo-Antigen Presentation in Murine Models of Primary and

Metastatic Malignancy, Australasian Society of Immunology National Convention,

Adelaide South Australia, December 12-16 2005

Postoperative Tumour Neo Antigen Presentation, Keystone Cancer Symposium,

Keystone Colorado USA, March 19-24 2005

Postoperative Tumour Antigen Presentation, Royal Australasian College of Surgeons

Annual Scientific Congress, Perth Western Australia, May 9-14 2005

Postoperative Tumour Antigen Presentation, Australasian Medical Science Research

Symposium (WA), Perth Western Australia, June 7 2005

Surgery Re-Sets the Anti-Tumour Immune Response, Australasian Society of

Immunology National Convention, Melbourne Victoria, December 4-7 2005

Post-operative tumour antigen presentation in murine models of malignancy. Urological

Society of Australasia Annual Scientific Meeting, Brisbane Queensland, March 26-30

2006

Intra-tumoural anti-CD40 agonist regresses local recurrence and metastasis after

surgery. Australasian Medical Science Research Symposium (WA), Perth Western

Australia June 9 2006

Sculpting the immune system with lymph node surgery. Royal Australasian College of

Surgeons Annual Registrar’s Day (WA), Perth Western Australia, July 22 2006


surgery. Royal Australasian College of Surgeons Annual Registrar’s Day (WA), Perth

Western Australia, July 22 2006

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surgery. Royal Australasian College of Surgeons State Meeting (WA). Bunker Bay

Western Australia, August 5–6 2006

Sculpting anti-tumour immunity with sentinel lymph node surgery. Australasian Society

of Immunology Annual Conference, Auckland New Zealand, December 2–7 2006

The immune benefit of cancer surgery: lessons from sinecomitant immunity.

Australasian Society of Immunology Annual Conference (Young Scientist Session),

Auckland New Zealand, December 2–7 2006

Locally delivered agonistic anti-CD40 antibody in murine models of post-operative

recurrence and metastasis. Urological Society of Australia and New Zealand Annual

Scientific Meeting, Adelaide South Australia, February 18-22 2007

Immune implications of sentinel lymphadenectomy. Urological Society of Australia and

New Zealand State Meeting (WA), Mandurah Western Australia, October 26-28th

2007

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Papers in Preparation

Brown MD, Vivian JB, Robinson BWS, Hall JC, Currie AJ. Immune implications of

sentinel lymph node surgery

Brown MD, Vivian JB, Robinson BWS, Hall JC, Currie AJ. The anti-tumour immune

benefit of surgery: conflicting paradigms and potential mechanisms

Brown MD, Vivian JB, Robinson BWS, Hall JC, Currie AJ. Post-operative tumour

antigen presentation

Brown MD, Vivian JB, Robinson BWS, Hall JC, Currie AJ. The immune benefit of

cancer surgery: lessons from sinecomitant immunity

Brown MD, Vivian JB, Robinson BWS, Hall JC, Currie AJ. Empirical evidence for

combined surgery and immune therapy approaches in solid malignancy

Brown MD, Vivian JB, Robinson BWS, Hall JC, Currie AJ. Anti-tumour immunity from

the surgeon’s perspective

Brown MD, Vivian JB, Robinson BWS, Hall JC, Currie AJ, Nelson D. Locally

delivered agonistic anti-CD40 antibody in murine models of post-operative recurrence

and metastasis

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Abstract

Only 50% of patients diagnosed with cancer can expect to survive more than five

years,1 and overall cure rates have remained static for some thirty years.

2,3 Within the

last decade, there has been resurgence in interest for the use of immune therapy to

improve oncologocial outcomes. While immune treatments have been disappointing as

monotherapies (responses are usually <20%),4-9

there is emerging evidence that immune

therapy can be effective when combined with surgery.10-19

Although there is clear

evidence that surgery impacts on general aspects of immunity, little is known about how

surgery affects key parameters of tumour-specific immunity. Until such insights are

obtained, the optimum strategy for combining surgery and immune therapy is likely to

remain unclear.

In this thesis, a haemagglutinin (HA) transfected murine mesothelioma tumour

(AB1HA)20

was employed to study the effects of surgery (primary resection and/or

sentinel node biopsy) on tumour-specific immunity. Using this tumour model, the HA-

specific immune response was tracked in vivo, to “spy” upon endogenous tumour-

specific immunity. Particular parameters of focus were: tumour antigen cross

presentation, tumour-specific cytotoxic T lymphocytes (CTL), sentinel nodes, and

resistance to re-challenge.

Antigen presentation was found to be highly efficient since both micro-metastases and

small primary tumours were associated with robust antigen presentation. Antigen

presentation was directly related to tumour size, but always confined to the sentinel

lymph nodes. Surgery, in the forms of primary resection and/or sentinel node biopsy,

had a profound effect on antigen presentation. Primary tumour resection produced a

gradual decline in antigen presentation, until it was no longer detectable at two weeks

after surgery. As was the case pre-operatively, tumour antigen presentation was

confined to the sentinel nodes for all post-operative time points. When sentinel node

biopsy was combined with primary resection, antigen presentation could be

immediately and completely ablated. If tumour remained in situ when sentinel nodes

were removed, antigen presentation shifted to more distant (or systemic) sites. It was

not possible to predict the nodes which presented tumour antigen after sentinel node

biopsy, but a lag phase of three to five days preceded the shift.

- 16 -

This thesis highlights the complex role of tumour antigen in the adaptive immune

response. While surgery could reduce tumour antigen, this reduction correlated with an

improvement in CTL function and tumour resistance. Not only did CTL function

improve, but unlike pre-operatively, it was detectable systemically after surgery. It was

postulated that tumour antigen presentation had a suppressive effect on tumour-specific

immunity, and may tether tumour-specific CD8+ to the sentinel node. By extension,

surgery could “slip the antigen tether”, releasing CD8+ from the sentinel nodes and

improving systemic CD8+-mediated tumour resistance.

The contribution of sentinel nodes was similarly complex. As previously described by

others,21,22

there seemed to be a topography of immunological function across sentinel

nodes. The sentinel nodes closest to a tumour exhibited poor tumour-specific CD8+

proliferation and tumour target lysis in vivo, but the nodes of intermediate distance

functioned well. Moreover, while sentinel nodes were a reservoir for tumour antigen,

and while a decline in antigen presentation correlated with enhanced tumour resistance

after surgery, sentinel biopsy was detrimental to tumour immunity. That apparent

paradox could be related to the disproportionate representation of tumour-specific CTL

within the sentinel nodes. Thus while sentinel node biopsy could reduce tumour antigen,

it may also remove the pool of CD8+ that would otherwise egress from the sentinel

nodes and acquire more effective cytotoxic characteristics.

To integrate the concepts of this thesis, the principle that surgery enhanced tumour-

specific immunity should be highlighted. This phenomenon, previously described as

“operation immunity”23,24

or “sinecomitant immunity”25

, provides a rationale for

combining surgery with immune therapy. Importantly, sinecomitant immunity was

strongest away from the surgical site and beyond the early post-operative phase. It

depended on three, logically linked factors: a decline in antigen presentation after

surgery, the presence of sentinel lymph nodes, and CTL. By uncovering these

requirements for sinecomitant immunity, and by identifying the effects of surgery on

tumour specific immunity more generally, this thesis provides an empirical framework

by which surgery and immune therapy may be combined.

Specifically, it is now hypothesised that the early and intermediate post-operative phase

may present a “window of opportunity” for immune therapy. During that period,

patients may be optimally responsive to immune treatments: tumour associated

- 17 -

suppressive factors may decline,26,27

and tumour antigen presentation is falling. The

importance of antigen decline during this phase suggests that tumour vaccines may

initially be inappropriate. In the early post-operative phase, it may rather be suitable to

aid the effector arm of the immune system, e.g. by activating anti-CD40 antibody (as

demonstrated in this thesis), CD4+ regulatory T cell (Treg)-targeting therapy (e.g.

cyclophosphamide), or adoptive transfer therapy. At delayed time points after surgery,

immune suppression and tumour antigen load may be re-constituted by recurrent

disease. In that setting, strategies to further debulk tumours (e.g. chemotherapy,

radiotherapy and surgery) and/or more complex immune interventions (e.g.

lymphodepletion + adoptive transfer + tumour vaccination) may be required.28

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Index of Figures

Figure 1.1. William Coley – the father of immunotherapy. ....................................... - 23 -

Figure 1.2. Simplified view of anti-tumour immunity ............................................... - 37 -

Figure 3.1. Growth kinetics of subcutaneous AB1HA in wild type and nude mice .. - 77 -

Figure 3.2. Location of HA-specific presentation in AB1HA tumour bearing mice . - 78 -

Figure 3.3. CL4 proliferation during tumour growth ................................................. - 79 -

Figure 3.4. Specificity of CL4 proliferation. ............................................................. - 80 -

Figure 3.5. Surgery for AB1HA tumours .................................................................. - 81 -

Figure 3.6. CL4 proliferation before and after surgery .............................................. - 82 -

Figure 3.7. Completeness of resection. ...................................................................... - 83 -

Figure 3.8. CL4 proliferation with locally recurrent or “metastatic” AB1HA. ........ - 85 -

Figure 3.9. HA-specific CD4+ proliferation before and after surgery. ...................... - 87 -

Figure 3.10. Flow cytometry for DC cell phenotyping. ............................................. - 88 -

Figure 3.11. DC phenotype before and after surgery. ................................................ - 89 -

Figure 3.12. Post-operative in vivo CTL ................................................................... - 90 -

Figure 3.13. Primed in vivo CTL after surgery .......................................................... - 92 -

Figure 4.1. Concomitant immunity in the AB1HA model. ........................................ - 99 -

Figure 4.2. Sinecomitant immunity in the AB1HA model. ..................................... - 100 -

Figure 4.3. Sinecomitant immunity in the surgical site. .......................................... - 102 -

Figure 4.4. Sinecomitant immunity: significance of re-challenge dosage. .............. - 103 -

Figure 4.5. Effect of surgical wounding on sinecomitant immunity........................ - 105 -

Figure 4.6. HA specific immunity does not dominate the sinecomitant response ... - 106 -

Figure 4.7. Sinecomitant immunity in BALB/c nu-/-

............................................... - 106 -

Figure 4.8. The effect of T cell depletion on sinecomitant immunity ...................... - 107 -

Figure 4.9. Incomplete surgery did not protect against new tumour challenges...... - 109 -

Figure 4.10. Tumour antigen persistence partially ablated sinecomitant immunity - 110 -

Figure 4.11. Distribution of HA-specific CD8+ T cells. .......................................... - 112 -

Figure 4.12. CD127 and CD44 analysis of CD8+Pentamer

+ cells ........................... - 113 -

Figure 4.13. Expression of CD44+ in Pentamer+ and Pentamer

- CD8

+ ................... - 114 -

Figure 4.14. Expression of CD127 in CD8+CD44

+ populations .............................. - 115 -

Figure 4.15. Representative flow cytometry for Treg quantification. ....................... - 116 -

Figure 4.16. Treg frequency pre- and post-operatively ............................................. - 117 -

Figure 4.17. Treg remained despite PC61 mAb. ....................................................... - 118 -

Figure 4.18. Correlation of Treg depletion with tumour emergence ......................... - 119 -

- 19 -

Figure 4.19. Effect of Treg depletion on local recurrence. ........................................ - 120 -

Figure 4.20. MSC in tumour bearing and post-operative mice. ............................... - 121 -

Figure 4.21. Response to immune therapy after surgery. ........................................ - 123 -

Figure 4.22. Response to immune therapy in the healthy flank and surgical site. ... - 124 -

Figure 5.1. Transit of methylene blue dye into the sentinel nodes. ......................... - 137 -

Figure 5.2. Traffic of DC to the sentinel nodes........................................................ - 139 -

Figure 5.3. Cross presentation and in vivo CTL function after surgery. .................. - 142 -

Figure 5.4. Tumour proximity and nodal function. .................................................. - 143 -

Figure 5.5. Viability and assay cell penetrance in the axillary and inguinal nodes. - 144 -

Figure 5.6. Nodal invasion: effects on antigen presentation and in vivo CTL. ........ - 145 -

Figure 5.7. Primary resection with sentinel node excision. ..................................... - 146 -

Figure 5.8. Antigen presentation after resection and sentinel node biopsy. ............ - 147 -

Figure 5.9. Cross presentation from local recurrence, after sentinel node removal. - 148 -

Figure 5.10. Effect of sentinel node removal on survival from re-challenge .......... - 150 -

Figure 5.11. Re-challenge after sentinel node sampling or delayed biopsy............. - 151 -

Figure 5.12. The post-operative CD8+ effector egress postulate. ........................... - 157 -

Figure 5.13. Predicted implications of sentinel biopsy: scenario 1.......................... - 158 -



Figure 6.1. Derivation of RencaHA sub-clone......................................................... - 166 -

Figure 6.2. RencaWT and RencaHAM in wild type and congenic BALB/c nu-/-

.... - 167 -

Figure 6.3. Presentation of HA from subcutaneous RencaHAM. ............................ - 168 -

Figure 6.4 Pulmonary morphology and histology post intravenous RencaHAM .... - 171 -

Figure 6.5. Antigen presentation from i.v. RencaHAM. .......................................... - 172 -

Figure 6.6. Antigen presentation from intra-cardiac RencaHAM............................ - 173 -

Figure 6.7. Gross morphology and histology of orthotopic RencaHAM. ................ - 174 -

Figure 6.8. HA presentation from orthotopic RencaHAM. ..................................... - 175 -

Figure 6.9. Pulmonary micrometastases from orthotopic RencaHAM. ................... - 176 -

Figure 6.10. Evidence of nodal invasion from RencaHAM..................................... - 177 -

Figure 7.1. The window of opportunity for post-operative immune therapy. ......... - 187 -

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Chapter 1

- 21 -

1. Introduction

Cancer surgery involves the removal of primary tumour mass, with or without a

regional lymph node procedure. Surgery is much maligned as an immune suppressive

treatment,29-33

but this seems at odds with several key phenomena. Firstly, the presence

of occult tumour cells and/or metastases in patients with clinically localised breast

cancer,34

colon cancer,35

melanoma,36

and prostate cancer37,38

has been well described.

In such instances, at least a proportion of patients may never present with recurrence

after primary resection, or they may enjoy a considerable period of remission - a

phenomenon known as “cancer dormancy”.3,39-41

This would seem impossible if surgery

was detrimental to tumour resistance.

Secondly, the spontaneous regression of metastases after primary resection has been

well described in melanoma and renal cell carcinoma.42

It has also been documented in

many other malignancies including: oesophageal cancer,43

gastric cancer,44

and

mesothelioma.45

If surgery was immune suppressive, such a phenomenon would be

unlikely.

Thirdly, long term survival is achievable if patients are able to undergo surgical

resection of metastases (metastectomy) in virtually every solid malignancy.3 While such

patients would have occult residual disease by definition, many enjoy prolonged

remission and some 15 – 20% are cured.3 If surgery was detrimental to the control of

micrometastases, this would seem inconsistent with the benefit of metastectomy.

Finally, there is increasing evidence that surgery boosts tumour-specific immunity by

numerous mechanisms, including: a reduction in myeloid-derived suppressor cell

(MSC) levels,46,47

a shift in CD4+ memory phenotype,

48 and a decline in the effector

requirements for tumour eradication.26

Given this immune benefit of cancer surgery,

combined surgery/immune therapy strategies for malignancy are advocated in this

thesis.

The concept of combining surgery with immune therapy dates back to the 19th

century.

In the 1800s, Verneuil recognised that post-operative infection delayed the onset of

recurrence and/or improved cure rates from resection.49,50

Typically, Verneuil would

leave cancer resections open or loosely approximated, intentionally facilitating post-

- 22 -

operative suppuration.49

As it is now recognised, bacterial infections elute molecular

danger signals (e.g. cytosine phosphorothioate guanine oligodeoxynucleotides, CpG-

ODN) which upregulate tumour immunity by numerous mechanisms (including the

activation of antigen presenting cells (APC).51

Indeed, in the era of Verneuil, the

practical application of infection (with or without surgical resection) became a popular

strategy for the amelioration and/or eradication of cancer.49,52

With the popularisation of aseptic technique, the use of bacterial infections to eradicate

cancer and/or improve results from cancer surgery declined.49

It was then in the late 19th

century, that the New York surgeon William Coley noted regression of sarcoma in a

patient who developed Streptococcal infection.49

This led Coley to embark on a

systematic study into the use of bacterial products with or without surgery, for the

treatment of malignancy.

Coley (see Figure 1.1), who is now credited to be the father of immunotherapy,49

developed a number of bacterial vaccines, including heat-killed Streptococcus pyogenes

and Serratia marcescens (Coley‟s Toxin), which was applied intra-tumourally and/or

topically.49

However, after Coley‟s death in 1936 and following the popularisation of

antibiotic use in surgery,49

there was a decline in efforts to combine surgery with

immune activating agents.

The later parts of the 20th

century and the early 21st century have seen a resurgence in

interest for the use of immune treatments in combination with surgery.3 There is

persisting research in the use of non-specific immune activating agents (like bacterial

toxins, as used by Coley),53

but many modern approaches employ a tumour-specific

approach (active specific immunotherapy).49

Indeed, the contemporary literature reports

a vast array of treatments that have been combined with surgery, in attempts to improve

the static cancer cure rates of the last 30 years.49

Most solid malignancies have been tackled with combined surgery/immune therapy,

including: melanoma54

and carcinomas of the breast,55

colon,56

prostate,57

lung,58

and

kidney.9,17

There are innumerable permutations in the type of immune treatment used,

the timing of immune therapy, mode of therapy (systemic,59

topical,60

or intra-

tumoural),61

extent of resection, accuracy/extent of staging,3 duration of immune

intervention, and whether the immunotherapy was combined with conventional

- 23 -

strategies. Some trials restricted therapy to cytokines,59

others trialled non-specific

immune activating treatments,62

some tried tumour vaccines as monotherapies,54

and

others used a combination of these approaches.63

Treatments were sometimes

commenced pre-operatively,64

sometimes started in the early post-operative phase,56

and

occasionally treatments were extended out to six months or more post-operatively.65

Figure 1.1. William Coley – the father of immunotherapy.

Dr William Coley practiced surgery at the New York Memorial (Sloan Kettering) Hospital between 1890

and 1936. He was the first to systematically study the use of immune treatments (in his case, heat-killed

bacteria) with or without surgery, for the treatment of solid malignancy.49,52

- 24 -

In this thesis, the immune benefit of cancer surgery is highlighted, and the essential

elements of this phenomenon are identified. This work further provides an empirical

framework by which surgery and immune therapy can be combined, rectifying

numerous deficits in our knowledge about the interaction of surgery with tumour-

specific immunity. Hopefully, the findings of this thesis will prove useful for the future

design of combined surgery/immune therapy strategies for malignancy, accelerating

developments in the field.

Subsequent discussion in this introduction will comprise three parts. In the first part, the

controversies about whether surgery boosts tumour immunity will be critiqued, because

improved tumour immunity would be the premise for combining surgery with immune

therapy. The dogma that surgery is immune suppressive will then reconciled with the

recent and historical evidence that resection benefits anti-tumour immunity. The

concept of sinecomitant immunity will also be highlighted, because the immunological

components of post-operative tumour immunity will be investigated in this work.

(Chapter 4).

In the second component of this Introduction, the evolution and anti-tumour immune

impact of sentinel node biopsy will be examined. Sentinel node biopsy is an

increasingly utilised technique of surgical prognostication, yet as will be explained,

little is known about the effects of sentinel biopsy on anti-tumour immunity. The

immune impact of sentinel node biopsy will be studied in Chapter 5 of this work.

The final aspect of Chapter 1 will provide a brief sketch of anti-tumour immunity,

highlighting the nexus of interaction between APC, CTL and helper T cells (TH). The

effects of surgery upon the tumour antigen presentation nexus have not previously been

studied in vivo, and examining this will be a major objective of this thesis.

The potential role of surgery in the disruption of tumour associated suppressive

networks will also be elucidated, because this phenomenon accounts for the anti-tumour

immune benefit of cancer surgery. Certain interactions between resection and tumour

associated suppression will subsequently be investigated in this thesis, including: the

role of MSCs, dendritic cell (DC) phenotypes, Treg, and antigen itself.

- 25 -

1 .1 . Rat ionale for Surgery / Immune Therapy

If surgery is to be combined with immune therapy, it is important to understand the

effects of operation on anti-tumour immune function. There is an existing body of

research that addresses the profound effects surgery can have on general immune

function. However, there is limited information about how surgery affects tumour-

specific immunity. In this section, current dogma about the effects of surgery on

immune function is dissected, and then reconciled with the central argument of this

thesis: that surgery improves tumour immunity.

1 . 1 . 1 . Effects of surgery on inf lammation

Surgery represents controlled trauma, producing inflammation proportionate to the

degree of the trauma.29,68

When trauma increases in magnitude, the inflammatory signal

shifts from immune stimulation to feedback loops of immune suppression.29,30

Thus the

effect of surgery on immune function may relate to numerous variables, including: type

of anaesthesia,69

presence/absence of blood transfusion,70-72

size of incision, amount of

tissue disruption/dissection, vascularity of the field, operation time, temperature

changes, and surgical approach (open or laparoscopic).73

To induce inflammation, surgery produces a flurry of endocrine, neural and cytokine

signals. These signals include local elution of PgE2,74

disseminated catecholamine

release,75

increase in adrenal corticosteroid production,74,76

and changes in cytokine

levels.74

Such cytokines are released from monocytes and macrophages at the wound

site, especially TNF, Il-1, and Il-6.68

Cytokine release occurs within hours of surgery,

and levels remain elevated for up to 3 days. An increase in hepatic acute phase reactant

production also occurs 77

(e.g. CRP, fibrinogen, haptoglobin) as well as elevated

systemic inflammatory markers (fever, elevated white cell count, and tachycardia).68,77

1 . 1 . 2 . Effects of surgery on innate immunity

Of the acute phase reactants released after surgery, CRP is perhaps the most studied. It

rises within hours of the surgery, peaks at 72 hours post-op, and remains elevated for 2

weeks.30,78

CRP may enhance neutrophil function and phagocytosis,78,79

but overall,

surgery is thought to depress neutrophil function.73

The cause of depressed neutrophil

function is unknown73

but a reduction in chemotaxis and phagocytosis has been

observed.80

- 26 -

Prostaglandin release from surgical wounds may also modulate natural killer cell (NK)

counts and function after surgery.81

One study suggests a 2 day nadir in natural killer

cell counts and cytotoxicity after hysterectomy,73

but a post-cholecystectomy82

study

paper suggested that decreased natural killer function could persist for up to 30 days

post-op. Therefore, the extent of impaired NK immunity after surgery remains

unknown.

1 . 1 . 3 . Effects of surgery on adapt ive immunity

Like innate immunity, surgery may have profound effects on adaptive immunity.

Several studies have assessed delayed type hypersensitivity (DTH) skin reaction after

surgery, demonstrating a depression in DTH, that was proportional to the extent of the

surgery (laparotomy versus laparoscopy or mini-laparotomy).83

The effect on DTH was

assessed early in each case: immediately,83

two days post-op,83

or three days post-op84

.

The time to recovery remains unclear, but is thought to be brief.85

The mechanism of depressed cell mediated immunity is undefined. Surgery and/or

trauma may be associated with a brief shift in the TH/Treg ratio (less than 7 days post-

op),85

with reduced production of Il-2,86

downregulation of MHC Class II expression on

macrophages,87,88

reduced induction of immature DC from peripheral blood

monocytes,89

and a reduction in the efficiency of antigen presentation.90

In addition to impairing antigen presentation and overall cell mediated immunity,

surgery may induce a preponderance to humoral immunity (a “TH1 to TH2 shift”).91

This

shift is mediated by a downregulation of TH1 cytokines (Il-2, Il-12, IFN, TNF, Il-

1)92

along with increased production of TH2 cytokines (Il-10, Il-1rA, sTNFr, sIL-

2r).93,94

Once again, the duration of the TH1 to TH2 shift is unknown.

1 . 1 . 4 . Surgery and overal l immune funct ion

The existing weight of literature suggest that surgery impairs general immune function.

By disrupting tissue, surgery may evoke a monocyte-derived cytokine profile that

enhances TH2-based inflammation, hinders antigen presentation, reduces cell mediated

immunity, and promotes a humoral response. However, this impairment is thought to be

brief, and reversible.85

- 27 -

Prevailing dogma also suggests that surgery impairs the anti-tumour immune response.

Many publications document accelerated tumour growth after surgery. For instance,

surgery accelerates pulmonary metastases from tail vein injection,31,95

produces faster

growth of spontaneous pulmonary metastases,32,33

enhances the growth of

intraperitoneal tumours,96

increases the number of hepatic metastases after portal vein

injection,97

and accelerates flank tumour growth.98

1 . 1 . 5 . Surgery and immunity: confl ic t ing paradigms

If surgery impairs anti-tumour immunity, the duration of that impairment is unknown.

Existing papers hint that surgical impairment of tumour immunity may be a short term

phenomenon (less than two weeks in rodents),96

and one group‟s data even suggests that

general immunity recovers to baseline at 24 hours post-operatively.99

Moreover, if surgery harms general aspects of immune function, modern techniques and

contemporary drug treatments might greatly attenuate that harm. In animal models,

blocking the immune mediators of surgery (e.g. non-steroidal anti-inflammatories100

and

corticosteroid inhibitors101

) can reduce surgical immune suppression. In addition,

changing to regional anaesthesia102

and minimally invasive techniques 73,103,104

could

further minimise the immunologic harm of surgery.

Finally, and more fundamentally, in almost every paper reported to describe accelerated

tumour growth with surgery, the surgery of interest was a sham procedure. As discussed

in 1.3.2, tumours normally suppress the immune response. Since cancer surgery reduces

tumour burden, it may improve the immune response rather than impair it. To assess the

effect of surgery on anti-tumour immune function more accurately, the surgery must

involve tumour resection – not irrelevant trauma. When this approach is taken, the

weight of literature shifts to suggest surgery improves anti-tumour immunity.

1 . 1 . 6 . Surgery improves ant i - tumour immunity

The anti-tumour immune benefit of cancer surgery was discovered nearly one hundred

years ago.23,24

Uhlenhuth and his co-authors reported that surgically treated rats could

resist a second challenge of the same tumour, and they dubbed this phenomenon

“operation immunity”. They noted that resection had to be complete, or else immunity

did not develop.24

- 28 -

Some 60 years later, Fisher et al proposed the term “sinecomitant immunity” to describe

the anti-tumour reaction against a tumour re-challenge after surgery.25

Sinecomitant

immunity was distinguished from “concomitant immunity”,25,105

where rejection of a

second tumour challenge is sometimes seen in animals with progressive primary

tumour. As such, the immune benefit of surgery can be understood as the excess of

sinecomitant immunity (immunity against re-challenge after primary tumour removed)

over concomitant immunity (immunity against re-challenge with primary tumour left in

situ).

Preceding research suggests the extent of sinecomitant immunity depends on the

antigenicity of the tumour, the size of the primary tumour, the time of re-challenge, and

the strength of the re-challenge.24,106,107

In previous publications, resistance to re-

challenge was best seen in immunogenic tumours (i.e. tumour models where protection

occurs after irradiated vaccination),108

relatively small tumours (<10mm in diameter),109

and when the re-challenge was delayed at least seven days after the surgery.110,111

Sinecomitant immunity has previously been thought to be weak, since inoculums >105

cells could overcome resistance in some models.108

1 . 1 . 7 . Other benef i t s of surgery/ immune therapy

Not only might surgery improve tumour immunity,112,113

but it may offer a number of

other benefits. For instance, de-bulking could improve a patient‟s overall function and

reduce symptoms.112,113

It can also eliminate the primary as a source of pain, para-

neoplastic syndromes, and haemorrhage.112

Primary resection also prevents further

metastases from that site,112

and reduces the number of tumour cells. Reduced tumour

burden may enable lesser doses of systemic therapy and better response.112

Moreover,

cancers are metabolically active and are associated with a degree of cachexia. By

removing metabolically active tumour and reducing the levels of cachexia-inducing

cytokines (e.g. Il-1)114

surgery could alleviate the catabolic state. Speculatively, this

might improve the availability of conditionally essential amino acids (e.g. glutamine)115

and improve immune function. Finally, tumour resection provides tissue. Not only does

this provide a definitive diagnosis and assist with prognostication, but it also facilitates

research (e.g. the preparation of tumour vaccines, tumour infiltrating lymphocyte

therapy etc.)112

- 29 -

1 . 1 . 8 . Against surgery/ immune therapy

There are numerous arguments against combining surgery with immune therapy, and

these should be acknowledged. Surgery exposes the patient to the risk of operative

mortality, and to the potential for considerable morbidity. The latter morbidity may

even preclude subsequent immune therapy.14,112,113

Most worryingly, many tumours

promote angiogenesis, but some tumours may release anti-angiogenic and non-specific

metastasis-suppressing factors107

(e.g. angiostatin, endostatin).116,117

Thus, in some

cancers, metastases may grow more rapidly after primary resection, because of

alleviated angio-suppression.107,116,117

Finally, surgical manipulation of tumours may

promote seeding and new tumours,34,118,119

, abrogating the benefit of immune therapy.

Indeed, surgical wounds are a rich environment for tumour seeding: hypoxia, fibroblast

activation, and paracrine factors promote tumour growth and/or suppress immunity.120

1 .2 . Immune Effec ts o f Sent ine l Node Biopsy

In the first section of this introductory Chapter, it was argued that surgery improves

tumour immunity rather than detracts from it. However, discussion has focussed on the

effects of tumour resection only. Oncological procedures may also entail a lymph node

procedure, and more recently, a sentinel node biopsy. Thus to understand the effects of

surgery on tumour immunity, it is also important to consider lymphadenectomy.

Lymph nodes are anatomically and functionally elegant collections of immune tissue,

strategically located on the efferent lymphatics of regional tissues. Lymph nodes are

frequently frequent sites for metastasis and for this reason, cancer surgery has

traditionally involved lymphadenectomy.22

The practice of lymphadenectomy has

undergone considerable evolution in recent decades, but the immune implications of

lymph node removal remain unknown.

1 . 2 . 1 . The evolut ion of lymph node surgery

In the early 1700s, Valsalva proposed that cancer was a local lesion which spreads via

the lymphatics to the regional nodes.121,122

This led to the practice of wide local

resection and regional node clearance, which has prevailed until the last few decades.122

However, tumours may spread by the haematogenous route, or they may bypass

regional nodes via lymphatico-lymphatic and lyphatico-venous shunts. Thus cancers

may avoid lymph glands and disseminate systemically, even at an early stage.123

From

- 30 -

the modern view-point, cancer is considered a highly complex, heterogeneous, and

systemic disease.122

Loco-regional lymph nodes are no longer thought as “barriers to

metastasis”, but simply organs that are frequently affected by cancer spread.122

Thus

node dissection is beneficial only to provide staging information about the cancer, or to

improve survival for particular cancers (e.g. testicular cancer,124

, melanoma,125

and

penile cancer)126,127

where nodes can be the solitary sites of metastasis. Given that

lymphadenectomy is primarily for prognostication, an assessment of node status without

extensive dissection is preferred. To this end, sentinel node biopsy has been

popularised.

1 . 2 . 2 . Sent inel lymph nod es

The sentinel node concept was first proposed by Cabanas in 1977.128

Sentinel nodes are

those lymph glands in direct lymphatic communication with the tumour site.129

The

value of sentinel node biopsy is that its tumour status (involved or not involved),

predicts the status of the entire regional node group.129

By sentinel biopsy, it is possible

to provide prognostication without exposing the patient to the risks and morbidity of

extended lymphadenectomy.

Notably, the sentinel nodes include not only the first lymph node seen on dynamic

lymphoscintigraphy, but all nodes in direct communication with the tumour site. Thus

identification of the sentinel node is best accomplished by visualising the lymphatic

channels130

(e.g. using isosulphan blue dye), together with radioactive isotope (to assist

in finding the nodes).131-133

Sentinel lymph node biopsy has been validated for a number

of malignancies, most rigorously in breast carcinoma,134

melanoma,135

and squamous

cell carcinoma. 132,136

1 . 2 . 3 . Immune funct ion of sent inel lymph nodes

By definition, the sentinel lymph node would encounter a tumour first. Therefore,

sentinel nodes are probably the site of initial tumour antigen presentation.137

Indeed, the

sentinel node has been shown to produce the greatest volume of IFN, GM-CSF and Il-

2.137,138

However, as the sentinel nodes are the earliest affected by the tumour, they

might also be most susceptible to immune suppression.21

Consistent with this, T cells of the sentinel lymph nodes have the highest levels of

TCR downregulation (evidence of T cell suppression) in nodes of patients with breast

- 31 -

cancer.139

Sentinel nodes also produce more Il-10 than non-sentinel nodes in

melanoma140,141

which may induce immune suppressive DCs142,143

, and reduce T cell

activation.22

Additionally, relative to mature DC, the DC of the sentinel nodes are less

dense,141,144

have reduced dendrite length,141,144

low co-stimulatory molecule

expression, 27

and decreased MHC Class II expression.145

A greater proportion of those

DCs also express indoleamine-2,3-dioxygenase (IDO),146

which reduces T cell

responses.147

1 . 2 . 4 . Immune consequences of sent inel node surgery

With numerous advances in tumour immunology, the immune impact of node removal

can be examined with new insight. Firstly, the tumour draining lymph nodes are the

principal site of cell associated tumour antigen presentation20,148,149

(although not soluble

antigens)150

. If tumour draining nodes are actually sentinel nodes (Chapter 5), then

sentinel biopsy might dilute antigen below immunogenic thresholds (through passage

into the vasculature),151

completely ablate antigen presentation,152

or force antigen

presentation to secondary lymph nodes.153

The validity nor sequelae of these hypotheses

have never been tested in vivo.

If tumour antigen can be eradicated by sentinel node biopsy, the impact of that

phenomenon is unknown. In one approach for colorectal cancer, dubbed “immune

corrective surgery”, (ICSTM

, Biocrystal, Columbus Ohio USA), investigators attempt to

resect all lymph nodes where tumour antigen is found.154

The technique uses radio-

isotype labelled anti-tumour antigen antibody (anti-TAG-72) to localise sites of antigen

presentation. Those nodes are then removed at the time of surgery. The authors report

an improvement in survival with this technique in a phase 1 study,154

but a larger study

has yet to be published.

Secondly, T lymphocytes localise in the sentinel lymph nodes of primary

tumours,48,155,156

possibly because of tumour-derived chemokine signals, inflammatory

signals, or persisting tumour antigen. Evidence for effector localisation to the draining

nodes includes not only modern data (adoptive transfer studies, tetramer staining etc.)155

but also the simple fact that draining lymph nodes (rather than naïve nodes and non-

draining nodes) convey protection against that tumour to syngeneic, naïve mice.153

Therefore, if sentinel node biopsy is performed, a patient may be depleted of tumour-

specific effectors. The result may be increased susceptibility to local recurrence and

- 32 -

metastases. Alternatively, the local lymph nodes might only be a component of a

generally activated lymphoreticular immune network.157

Removing the sentinel nodes

might therefore be harmless, as remaining components of the network provide tumour

surveillance. Which of these theories are correct, remains unknown.

Sentinel node biopsy may also impair immunological memory, particularly the

generation of memory CD4 cells. While established memory cells are maintained in the

absence of lymphoid tissue, CD4+ cells require secondary lymphoid organs for memory

differentiation.158

Only a handful of studies from the 1960s and 1970s have examined the role of lymph

node dissection on tumour growth using syngeneic models. Sentinel node biopsy had

not been established at that stage and results were highly variable. Several publications

suggested that lymph node dissection had an insignificant effect on the growth of

primary tumours, local re-challenges, distant re-challenges, or metastases.152,153,157,159

Others reported that node dissection accelerated the growth of nearby re-challenges, but

not distant tumours.160

Further publications suggested that node dissection slowed the

onset of concomitant immunity161

and impaired the response to re-challenge (local and

distant).161-163

In humans, the picture is even more confusing. In some tumours, node

dissection may reduce local recurrence164

and/or metastases.164,165

In other tumours (e.g.

breast) variations of node dissection made no difference to recurrence and/or survival

from metastasis.166-169

Inconsistencies between the findings may relate to whether

lymph node metastases were present or not, whether primary resection was complete or

not, the extent and timing of node dissection, species and subspecies variation,

immunogenicity of the tumours,170

size of the primary, sites of re-challenge, and the

strength of the re-challenge inoculum.161

1 . 2 . 5 . Nodal invas ion and tumour proximity

The location and tumour status of sentinel nodes may affect their contribution to tumour

resistance, and by extension, the effects of sentinel node removal. Numerous studies

demonstrate that tumour proximity affects node function. Those lymph nodes closest to

a tumour have less mitogenic reactivity for allo-reactive CTL,171

reduced CD4+:CD8

+

ratios,172,173

higher concanavalin-A induced suppressor cells,174

and less tumour

reactivity.175

- 33 -

Based on immune reactions from melanoma and breast cancer node specimens, Cochran

and colleagues proposed a topography of anti-tumour immunity across the lymph node

basin.21,22

Specifically, those nodes closest to a tumour may be exposed to the greatest

antigen stimulation, but also the strongest tumour-derived suppression. Those nodes

furthest away react poorly also, because they are too distant to be engaged by the

tumour. Thus the intermediate zone lymph nodes function best – encountering tumour

antigen, but relatively spared from tumour-derived suppression.21,22

The effect of node

dissection may therefore depend on the design of the resection: removal of closest nodes

may alleviate suppression, but removal of more distant nodes might impair immunity.

Secondly, tumour invasion may impact on the contribution of nodes to tumour

immunity. By invading the nodes, tumours may tip the profile of antigen processing

away from cross presentation and towards direct presentation. Since tumour cells have

poor MHC class I expression and low co-stimulatory molecule expression,176

the result

may be reduced priming and/or anergy. Supporting this notion, previous research

demonstrates that nodal metastasis are associated with the accumulation of suppressor

cells174

,T lymphocyte anergy, 177-179

, and the ablation of concomitant immunity.180

However, tumour-specific parameters of in vivo CTL function and antigen presentation

by tumour-invaded nodes have not been tested previously.

1 . 2 . 6 . Lymphadenectomy and immune therapy

As the immune impact of sentinel node dissection has not previously been elucidated, it

is unsurprising that little is known about how sentinel dissection affects response to

immune therapy. There are only two publications, neither of them looking at sentinel

node biopsy. Specifically, Harada and colleagues found a reduced response to Il-12

therapy after node dissection in the B16 melanoma model.181

Then, in a small clinical

trial of recurrent head and neck cancer, a reduced response to local Il-2 was observed in

patients after bilateral lymph node dissection.182

1 .3 . Surgery and Tumour-Speci f i c Immuni ty

The anti-tumour immune response provides a “last line of defence” against malignancy,

swinging into action once the normal intracellular and genetic safety mechanisms have

failed to combat carcinogenesis.39

Innate immunity may contribute to tumour

immunity,183,184

particularly through inflammation.185

However, this thesis emphasises

the effects of surgery on adaptive immunity, because tumour-specific immunity is the

- 34 -

basis for most successful immune strategies to date.186-188

Understanding adaptive

tumour immunity requires an appreciation of the antigen presentation nexus, which

consists of APC, TH, and CTL. However, no previous study describes the effects of

surgery upon the levels of antigen presentation in vivo, and in this thesis, a primary aim

is to rectify that deficit. Potential interactions of surgery with the antigen presentation

nexus are now explained, followed by a critique of the potential mechanisms by which

surgery boosts tumour immunity.

1 . 3 . 1 . Surgery and ant igen presentat ion

The antigen presentation nexus involves the processing and recognition of tumour

antigen. Those tumour antigens may be unique to an individual tumour (a new antigen,

or “neo-antigen”), tumour-specific shared antigens (e.g. MAGE), antigens common to a

particular type of tissue (e.g. PSA), tissue differentiation antigens (e.g. CEA), or

ubiquitous antigens (e.g. HER-2/neu).66

The effects of surgery upon tissue

differentiation and normal tissue antigens has been well described for various tumours

(e.g. PSA after prostatectomy,189

post-operative CEA in colon cancer190

). However,

such antigens are not foreign to the host, and are therefore subject to tolerance. Rather it

is the foreign (neo-antigens) that are most crucial to tumour rejection, and therefore

potentially the most important to consider. Unfortunately, the kinetics of post-operative

tumour neo-antigens have not previously been studied in vivo, and until this deficit can

be rectified, our understanding of post-operative tumour immunity remains deficient.

Neo-antigens may be directly presented to CD8+ T cells by the endogenous or “classical

MHC Class I” pathway.191

As with healthy cells, tumour cellular proteins are

continuously turned over. Tumour proteins marked for degradation are tagged with

ubiquitin molecules, taken up by proteosomes, and degraded into oligopeptide residues.

Those tumour oligopetides are hydrolysed by cytosolic peptidases into peptides. Most

of those peptides are recycled by the cell for protein synthesis and energy, but a

proportion are transported into the endoplasmic reticulum (ER) via the “transporter

associated antigen processing protein” (TAP). Once there, the peptides are trimmed by

ER aminopeptidase into 8 or 9 amino acid peptides, associated with MHC Class I, and

transported to the cell surface.192-197

Once on the cell surface, the tumour peptides are

presented in the context of MHC Class I. The tumour peptide is then recognised by

CD8+ cells, and that cell is targeted for destruction.

- 35 -

For direct presentation to predominate in priming, intra-lymphatic and intra-nodal

tumour must occur.198-200

However, antigen presentation can be robustly detected in the

absence of intra-lymphatic tumour and nodal metastases, suggesting a cross priming

predominance.198

Additionally, direct presentation from tumour cells is probably a weak

phenomenon, due to downregulation of co-stimulatory molecules201

and low MHC class

I expression.202

Indeed, there are many examples where direct presentation is shown to

have a minor role in tumour surveillance. For instance, chimeric mice lacking functional

APCs are unable to generate CD8+ T cell responses against tumours in numerous

models. In contrast, when APCs are provided to those mice (facilitating cross

presentation), antigen presentation is evident. 203-205

Thus while direct presentation may contribute in the process of immune surveillance,

another mechanism of antigen presentation seems to be important. This form of neo-

antigen presentation, “cross presentation”, involves the uptake and processing of

exogenous (class I) tumour antigens, using the machinery normally reserved for the

endogenous (class II) pathway. Cross presentation occurs predominantly by

professional APC (DC, macrophages),206

although B cells, endothelial cells, and

neutrophils may also cross-present.191,203,207-212

The downstream consequence of cross presentation is the delivery of tumour antigen

epitopes on class I, to CD8+ T cell receptors (TCRs). For the naïve CD8

+, the outcome

of cross presentation may be tolerance (“cross-tolerance”) or priming (“cross-

priming”).213

Which of the two occurs depends on the activation status of the APC.

That, in turn, depends on the presence of immune stimulatory (danger) signals. Those

danger signals may be from cellular products (e.g. heat shock proteins), from CD4 cells

(e.g. CD40 ligand), or microbial derivatives (e.g. CpG).191,214-216

To unify these concepts, and to understand how surgery might affect antigen

presentation, a simplified model is proposed (Figure 1.2). The first step of the process is

infiltration of the tumour by APCs, of which the DC is probably the most important.217-

220 Those APCs phagocytose tumour antigen from dead or dying tumour cells, or by

“nibbling” tumour cells.217-220

Laden with tumour antigen, APCs traffic to the draining

lymph nodes, 221

where they present to both CD4+ and CD8

+ T cells. Providing

appropriate co-stimulation is present, CD8+ cells will become activated, proliferate, and

differentiate into CTL effectors.214,215

Those CTLs then traffic back to the tumour,

- 36 -

exiting the tumour vasculature and accessing the neoplastic parenchyma. The CD8+

cells then effect tumour cytolysis by numerous mechanisms (e.g. FasL). 222,223

Helper T

cells may augment the process at numerous steps (Figure 1.2), e.g. activation of the

APC,224-229

assisting CD8+ differentiation,

230,231 and/or exerting direct anti-tumour

activity.232-234

Surgery may impact on numerous facets of this process. Firstly, by definition, cancer

resection will reduce tumour mass. While antigen presentation has been related to

certain threshold values of antigen in previous work,235

the correlation between antigen

presentation and tumour mass has not previously been studied in vivo. Also the

relationship between surgery and tumour antigen presentation in vivo has only been

mentioned in one conference abstract (no formal publications as yet).236

This shortfall in

knowledge presents a conundrum to planning post-operative immune therapy. For

instance, 50% of patients suffer progressive renal cancer after resection of apparently

localised tumour.237

In this situation, immune therapy may be useful to aid tumour

clearance. However, it is unknown whether sufficient tumour antigen remains after

macroscopic tumour clearance (i.e. an immune boosting adjuvant is most appropriate),

or whether antigen signal is a limiting factor after surgery (i.e. a vaccine is required).

Secondly, from the model presented in Figure 1.2, antigen presentation occurs within

so-called “tumour draining” lymph nodes. However, it has yet to be established whether

tumour draining lymph nodes are regional nodes (i.e. all adjacent lymph nodes to the

tumour), sentinel nodes, or other nodes entirely. Understanding this relationship

becomes increasingly important, as cancer surgery may involve removal of some (or all)

of these nodes. In addition, if the nexus of priming occurs in the sentinel lymph nodes,

then sentinel node biopsy could have profound implications for tumour immunity. To

date, no-one has examined the effects of sentinel node biopsy on tumour antigen

presentation or even tumour resistance in vivo. Finally, the interaction of CTL with the

tumour itself may be affected by surgery. If tumour associated CTL are predominantly

located in sentinel nodes, then removing sentinel nodes could have dire effects on

tumour resistance and anti-tumour immune memory. These issues are tackled in

Chapter 5.

- 37 -

Figure 1.2. Simplified view of anti-tumour immunity

Modified from reference 238. Adaptive anti-tumour immunity is thought to revolve around the nexus of

antigen presentation and CTL priming. CD4+

“help” the process at multiple steps. A: DCs acquire tumour

antigen by phagocytosis, and traffic to the draining lymph nodes. B: DCs present tumour epitopes to both

CD4+ and CD8

+ lymphocytes. C: CD8

+ cells become activated and differentiate to effector CTL. D:

CD8+ cells traffic back to the tumour and egress the tumour vasculature. E: CTL infiltrate the tumour and

effect cytolysis.

In summary, the nexus of interaction between APC, CTL, and DC has previously been

localised to the tumour draining lymph node. As surgery removes tumour bulk and/or

sentinel lymph nodes, this will likely impact on antigen presentation and tumour

immunity. By uncovering the effects of surgery on key elements of anti-tumour

immunity in vivo, particularly antigen presentation and in vivo CTL function, this thesis

provides an empirical framework for combining surgery with immune therapy. This

- 38 -

work may also assist in understanding the mechanisms by which surgery boosts tumour

resistance, further validating the principle of combining immune therapy with surgery.

Current insights into the mechanisms by which surgery boosts tumour immunity are

now discussed.

1 . 3 . 2 . Surgery and immune suppress ion networks

The anti-tumour immune benefit of surgery may arise from the disruption of “tumour

associated immunosuppressive networks”.27

While cancers may engage the host

immune system by the mechanisms discussed in 1.3, from the regrettable frequency of

cancer it is clear that tumours have the means to frustrate tumour resistance, including:

ignorance,179,206,239-243

selection of non-immunogenic clones,39,201,202,244,245

production of

immune suppressive agents (e.g. PgE2, soluble phosphatidylserine, VEGF),27

induction

of humoral TH (TH2 predominance),246-248

MSC27,246,249-256

tumour associated

macrophages (TAM),27,146,254,257

IDO+ plasmacytoid DC,

258 Treg,

259-266 expression of Fas

ligand,222,267-270

exclusion of effector cells/altered tumour vasculature,27,271-274

blocking

of effector function (e.g. release of soluble phosphatidyl serine etc.),27,39,275-279

and the

induction of immune anergy.176

Surgery may impact on numerous elements of these

“escape” mechanisms.

1 . 3 . 2 . 1 . A l l e v i a t e t u m o u r - d e r i v e d s u p p r e s s i v e f a c t o r s

Tumours are known to produce a number of factors that suppress the immune system,

including soluble phosphatidylserine, Il-10, Il-4, VEGF, and TGF.27

The accumulation

of those factors is thought to parallel tumour burden27

and by extension, reducing

tumour burden should decrease the production of those factors.3,26,27

Whether this

occurs is yet to be determined, but theoretically, a reduction in tumour derived soluble

factors should produce downstream disruption of the tumour‟s immune suppressive

network27

(MSC, IDO+ plasmacytoid DC, Treg and TAM).

1 . 3 . 2 . 2 . R e d u c e e f f e c t o r r e q u i r e m e n t s

A certain critical ratio of lymphocytes to tumour cells may be required to control

tumour growth. In theory, reducing tumour cell burden tips the ratio favourably,

increasing the probability that host immunity can control tumour.26

Furthermore, fewer

tumour cells should mean fewer mutations,26

and therefore less probability that immune

evasion characteristics will develop. This has not been tested in vivo.

- 39 -

1 . 3 . 2 . 3 . R e d u c e M S C

MSC were formerly known as “early myeloid cells”249,250

or “inhibitory

macrophages”.250,251

These are a heterogeneous population of immature, bone-marrow

derived cells that characteristically are CD11b+

and Gr-1+.246

While additional markers

for MSC have been described (CD31+

251 and Il-4R

+

252), they have yet to be

definitively typed.

MSC are thought to be recruited by the production of VEGF, GM-CSF, Il-3, and Il-6.246

They progressively accumulate in the blood and spleen, in parallel with tumour

burden.253

MSC may also localise in the tumour and in secondary lymphoid organs in

response to chemokine signals, forming an “immunosuppressive network”.27

MSC

mediate immune suppression by several mechanisms. First, MSC may produce T cell

anergy by the uptake of soluble tumour antigens and the presentation of those antigens

in a tolerogenic manner.254

However, MSC may also produce CD8+ inhibition by

antigen independent mechanisms246

that require cell to cell contact.280,281

Such mechanisms include IDO27

and arginase I.27,255

Those enzymes deplete tryptophan

and arginine in the tumour microenvironment respectively, impairing CD8+

proliferation and maintenance of the CD3 chain.256

Moreover, MSC produce

peroxynitrites (which induce CD8+ apoptosis).

246 In addition to hampering the anti-

tumour immune response, MSC can support tumour growth through the release of

tumour nutritive polyamines.282,283

Surgery reduces MSC counts in mice and humans with cancer. This occurs somewhere

between 10 days46

and 4 weeks47,284,285

after surgery. In a chemically induced BALB/c

sarcoma model, reduction in MSCs was paralleled by an improvement in cell mediated

immunity and resistance to tumour.285

However, a reduction of MSC alone is probably

insufficient to convey effective anti-tumour immunity after surgery.281

1 . 3 . 2 . 4 . P r o v i d e “ a n t i g e n h o l i d a y ”

Tumours characteristically induce anergy of the tumour-specific CD8+ repertoire.

176

One mechanism for that anergy is by the failure to provide “second signal”.176

Tumour

cell death may occur in an immunologically bland manner,238

such that “danger signals”

are not delivered and APC are not activated. Co-stimulation is probably similarly absent

with direct presentation by the tumour itself,201

adding to the problem.

- 40 -

A second postulated mechanism of tumour-induced anergy is the release of a

continuous stream of antigen. Continuous antigen exposure has been shown to induce a

state of immune hyporesponsiveness in viral models. 286-291

Indeed, maintenance of that

hyporesponsiveness requires persistence of the antigen, and T cells may recover when

removed from that chronic antigen.288,291-293

Numerous associations of antigen

associated anergy have been identified, including: TCR downregulation,286,291,294-296

TCR desensitization,293

and increased expression of negative regulatory molecules

(CD5291

and PD-1297

).

In recent years, based on findings in virology and by the detection of up-regulated PD-

L1 (B7-H1) on numerous tumours, there has been increased interest in the role of PD-1.

With acute infectious disease, the immune system is exposed to a burst of foreign

antigen. This is followed by an immune response, a wane of infection, and a reduction

in antigen levels. In this scenario, CD8+ cells are activated, produce cytokines, and

ultimately form memory.297

The immune response to chronic viral infection is different.

With chronic antigen exposure, cells are tolerised and gradually acquire an “exhausted”

phenotype.298

The exhausted phenotype features an up-regulation of the inhibitory

receptors PD-1 and CTLA4, downregulation of co-stimulatory proteins, defects in

accessory and cytokine signals, and disrupted proximal T cell receptor signalling.298

Exhausted cells function less well as effectors, do not form useful memory cells, and are

unable to control the viral infection.297

Of the two inhibitory receptors, PD-1 signalling seems critical to the exhaustion

process.299

PD-1 acts in peripheral tissues, binding two receptors: PD-L1 (B7-H1) and

PD-L2 (B7-DC).299

PD-L1 is thought to be inhibitory and PD-L2 can be inhibitory or

activating.297,299

The importance of PD-1/PD-L1 signalling is highlighted by the

recovery of exhausted cells to full effector function using a blocking monoclonal

antibody. After PD-1/PD-L1 blockade, despite chronic viral infection, there is increased

CD8+ proliferation, increased viral control, and improved cytokine production.

300

In short, tumours may induce anergy/tolerance by providing a continuous source of

antigen and the absence of a danger context. Mechanisms that reverse this phenomenon

(e.g. Il-2 and “antigen rest”) may be beneficial in overcoming the exhaustion of

continuous tumour antigen exposure.300,301

Surgery might affect antigen rest by reducing

- 41 -

the tumour burden. The degree and duration of antigen rest for the recovery of immune

responsiveness is unclear, and it may differ for memory302

and effector292

CTL.

Memory CTL may recover if antigen is absent for two days,302

whereas effector CTL

may require between 26 days and 2 months of an “antigen-free” environment.292

It is unresolved whether surgery can in fact produce antigen rest. At least in theory, if

some tumour cells are left behind, they may be below the threshold required for antigen

presentation.235

Thus an antigen-free environment may be possible after surgery, even in

the setting of residual disease. This has never been confirmed or refuted in vivo.

1 . 3 . 2 . 5 . C h a n g e m e m o r y p h e n o t y p e

The majority of antigen specific T cells that participate in an initial antigen response

will undergo apoptosis,303

leaving a small subset of memory T cells. Memory cells have

a great avidity for antigen,304

are capable of responding quickly to a recurring threat,305

have faster cell divisions,305

and more rapidly acquire effector function.306

Memory

subsets remain an area of intense research and debate.307

However, the classification

into central and effector memory subtypes seems well accepted. Central memory (CM)

cells have lymph node homing molecules expressed on their cell surface (CCR7 and

CD62L) and are found in the lymph nodes, blood, and spleen. Effector memory (EM)

cells do not have these molecules, and are found in the peripheral tissue.308

The significance of this distinction is somewhat contentious. Whether central and

effector memory develop independently308

or inter-convert is unresolved.309,310

However, central memory tends to predominate in relatively low antigen

environments.48,298,299,308

In some models at least, central memory cells control

infections more effectively, proliferate more rapidly, and generate more delayed type

hypersensitivity.299,311

In the case of tumours, antigen titres are (possibly) high and

persistent. This may mould the memory response towards an effector phenotype.

Effector memory cells may be inferior for tumour control because they tend to

proliferate more slowly, and home to regional lymph nodes more avidly.299,311

It has

already been demonstrated that surgery enables the predominance of central memory

phenotype in the CD4 compartment.48

However, this study is based on a transgenic

mouse strain that had a high frequency of tumour-specific CD4+ cells. Additionally, the

effect of surgery on post-operative CD8+ memory phenotypes has never been

investigated.

- 42 -

1 . 3 . 2 . 6 . R e l e a s e e f f e c t o r s

In response to tolerogenic priming,176,230

persistent antigen,312

inflammation,313

and/or

chemokine signals,230

tumours might trap effector cells in the draining lymph nodes.

Surgical resection might remove that trap for activated lymphocytes,112,113

enabling

dissemination of T cells,48

and enhanced systemic immunity. Systemic release of CTL

has previously been seen with CD40 therapy155,156

and CD4+ adoptive transfer

therapy.314

However, it is unknown whether this occurs after surgery.

1 . 3 . 2 . 7 . I m p r o v e o v e r a l l c e l l m e d i a t e d i m m u n i t y

Theoretically, the combination of surgical-associated improvements in immune function

should translate into enhanced tumour-directed cell mediated immunity. Direct evidence

for this remains limited, but Salvadori and colleagues demonstrated improved

allogeneic tumour rejection after primary resection when metastases were present.285

This encouraging finding suggests that surgery could be beneficial to tumour-specific

immunity, even when disease is extensive.

1 .4 . Aims and Hypotheses

The notion that surgery boosts tumour immunity remains the rationale for combining

surgery with immune therapy. The mechanisms by which surgery improves tumour

resistance remain largely hypothetical, and relate to the disruption of tumour associated

suppression networks. The impact of sentinel node biopsy on the tumour-specific

immune response is also unknown, and there has been no previous in vivo study of this

issue. These shortcomings in current knowledge will be rectified in the four

experimental Chapters of this thesis. Collectively, these studies describe the effects of

surgery (primary resection and sentinel node biopsy) upon key elements of tumour-

specific immunity, providing an empirical framework for the combination of surgery

and immune therapy in the treatment of solid malignancy.

Chapter 3 aims to determine the effects of surgery on tumour antigen presentation, and

the implications of those antigen changes on CTL function, phenotype, and distribution.

It is hypothesised that surgery reduces cross presentation and that reduced post-

operative tumour antigen presentation could improve CTL function and tumour

immunity.

- 43 -

In Chapter 4, the effects of surgery on key mediators of the immune suppressive

network (MSC, plasmacytoid DC, Treg) and overall anti-tumour immunity are explored.

Furthermore, the possible relationships between tumour antigen presentation and overall

tumour resistance is examined. Building on the findings of Chapter 3, it is hypothesised

that tumour antigen presentation and tumour resistance are inversely proportional. It is

also postulated that tumour resistance is inversely related to post-operative MSC, Treg,

and plasmacytoid DC levels.

Chapter 5 examines the immune significance of sentinel node excision. Building on

Chapters 3 and 4, it is hypothesised that sentinel node biopsy will shift antigen

processing to the next most proximal nodes, and that this will be detrimental to tumour

resistance overall. As a secondary objective, the hierarchy of antigen presentation and

CTL function of the sentinel nodes, based on tumour proximity, are explored.

Chapter 6 discusses an orthotopic murine kidney cancer model, providing insights into

tumour-specific immunity that are not possible with AB1HA. The model is especially

useful for future research because kidney cancer is a promising target for combined

surgery/immunotherapy, as facilitated by this thesis. The orthotopic model should allow

analysis of combined surgery/immunotherapy strategies in kidney cancer, and the direct

testing of principles outlined in Chapters 3 – 5.

The main findings of this thesis are summarised in Chapter 7, which is the final chapter.

Conclusions are also stated, and some future directions for research are identified. The

methods used in these experiments are detailed in the next chapter, and then the results

are presented. Secondary aims and hypotheses of each experiment are detailed in the

relevant chapters.

- 44 -

Chapter 2

- 45 -

2. Methods

In this section, the methods used in Chapters 3 – 6 are detailed. This research

emphasises the tracking of tumour specific immunity in vivo. HA tumour transfectants

and the corresponding, HA-specific transgenic mice are used extensively. Most of the

methods have been previously published, and these are referenced accordingly.

2 .1 . Cel l l ines and cul ture techniques

Cell lines were cultured in standard culture medium (denoted “R10”), comprised of

RPMI 1640 (Gibco/BRL, Grand Island, New York USA) supplemented with 20 mM N-

2-hydro-2ethylpiperazine-N-2ethane (HEPES, Gibco/BRL), 0.05 mM 2-

mercaptoethanol, 100 µ/mL (Sigma-Aldrich, St Louis, Minnesota USA),

benzylpenicillin 60 mg/L (CSL, Melbourne, Victoria, Australia), 48 µg/mL gentamicin

(Sigma-Aldrich) and 5-10% foetal calf serum(FCS, CSL Melbourne, Victoria

Australia). The cell culture media were prepared sterile, and adjusted to 300 mOsm.

Where HA transfectants were used (RencaHA and AB1HA), culture medium was

supplemented with the neomycin analogue Geneticin (Invitrogen, Mount Waverley,

Victoria Australia) at the concentration of 400 g/mL. All cell lines were maintained at

37°C and 5% CO2 in monitored incubators.

2 . 1 . 1 . Cel l harves t

Adherent cells were detached by brief exposure to gamma irradiated trypsin (JRH,

Lenexa, Kansas USA), and washed twice with sterile phosphate buffered saline

(PBS, Invitrogen). Cell counts and viability were assessed by trypan blue exclusion

(Sigma-Aldrich). Viabilities of 95% or greater were deemed acceptable for

inoculation. Unless otherwise stated, tumour inoculums consisted of 1x106 cells,

suspended in 100L of 0.9% sterile normal saline (Astra Zeneca, North Ryde, New

South Wales Australia).

2 . 1 . 2 . Mycoplasma screening

All cell lines were screened for mycoplasma on a three monthly basis. Once cell lines

were growing in antibiotic-free medium for 72 hours, they were trypsinised, washed

twice in normal saline (Astra Zeneca) and re-suspended at a concentration of 1x106 cells

- 46 -

per 100L sterile saline (Astra Zeneca). Samples were tested by Pathcentre (SCGH

Hospital, Nedlands, Western Australia) for the presence of mycoplasma DNA by PCR.

If two successive PCR results for mycoplasma were negative and the cell culture

remained healthy, experimentation proceeded until the next screening.

2 . 1 . 3 . AB1

The AB1 mouse mesothelioma model has been described previously.315

Briefly; IUCC

reference samples of Wittenoom Gorge crocidolite (asbestos) were injected

intraperitoneally (i.p.). After a latency period of 7-25 months, 35% of BALB/c subject

mice developed ascites. At that stage, disease affected mice were culled, exudates

harvested, and cell lines maintained in culture. The AB1 mesothelioma tumour line

shared biological characteristics of human mesothelioma: variable and long latency,

asbestos as a causative agent, ultra-structural microvilli, and low immunogenicity.

Similar to human mesothelioma, AB1 was class I positive and had low expression of

MHC class II.315

After subcutaneous inoculation of 1x106 AB1 cells into BALB/c mice, solid and

vascularised tumours were reliably produced. When the tumour was inoculated

intraperitoneal, a plaque lesion forms at the site of inoculation, together with exudates

consistent with malignant ascites.

2 . 1 . 4 . AB1HA

AB1HA was produced as previously described.148

In short, the Mount Sinai PR8

haemagglutinin (HA) gene was sub-cloned into the expression vector phBApr-1-neo,

and transfected into AB1 cell line using DOTAP (a cationic lipid). Stable transfectants

were selected using geneticin (neomycin analogue) in serial dilutions. HA expression

was verified using biotinolyated HA-specific monoclonal antibody (H18), originally

donated by Dr Walter Gerhard (Wistar Institute, Philadelphia, Pennsylvania USA).

Three clones of HA expression profile were isolated: ABHAHI

, AB1HAMEDIUM

, and

AB1HALO

. The sub-clone AB1HAMEDIUM

was used throughout this thesis, and was

denoted as “AB1HA”.

- 47 -

2 . 1 . 5 . Renca

The Renca cell line was provided by Dr Mark Smyth and Mr Jeremy Swann (Peter

MacCallum Cancer Centre, Melbourne, Victoria Australia). Renca arose spontaneously

as a renal cortical adenocarcinoma in a BALB/c mouse, and was subsequently passaged

subcutaneously and in vitro. The tumour formed solid, grey and vascular tumours, often

undergoing central necrosis and/or haemorrhage.316

Renca has been successfully transplanted intra-renally, subcutaneously, intravenously,

and intraperitoneal. However, the behaviour of Renca is affected strongly by its route of

transplantation. When Renca is implanted subcutaneously, it does not metastasise to the

lungs. However, when placed orthotopically (i.e. into the kidney), Renca grows rapidly

and metastasises aggressively.317

Renca is similar to human renal cancer in its metastatic targets, histological appearance,

immunogenicity (displays sinecomitant immunity), and tendency to produce

polycythaemia.316

When implanted intra-coelomically, Renca has a tendency to invade

adjacent structures and metastasise to the lymph nodes, lung, liver, spleen,

mediastinum, bladder, and the serosa of the gastrointestinal organs.316

Like AB1, Renca

expresses MHC Class I and not MHC Class II.198

It also does not express CD80 (co-

stimulatory molecule).198

2 . 1 . 6 . RencaHA

RencaHA was kindly donated by Dr Eduardo Sotomayer and Dr Fengdong Cheng (H.

Lee Moffitt Cancer Centre & Research Institute, Tampa, Florida USA) and was

originally developed by Dr Linda Sherman and colleagues (The Scripps Research

Institute, La Jolla California USA).318

Briefly, wild type Renca was transfected with

haemagglutinin (HA) of PR8 influenza H1N1 (A/PR/8/34) using calcium phosphate

mediated plasmid transfection. HA positive cells were selected by culture in 400 g/mL

G418 (a neomycin analogue), and tested for HA expression using H18 mAb.198

Like

AB1, RencaHA expresses MHC Class I and not MHC Class II. It also does not express

CD80 (co-stimulatory molecule).198,318

Due to possible contamination of the RencaHA

cell line during transport, it was passaged in vivo by subcutaneous administration into 5

BALB/c mice. In each instance, the tumour grew, but only one had retained HA

positivity on H18 staining and flow cytometry. Previously, RencaHA had been grown

unreliably subcutaneously, and was associated with loss of HA expression.318

- 48 -

The stably expressing HA cell line was then cultured in vitro, using R10 culture

medium supplemented with 400 g/mL G418. After one passage, repeat H18 staining

and flow cytometry were performed, revealing a broad HA expression profile. After

further passage and expansion, the cell line was sorted thrice on HA expression levels,

with the assistance of Dr Kathy Heel (BIAF, University of Western Australia, Nedlands

Australia) and the FACS Vantage Cell Sorter (Becton Dickinson, Mountain View,

California USA).

The resultant clones of RencaHA (RencaHALO

, RencaHAMEDIUM

, and RencaHAHI

) were

passaged in vitro and frozen at low passage numbers of 3 – 7. The RencaHAMEDIUM

cell

line was chosen for experimentation because of its favourable in vitro and in vivo

growth characteristics. Herein, RencaHAMEDIUM

was denoted as RencaHAM.

RencaHAM, was successfully transplanted by intracardiac, tail vein, intraperitoneal and

intra-renal administration. The relative growth properties of RencaHAM are detailed in

Chapter 6, Sections 6.2.2 - 6.2.4.

2 .2 . Murine Species

2 . 2 . 1 . BALB/c and BALB/c nu- / -

mice

Female BALB/c and congenic BALB/c nu-/-

mice were obtained from the Animal

Resource Centre (Perth, Western Australia). BALB/c mice are a common in-bred

laboratory mouse strain, with MHC Class I and Class II denotations of 1-Ad and H-

2Kd respectively. BALB/c nu

-/- have both BALB/c and nude characteristics. As

BALB/c nu-/-

lack a thymus, they are unable to produce T cells.

All animals were maintained under standard clean conditions at the University of

Western Australia Animal Care Unit in Sir Charles Gairdiner Hospital.

All protocols were approved by the University of Western Australia Animal Ethics

Committee, and were compliant with National Health and Medical Research

Committee (NHMRC) and Office of Gene Technology Regulation (OGTR)

regulations.

- 49 -

2 . 2 . 2 . CL4 TCR transgenic mice

The CL4 TCR transgenic murine line has been previously described.319

CL4 mice are

class I restricted, recognising the dominant H-2d restricted HA epitope

(IYSTVASSL). Breeding pairs were obtained from Dr Linda Sherman (The Scripps

Research Institute, La Jolla, California) and backcrossed for at least five generations

onto the BALB/c genetic background at the University SPF animal facility. Purity of

CL4 transgenic progeny was typed prior to experimental use, using mAbs anti-CD8-PE

(53-6.7) and anti-Vb8.1-FITC (MR5-2) (Pharminogen, San Diego, California).

2 . 2 . 3 . HNT TCR transgenic mice

The derivation of the HNT TCR transgenic murine line has similarly been described

previously.320

HNT lines are class II restricted, recognising the HNTNGVTAACSHE

epitope of HA in the context of H-2Kd. Breeding pairs were obtained from Dr D Lo

(The Scripps Research Institute, La Jolla, California) and backcrossed for at least five

generations. HNT mice were typed using the mAbs anti-CD4-PE (RM4-5) and anti-

Vb8-FITC (F23.1) (Pharminogen, San Diego, California)

2 .3 . In Vivo Procedures

2 . 3 . 1 . Anaesthes ia

For prolonged surgery (nephrectomy and lymph node dissection), chloral hydrate

(Orion Laboratories, Balcatta, Western Australia) was used as primary agent. The syrup

preparation was used, diluted in sterile normal saline to a concentration of 3.5%. Each

mouse was weighed and administered 0.1mL/10g intra-peritoneally. Over a period of

five to ten minutes, general anaesthesia would occur. This was supplemented with

inhalational methoxyflurane (Medical Developments International Limited, Springvale,

Victoria Australia)

Methoxyflurane (Penthrane, Medical Developments International Limited) was used as

primary agent for anaesthesia for all other surgical procedures. Briefly, animals were

placed in a glass jar, with paper tissues as a base. The tissue base was impregnated with

approximately 0.5mL of methoxyflurane, and the jar sealed. Animals were monitored

- 50 -

for the onset of anaesthesia, evidenced by depressed respirations and the absence of

movement.

No procedure was commenced unless the animals were deeply anaesthetized. The depth

of anaesthesia was deemed adequate if the following applied: no righting or startle

reflex, no response to pinprick, and slow respiratory rate. Animals were carefully

monitored during anaesthesia for evidence of lightening. In that instance, supplementary

methoxyflurane (Penthrane, Medical Developments) was administered via nose cone.

During anaesthetic maintenance, the operator monitored the reflexes and respiratory rate

regularly. The nose cone was shifted on and off to maintain a steady depth of

anaesthesia.

Once anaesthesia was complete, animals were shifted to a recovery area. Mice were

placed on their sides, on top of a heat pad. The head, neck and torso were maintained in-

line. A moist dab was placed on each exposed eye. The animal was watched for

adequate respiratory effort and occasionally stimulated (by rubbing) if under-breathing.

Once mobilizing and capable of shifting posture, mice were returned to the cage.

Animals were further monitored for an hour, then twice daily for the first three days.

2 . 3 . 2 . Analgaes ia

Intraperitoneal 0.05 mg/kg buprenorphine (Reckitt & Coleman, Chiswick, UK)

analgaesia was administered intraperitoneal (i.p.). Analgaesia was routinely

administered to all animals undergoing laparotomy or lymph node dissection, just prior

to the completion of anaesthesia. All other animals were monitored in the early

postoperative phase (days 0 – 3). During that period, analgaesia was administered at a

dosage of 0.05 mg/kg four times daily on a discretionary basis. Few animals required

such analgaesia.

2 . 3 . 3 . Subcutaneous inoculat ion

Subcutaneous injections were undertaken to administer immune therapies or transplant

tumour cells. Prior to injection, the flank region was shaved and prepared with 70%

ethanol. The most common site of inoculation was the caudal flank region,

approximately in the mid axillary line. A 29 gauge needle (0.5mL, BD UltraFineTM

,

Becton Dickinson Pty Ltd, Scoresby, Victoria Australia) was placed into the subcutis of

- 51 -

this area and a bleb of inoculum was gradually raised. Animals were monitored in the

post-procedural period for distress and bleeding.

2 . 3 . 4 . Axil lary inoculat ion

To study the effects of tumour proximity on node function, a near-axillary injection site

was chosen. Animals were restrained supine, and an area just ventral and caudal to the

axilla was chosen. The skin was depilated by clippers, and prepared with 70% of

alcohol. As with flank inoculums, a 29 gauge needle (0.5mL, BD UltraFineTM

, Becton

Dickinson Pty Ltd) was placed at 30 degrees to the skin, and an injection bleb raised in

the subcutis.

2 . 3 . 5 . Intranodal inoculat ion

To determine the effect of nodal invasion on antigen presentation and in vivo CTL

function, tumour was transplanted into axillary nodes. Animals were subject to general

anaesthesia, and the axillary region was shaved. After preparation with 70% alcohol, an

oblique incision of 5mm was centred over the axilla. Superficial fascia was dissected

clear to demonstrate the free border of pectoralis major. That border was grasped with

haemostat artery forceps, and retracted superiorly and towards the midline. By blunt

dissection and with the aid of 3x prism operating loupes, the axillary node was exposed.

The axillary node was bi-lobulate or somewhat pear shaped in appearance, and

invariably plastered onto the axillary vein. By careful traction on the capsule, a second

operator injected the node with tumour cells using a 29 gauge needle (0.5mL, BD

UltraFineTM

, Becton Dickinson Pty Ltd). The concentration of inoculum was 1x106 cells

per 50 L of sterile saline (Astra Zeneca). Invariably the node swelled with inoculum,

and some cells leaked into the fossa. The wound was closed using four cutaneous 5-0

vicryl (polyglactin 910, Ethicon Australia) interrupted sutures.

2 . 3 . 6 . Intravenous inoculat ion

Intravenous inoculation of immune therapies and tumour cells was performed using the

lateral tail vein. Animals were heated under a heat lamp, and restrained using a standard

restraint cylinder. The lateral tail vein was then identified and injected under vision with

a 29 gauge needle (0.5mL, BD UltraFineTM

, Becton Dickinson Pty Ltd). Flashback into

the bevel of the syringe, the absence of resistance to plunger depression, and a lack of

tail swelling were indicators of successful injection.

- 52 -

2 . 3 . 7 . Intracardiac inocu lat ion

Intracardiac injections were occasionally required when intravenous inoculation was not

possible and/or to study the RencaHAM system. Animals were subject to general

anaesthesia, and secured in a supine position. The midpoint of the sternum was located

by digital palpation. A 29 gauge needle (0.5mL, BD UltraFineTM

, Becton Dickinson Pty

Ltd) was then slowly inserted perpendicular to the skin, in the mid-clavicular line, at the

level of the sternal midpoint. Steady aspiration was continued as the needle was

advanced into the mediastinum. Ready flashback of dark red blood indicated entry of

the needle into the right ventricle, at which time the required volume was slowly

injected. The needle was then gradually withdrawn from the animal, and the animal was

monitored closely for complications. Animals were euthanased if respiratory distress or

cardiovascular compromise occurred.

2 . 3 . 8 . Intrarenal inoculat ion

To study the behaviour of intra-renal RencaHAM, orthotopic (i.e. into the kidney)

tumour implantation was required. Animals were subject to general anaesthesia using

methoxyflurane. The right flanks of the animals were shaved and prepared with 70% of

alcohol. Animals were taped into the right lateral position using masking tape. A

transverse loin incision was fashioned, 3 – 4 mm caudal to the lowermost limit of the

thoracic cage and centred over the mid-axillary line. Incision of the skin and abdominal

musculature exposed the erector spinae muscles posteriorly, and peritoneum.

Peritoneum was incised, and the ovaries swept clear of the operative field.

Where necessary, the right lobe of the liver was gently supported off the lateral aspect to

the kidney. Invariably with this technique, the anterior and lateral surfaces of the kidney

were seen well. Under direct vision, a 29 gauge needle (0.5mL, BD UltraFineTM

, Becton

Dickinson Pty Ltd) was advanced deep to the renal capsule. Cell suspensions were

gradually injected: 1x106 Renca or RencaHA cells in 50 L of sterile saline (Astra

Zeneca). The kidneys swelled slightly from the injection, and sometimes took on a

slightly mottled appearance. The capsule of the kidney dissected off the parenchyma

from the injection, particularly adjacent to the puncture. Most inoculum seemed to

collect under the capsule.

- 53 -

2 . 3 . 9 . Resect ion s tudies

Subcutaneous tumours were excised on day 16 after tumour cell inoculation. At that

time point, tumours were solid and vascularised, with a diameter of 5.57 0.30 mm.

Incisions were elliptical, longitudinal, and centred over the lesion. Incisions were

approximately 15mm in length, with lateral margins of 3 – 4mm. Once the incisions

were fashioned, the rostral tip of the cutaneous flaps were elevated and teased clear of

deep fascia. With steady outwards and caudal tension, skin and adherent tumours were

removed en bloc. Tumour pedicles were dissected clear of the adjacent deep fascia, and

ligated with 5-0 vicryl. Inguinal nodes were identified and preserved, unless

lymphadenectomy was to be included in the procedure. Once the tumour and cutaneous

flap were removed, adjacent skin was undermined and approximated with 5-0 vicryl

(Polyglactin 910, Ethicon) sutures.

2 . 3 . 10 . Lymphadenectomy

Lymphadenectomy was undertaken for axillary and/or inguinal nodes. For the axillary

node, the axillary area was shaved and prepared with 70% ethanol. An oblique incision

of 5mm in diameter was centred over the axilla, and superficial fascia was dissected

clear of the free border of pectoralis major. That border was then retracted, and blunt

dissection into the axillary fossa was undertaken to expose the axillary node and vein.

Gentle traction on the axillary node capsule, together with delicate dissection enabled

delivery of the axillary node off the vessels. The skin was closed with four 5-0 vicryl

(Polyglactin 910, Ethicon) interrupted sutures.

The inguinal node was removed with or without tumour. When the tumour was absent

or left behind, a longitudinal inguinal incision was performed. The incision was 10mm

in length, located in the anterior axillary line, and just rostral to the hindquarter. The

skin edges were elevated and retracted to expose fascia and the lateral thoracic vessels.

The inguinal node was located at the confluence of the inferior epigastric and lateral

thoracic vessels. Blunt dissection was used to carefully remove the node, without

damaging the closely applied vessels. Skin was closed with 5-0 vicryl (Polyglactin 910)

suture. Where tumour was present, the skin and adherent tumour were elevated as

previously described. The inguinal node was identified and the pedicle was ligated and

incised, ensuring the inguinal node was included in the resected pedicle.

- 54 -

2 . 3 . 11 . Monitor ing

Animals with tumour in situ were monitored every three days for weight loss, poor

grooming, huddled behaviour, elevated or depressed respirations, and reduced activity.

These signs were triggers for euthanasia.

Animals were also monitored post-operatively, initially twice daily and then once daily.

All checks were done by the author, who having performed the procedure, was familiar

with potential problems. For instance, the potential complications of lymph node

dissection were swelling of the foreleg (on the affected side), ischaemia of the foreleg,

and reduced mobilization of the nearby joints. The more general aspects of animal well-

being were also checked post-operatively: behaviour/activity (especially huddling), free

mobilization of all limbs and head, feeding, interactions with the other members of the

colony, grooming, and the presence of subcutaneous fat. In addition, tumours were

closely examined for size and evidence of necrosis, bleeding, inflammation and/or

infection.

2 . 3 . 12 . Tumour s ize assessm ents

Tumour sizes were assessed at a minimum of three daily intervals, via standard

callipers. Measurements were taken in millimetres, using two perpendicular directions

that were denoted length (l) and width (w). Tumour product was calculated by the

multiplication of (l) by (w), and tumour diameter represented the square root of that

value.

Tumour diameters of each cohort were expressed as mean SEM. Differences in tumour

diameter between any one intervention group and its matched control arm were tested

using the student‟s t test. Where relevant, tumour growth was compared between

cohorts by two way analysis of variance (ANOVA), removing cured animals from

analysis and considering the interactions of time and treatment. Differences were

considered significant for all statistical tests when P values were < 0.05.

2 . 3 . 13 . Survival analys is

Euthanasia was considered “mortality” and was necessary when tumour diameters

reached 10mm, and/or animals showed any indicators of distress (2.3.11). Survival was

compared between groups using the Log Ranks test, and differences were considered

significant when P < 0.05. Survival benefits for treatments were expressed as hazard

- 55 -

ratios (H.R.) relative to untreated or placebo control groups, and 95% confidence

intervals for H.R. were provided. When follow-up was short (dual-tumour experiments:

see Chapter 4) tumour-free survival was used as proxy for overall survival. Tumour-free

survival was similarly compared using the Log Ranks test, with the same conditions for

statistical significance.

2 . 3 . 14 . Adopt ive cel l t ransfers

CL4 cells were prepared for adoptive transfer as per “CFSE Proliferation Assay” and

HA peptide pulsed targets were formulated as per the “In Vivo CTL Assay” (Sections

2.4 and 2.5). Cells were suspended at a concentration of 2x107 per 100 L of sterile

normal saline. Intravenous access was via the lateral tail vein (or failing that,

intracardiac injection) by 29 gauge needle (0.5mL, BD UltraFineTM

, Becton Dickinson

Pty Ltd)

2 .4 . In v ivo CFSE prol i ferat ion assay

The CFSE proliferation assay was based on the principle that adoptively transferred

tumour-specific CD8+ cells proliferate on antigen encounter. The technique for in vivo

antigen presentation analysis has been previously described,321

but is accounted below.

2 . 4 . 1 . Preparat ion of CL4 cel l s for adopt ive t ransfer

One CL4 transgenic mouse was sacrificed for every three subject BALB/c mice

assayed. Extensive lymph node harvest was undertaken for each CL4, including the

axillary, brachial, inguinal, cervical, facial, jugular, para-aortic, iliac, mediastinal,

celiac, and renal nodes.

CL4 nodes were collected into chilled PBS (Invitrogen) on ice, and mechanically

digested using sterile glass slides. Resultant single cell suspensions were filtered via

40m nylon cell strainer (Falcon Cell Strainer, Becton Dickinson and Company,

Franklin Lakes, New Jersey USA), and centrifuged.

Pellets were subject to red cell lysis by incubating and agitating with in vivo red cell

lysis buffer (5% NH4Cl in PBS, pH 7.2; BDH, Victoria Australia). A further two

washes with PBS followed, and then cells were suspended in RPMI/HEPE

- 56 -

(Gibco/BRL) at a concentration of 2x107 per mL.

322 10L of 5mM 5,6-Carboxy-

fluoroscein-succinimidyl-ester (CFSE; Molecular Probes, Eugene, Oregon, USA) was

added for each 20mL of suspension. CFSE was incubated with the cells for a total of 10

minutes at room temperature. Solutions were then centrifuged through FCS (CSL)

underlay.

A further two spins of RPMI/HEPE (Gibco/BRL) with FCS (CSL) underlay were

performed, and the solution were washed twice with PBS (Invitrogen). The pellet was

then re-suspended in normal saline (Astra Zeneca) and adjusted to a concentration of

2x108 per mL. The adequacy of CFSE staining was checked before adoptive transfer by

flow cytometry (FACScan; Becton Dickinson, Franklin Lakes, New Jersey USA).

2 . 4 . 2 . Adopt ive t ransfer for ant igen presentat ion

2x107 CFSE stained CL4 cells (suspended in 100 L of sterile saline) were adoptively

transferred into each subject mouse and each relevant control. Cells were administered

by tail vein (2.3.6) and/or direct intracardiac (2.3.7) injection.

2 . 4 . 3 . Analys is for in v ivo ant igen presentat ion

Total lymphadenectomy and splenectomy were performed on each subject mouse, three

days after CL4 transfer. Those nodes were similarly collected into chilled PBS

(Invitrogen), mechanical digested, filtered, washed, and red cell lysed. Samples were

suspended in PBS/5% FCS (Invitrogen & CSL) and incubated with 1:200 anti-CD8a-

PE-Cy5 (BioLegend, San Diego, California USA), or rat IgG2aK-PECy5 (BioLegend)

isotype control. All samples were processed using FACscan (Becton Dickinson), Cell

Quest V3.1 (Becton Dickinson) and FlowJo V7.1.3 (Tree Star Inc, Ashland, Oregon

USA). Samples were gated to the lymphocyte population, and the forward scatter and

side scatter distribution. A minimum of 2,500 CFSE positive events were collected in

any one sample. Thresholds were set by CD8+ single stain and isotype controls, and

lymphocyte populations were sub-gated for CD8 positivity. CD8+ cells were examined

for CFSE intensity using a histogram distribution. Lymphocyte proliferation, as a

readout of in vivo antigen presentation, was quantified using the technique described by

Lyons and Parish.321

Subject samples were compared to normal BALB/c, sham operated

BALB/c, and unstained BALB/c controls.

- 57 -

Antigen presentation was quantified as the proportion of CFSE positive cells of lesser

fluorescence than the brightest (parental) population. Antigen presentation in subject

animals was compared to naïve and sham surgery controls. A background proliferation

rate was considered normal, possibly attributable to the division of transfused B cells.321

2 . 4 . 4 . Stat is t ical analys is for ant igen presentat ion

The proportion of lymphocytes proliferating was treated as a continuous variable,

representative of cross presentation levels. Different mouse groups were considered

categorical variables. Explanatory variables included: presence/absence of tumour,

tumour size, post-operative time point, presence/absence of lymph node dissection, and

tumour location. Groups were compared using matched pairs student t tests, using

Prizm 3 statistical software (Graphpad Software, San Diego, California USA).

Differences were considered significant when the two tailed P value was < 0.05.

2 .5 . In v ivo CTL assay

Analysis of in vivo CTL activity was performed similarly to that previously

described,323

and is outlined below.

2 . 5 . 1 . Pulsed and target reference peaks

Full lymphadenectomy and splenectomy were performed on one BALB/c for every

three mice studied. Tissues were collected into chilled PBS (Invitrogen), mechanically

digested, filtered, and red cell lysed as described (Section 2.4.1.) Single cell suspensions

were made up to 4mL in volume and divided into two equal volumes. After

centrifugation, each pellet was re-suspended in R10. CL4 peptide from HA (Chiron

Technologies, Clayton, Victoria Australia) was added to one fraction (pulsed peptide

fraction) at a concentration of 1 g/mL. The other fraction was left unpulsed. Both

fractions were incubated at 37°C for 90 minutes, and periodically agitated. The two

suspensions were then topped up with R10 and centrifuged. Pellets were re-suspended

in RPMI/HEPE (Gibco/BRL) to a final concentration of 2x107 cells per mL.

The two fractions were labelled as either 2.5 M CFSE (pulsed peak) or 0.50 M CFSE

(non-pulsed peak). Following CFSE incubation, solutions were centrifuged thrice over a

FCS (CSL) underlay, and washed twice with PBS (Invitrogen). Each pellet was re-

- 58 -

suspended in sterile saline (Astra Zeneca) and cell counts were performed to ensure

equal numbers. The two fractions were combined in equal portions, and flow cytometry

was performed (FACSscan, Becton Dickinson) to ensure adequate peak separation and

equivalence. A final suspension of 2x108 cells per mL was formulated using sterile

normal saline (Astra Zeneca).

2 . 5 . 2 . Adopt ive t ransfer for in v ivo CTL lys is assay

2x107 cells were then administered to each subject animal by lateral tail vein (2.3.6)

and/or intra-cardiac (2.3.7) injection. Several naïve BALB/c mice were injected with

labelled targets each time the experiment was performed, to provide controls for non-

specific target killing (see also, 2.5.3).

2 . 5 . 3 . Analys is for in v ivo CTL lys is

After a period of 16 hours, total lymphadenectomy and splenectomy was performed on

each recipient mouse. Nodes and spleens were prepared into individual single cell

suspensions, by collection into chilled PBS (Invitrogen). Mechanical digestion,

filtration, washing, and red cell lysis followed, as per 2.5.1. Samples were suspended in

PBS/5% FCS (Invitrogen and CSL), at an approximate concentration of 1x107 per mL.

Flow cytometry was performed using FACScan (Becton Dickinson), Cell Quest V3.1

(Becton Dickinson), and FlowJo V7.1.3 (Tree Star Inc). The percentage of tumour

targets lysed was calculated as follows:

Calculated % Lysis = (Calculated #Lysed)/(Calculated #Pulsed) x 100

Calculated lysed and pulsed values were obtained via correcting assay values for

background killing. Specifically, the ratio of pulsed to non-pulsed cells was calculated

for normal animals. This ratio was then used to calculate the theoretical number of

pulsed cells that were present, prior to CTL lysis, in any one subject animal tissue:

Calculated # Pulsed = (Observed #Pulsed) x (Ratio from Normal Animal)

The number of cells lysed could then be determined by subtracting the number of

pulsed cells at flow cytometry from the calculated number of cells originally present:

Calculated # Lysed = (Calculated #Pulsed) - (Observed #Pulsed)

- 59 -

Those values were then substituted into “Calculated % Lysis” equation above, to

determine in vivo CTL killing for each animal and tissue. Since values were corrected

against normal animals in each assay, it was possible to compare cytotoxicity

percentages between experiments across time.

2 . 5 . 4 . Stat is t ical analys is for in v ivo CTL Assay

The primary outcome for statistical analysis was the percentage cytotoxicity of HA

pulsed cells in each tissue studied (axillary, inguinal, non-draining nodes, spleen). As

subject animals were matched with sham surgery controls, explanatory variables were

primary tumour, cancer surgery, and the presence/absence of nodal invasion.

Cytotoxicity was analysed as a continuous variable, with different groups of mice

examined as categorical variables. Statistical analysis was performed using the Prism 3

statistical programme (GraphPad Software) and the matched pairs student‟s t test.

Differences were considered significant when the two-tailed P value was < 0.05.

2 .6 . DC phenotyping

DCs were extracted from lymph nodes as published previously,324,325

and detailed

below.

2 . 6 . 1 . Isola t ion of DCs from lymph nodes

Lymph nodes were collected into cold RBMI (Invitrogen) with 2% FCS (CSL), “R2”,

and then diced in minimal volume using sterile scalpels. Segments were re-suspended in

2mL of R2 with 1 mg/mL Collagenase (Worthington Biochemical Corporation,

Lakewood, New Jersey USA) and 5 g/mL DNase (DNase I, grade 2, Roche Applied

Science, Basel, Switzerland) and digested for 20 minutes at room temperature, on a

shaking roller. Next, 430 L of 0.1M EDTA (Sigma-Aldrich) was added to each

sample, and further homogenised by repeated pipetting through glass transfer pipettes.

Undigested fragments were removed by passage through 40 m nylon cell strainer

(Falcon Cell Strainer, Becton Dickinson and Company), and suspensions were topped

up with 4.5mL of R10 with 20mM HEPES (Gibco/BRL). Samples were underlaid with

1:9 0.1M EDTA (Sigma Aldrich) and FCS (CSL) and centrifuged. A single wash with

FACS/EDTA solution preceded antibody staining.

- 60 -

2 . 6 . 2 . Staining of DCs

Samples were incubated in 50 L of FACS/EDTA with 1:200 CD45RA-FITC

(0.2mg/mL BD PharMingen, San Diego, California USA), 1:50 CD11c-PE

(eBioscience, San Diego, California USA), and 1:200 CD8PECy5 (BioLegend).

Additional samples were unstained, incubated with single stain, or stained with the

appropriate isotype controls: rat IgG2bK-FITC (BD PharMingen), Armenian hamster

IgG1K-PE (eBioscience), and IgG2aK-PECy5 (BioLegend).

2 . 6 . 3 . DC flow cytom etry

Samples were processed for four colour flow cytometry with the assistance of Dr Kathy

Heel (BIAF, University of Western Australia, Nedlands Australia) using the FACS

Vantage (Becton Dickinson) flow cytometer.

Flow cytometry voltages and compensation were set with single stain samples, and

specificity was verified using unstained and isotype controls. Analyses were performed

with FlowJo V7.1.3 (Tree Star Inc). CD11c positive cells were sub-gated from the

lymphocyte population on forward scatter and side scatter. CD11c positive cells were

plotted for CD45-RA positivity (x-axis) against CD8a positivity (y-axis). As has been

published previously,326,327

plasmacytoid, CD8a positive, and double negative DCs were

then assessed.

2 . 6 . 4 . Analys is of DC phenotypes

DC samples were pooled from five animals for each experimental group. Naïve animals

were compared to tumour bearing and post-operative subjects. In the absence of

individual data points, it was impossible to make statistically reliable comments on the

significance of the trends.

2 .7 . T r e g assays

Treg frequency (as a proportion of lymph node CD4+ cells) was assessed in naïve,

tumour bearing, and post-operative animals. Treg depletion was also verified in depletion

experiments. Peripheral blood and lymph node samples were examined as single cell

suspensions, using the currently recognised Treg cell surface labels: CD4, CD25, and

Foxp3.328,329

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2 . 7 . 1 . Cel l sur face and intracel lular s taining for T r e g

Lymph nodes were dissected from subject animals (via biopsy or at the time of

euthanasia) and collected into chilled PBS (Invitrogen). Nodes were mechanically

digested using frosted glass slides, and prepared into single cell suspension. Cells were

filtered with 40 m nylon filter (Falcon Cell Strainer) and centrifuged. Pellets were

washed twice with PBS (Invitrogen) and suspended into 200 L of PBS (Invitrogen),

with 10% FCS (CSL). Cell surface stains of CD4-FITC (0.5mg/mL, PharMingen) and

CD25-PE (0.2mg/mL, eBioscience) were incubated with the samples at concentrations

of 1:500 and 1:200 respectively. Specificity of staining was validated, using the isotype

controls of mouse IgG1–FITC (PharMingen) and rat IgG1–PE (eBioscience).

After half an hour of incubation at 4°C, samples were washed once with PBS/10% FCS

(CSL), followed by one wash with PBS (Invitrogen). Following surface staining, cells

were stained for intracellular Foxp3, according to manufacturer‟s instructions

(BioLegend). Wash buffer was comprised of PBS (Invitrogen) with 2% FCS (CSL), 1%

BSA (Sigma-Aldrich) and 0.01% sodium azide (Sigma). Samples were incubated with

200 L of Fix/Perm Buffer (BioLegend) and then centrifuged for five minutes. Samples

were then washed with Wash buffer, followed by Perm Buffer (BioLegend). Samples

were then incubated with Perm Buffer (BioLegend) for 15 minutes at room temperature.

After incubation with Perm Buffer (BioLegend), samples were centrifuged and stained

with 1:50 Foxp3-Alexa Fluor® 488 (BioLegend) for 30 minutes. Foxp3 labelled

samples were washed twice with Wash buffer, and then fixed with 4%

paraformaldehdye (BDH Chemicals, Kilsyth, Victoria Australia) for ten minutes.

2 . 7 . 2 . Flow cytometry for T r e g

Flow cytometry was performed on the FACSVantage (Becton Dickinson) flow

cytometer, with calibrations performed using single stain and isotype controls with the

assistance of Dr Robert van der Most. CD4, CD25 and Foxp3 were considered markers

for Treg cells, as generally accepted.328,329

Using FlowJo V7.1.3 (Tree Star Inc), Treg

were identified by triple positivity for these markers, along with appropriate cell size

and granularity. In some instances, combinations of CD4+ and Fox P3

+, Foxp3

+ alone,

and/or CD4+ and CD25

+ were used to identify Treg.

- 62 -

2 . 7 . 3 . Stat is t ical analys is o f T r e g

Treg relative frequencies (proportions) were calculated by dividing the number of

CD25+CD4

+Foxp3

+ cells, by the number of CD4

+ cells in each sample. Mouse groups

were treated as categorical variables, and Treg proportions were treated as continuous

variables. Explanatory variables included the presence/absence of tumour, post-

operative time point, and the presence/absence of Treg depletion. Differences between

groups were examined using the matched pairs t test in Prism 3 (GraphPad Software).

Differences were considered statistically reliable if they were significant at the 5% level

or better.

2 .8 . MSC Studies

Splenic MSC were identified in subject animals using flow cytometry and the

characteristic markers of CD11b and Gr-1. Spleens of normal animals, subject animals,

and controls, were collected into chilled PBS (Invitrogen), mechanically digested (using

glass slides), filtered with 40 m nylon cell strainer (Becton Dickinson and Company),

red cell lysed with 5% NH4Cl (BD) and washed twice with PBS (Invitrogen). A tenth of

each sample was re-suspended in 250 L of PBS (Invitrogen), with 5% FCS (CSL).

Each sample was incubated for 30 minutes with 1:200 Gr-1/Ly-6G/Ly-6C-PECy5

(BioLegend, San Diego, California USA) and 1:200 CD11b-APC (BioLegend),

centrifuged and washed in PBS/Turbo (Invitrogen/CSL). Flow cytometry was

performed using the BD FACSCalibur Flow Cytometer (Becton Dickinson), Cell Quest

V3.1 (Becton Dickinson) and FlowJo V7.1.3 (Tree Star Inc). Flow cytometer voltages,

threshold fluorescence values and compensation levels were set with reference to

unstained, single stain CD11b-APC (BioLegend) and single stain Gr-1- PECy5

(BioLegend) samples. Specificity of staining was verified by irrelevant isotype control

mAb; specifically rat IgG2bK-PECy5 (BioLegend) for Gr-1, and rat IgG2bK-APC

(BioLegend) for CD11b.

In accordance with previously published work, the Gr-1+CD11b

+ population was a

heterogeneous cell group.246

As it is unknown which cell type of the Gr-1+CD11b

+

population is responsible for immune suppression,246

the group was quantified as a

whole. Quantification was performed by dividing the number of Gr-1+CD11b

+ cells by

the number of viable, non red cell splenocytes collected over the same period, in that

- 63 -

sample.281

Percentage splenic MSC were expressed as mean values SEM where

relevant. Percentages of MSC were considered continuous variables and compared

between post-operative, normal, and tumour bearing animals. Explanatory variables

were the presence/absence of tumour, tumour size, and post-operative time point.

Statistical analysis was performed using Prism 3 (GraphPad) and the matched pairs

student t test. Differences were considered significant at the singled tailed 5% level.

2 .9 . HA-Speci f i c CD8+: de tec t ion/phenotype

Tumour-specific CD8+ cells were identified by fluorescently labelled IYSTVASSL-

MHC I pentameric complexes (Pro5® MHC Pentamer, ProImmune, Oxford United

Kingdom) in conjunction with CD8 staining in the HA tumour transfectant system

(AB1HA). Tumour-specific CD8+ cells were further characterised by the memory

marker CD44330

and the CD127 homeostatic proliferation receptor.298

Homeostatic

proliferation is canonical to a true memory cell,298

and the combination of HA

Pentamer, CD8, CD44 and CD127 enables a precise delineation of true, tumour-specific

memory cells.

2 . 9 . 1 . Pentam er cal ibrat ion

CD44, CD8, CD127, and IYSTVASSL Pro5® MHC Pentamer staining was calibrated,

prior to examining for tumour-specific CD8+ cells. 1x10

6 tumour-specific CD8

+ cell

samples (obtained from preparations of CL4 lymph nodes), were used to test varying

dilutions of pentamer (4:100, 8:100, 12:100, 16:100, 1:5, and 1:4). Of these, a 1:5

pentamer concentration (maximum of 1x106 tumour-specific cells) was selected, as it

was associated with minimum background binding and near maximal detection of

IYSTVASSL-specific CD8+ cells.

2 . 9 . 2 . Assessm ent of tumour -speci f i c memory cel l s

Samples of axillary, inguinal and non-draining (e.g. mediastinal, mesenteric) lymph

nodes and spleens were harvested from naïve, tumour bearing, post-operative, and post-

sham surgery BALB/c mice of various time points. Lymph nodes were harvested into

chilled PBS (Invitrogen), mechanically digested (using frosted glass slides), filtered

with 40mcm nylon cell strainer (Becton Dickinson and Company), red cell lysed with

5% NH4Cl (BD) and washed with wash buffer. The wash buffer was comprised of

- 64 -

0.1% sodium azide (Sigma-Aldrich), 0.1% BSA (Sigma-Aldrich) and 2% FCS (CSL) in

PBS (Invitrogen).

Residual fluid on the pellet (approximately 50L) was used as suspension volume for

pentamer staining. To remove protein aggregates, the pentamer reagent was micro-

centrifuged at 14,000g, and 4°C for 3 minutes prior to staining. Pentamer was added to

the concentrated cell suspensions at a dilution of 1:5, agitated, and incubated at room

temperature (22 degrees) for 10 minutes. Solutions were shielded from light during

incubation. After pentamer incubation, cells were washed and re-suspended in 100 L

of wash buffer for antibody staining. Samples were incubated with 1:200 CD8a-PECy5

(BioLegend), 1:200 CD44-FITC (BioLegend) and 1:50 CD127-APC (eBioscience) for

20 minutes, shielded from light.

Unstained, single stain samples, and isotype controls were used to verify specificity of

the staining procedure: rat IgG2b-FITC (BioLegend), rat IgG2b-PECy5

(BioLegend), and rat IgG2a-APC (eBioscience). Samples were processed by four

colour flow cytometry (BD FACSCalibur, Becton Dickinson) with the assistance of Dr

Andrew Currie (Tumour Immunology Group, Sir Charles Gairdiner Hospital).

Samples were analysed using FlowJo V7.1.3 (Tree Star Inc). Tumour-specific CD8+

cells were expressed as a proportion of lymph node CD8+ cells, to a maximum of two

decimal places. CD44+ and/or CD127

+ were also calculated proportionately to a

denominator of Pentamer+, CD8

+ lymphocytes. CD44

+ and CD127

+ percentages were

expressed to a maximum of two decimal places, and denoted as means SEM, where

pertinent.

As outcomes were proportionate and all samples were processed concurrently,

comparisons could be made between subject groups. Tumour-specific CD8+

proportions, CD44+ proportions, and CD127

+ proportions were all considered

continuous variables. Different mouse groups were considered categorical variables.

Proportions were compared between subject groups and controls, using the paired

student t test and Prism 3 (GraphPad). Differences were considered significant at the

5% level.

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2 .10 . Therapies

2 . 10 .1 . Tol l Like Receptor (TLR) l igand therapy

TLR therapy doses were in accordance with previously published values, and were

optimised in the Tumour Immunology Group at Sir Charles Gairdner Hospital

laboratory by Dr Andrew Currie and Mr Steven Broomfield.

2 . 10 .2 . poly I :C

The TLR3 ligand, polyriboinosinic acid-polyribocytidylic acid (poly I:C, InvivoGen,

San Diego, California USA) was administered in the scar of postoperative animals, or

into experimental metastases after surgery. The maximum dosage was 10 g in 100 L

of saline (saline), every day, for up to six treatments. This was consistent with the range

of dosages seen in the literature, including a clinical trial of intramuscular poly I:C for

malignant glioma in humans.331

When tumours were being treated, therapy was

delivered intra-tumourally once the tumour was visible and palpable. If tumours were

not present, doses were skipped until such time as they reappeared. The maximum

frequency of treatments was daily, and the maximum number of treatments was six.

Saline and inactive nucleic acid, CpG 1720 (nCpG, Tib-Molbiol, Hamburg, Germany)

were administered at comparable frequency and dosage, to matched control animals.

Comparisons of survival were made between poly I:C treated animals and controls

using the log rank test.

2 . 10 .3 . CpG-ODN 1668

The TLR9 ligand, cytosine phosphorothioate guanine oligodeoxynucleotide 1668 (CpG-

ODN 1668, Tib-Molbiol, Hamburg, Germany) was administered intra-tumourally to

experimental post-operative metastases, or intravenously at the time of surgery. When

CpG was administered intravenously, a single injection of 10 g was provided at the

time of surgery. This dosage (0.5mg/kg) approximates the upper range of published

intravenous treatments in humans, but is low in comparison to other published dosages

in mice.332

Intra-tumoural CpG was given at a dosage of 10 g in 100 L of saline,

comparable to previously published regimens for mice.53

When tumours were absent,

doses were skipped. The maximum frequency of dosage was daily, and each mouse

received up to six treatments.

- 66 -

As with poly I:C experiments, saline or inactive nucleic acid nCpG (Tib-Molbiol) was

administered at comparable frequency and dosage to matched control animals.

Comparisons of survival were made between CpG treated animals and controls using

the log rank test.

2 . 10 .4 . 3M019T M

3M019 (3M Pharmaceuticals, St Paul, Minneapolis USA) is a recently developed

imidazoquinoline. The intra-tumoural mode of administration in this research (rather

than topical) differs significantly to all previously published literature on TLR7

analogues. In collaboration with Mr Steven Broomfield (TIG) and 3M Pharmaceuticals,

3M019 was injected into post-operative experimental metastases. A dosage of 50 g

was used, suspended in 100 L of sterile saline (Astra Zeneca). When tumours were

absent, doses were skipped. The maximum frequency of treatment was every second

day, and each mouse was treated up to six times. As with the other treatment

experiments, comparisons were made between 3M019 treated animals and controls

using the log rank test.

2 . 10 .5 . Act ivat ing ant i -CD40 ant ibody therapy

Murine agonistic anti-CD40 monoclonal antibody (FGK45 mAb, Monoclonal Antibody

Facility, Perth Western Australia) was administered intra-tumourally or peri-tumourally

at a dosage of 40 g in 100 L of sterile saline, every 2nd

day, for a maximum of six

treatments. FGK45 was also given intravenously at the dosage of 100 g every third

day, once tumours had emerged. This was consistent with previously published doses

for systemic FGK45 (e.g. 100 g q2dx311,333

and 100 g q2dx5334

). Survival amongst

FGK45 treated animals and controls were compared using the log rank test.

2 .11 . In v ivo deple t ion s tudies

CD4+, CD8

+, and Treg depletions were performed in vivo. Previously described mAb

reagents were used for all depletions. Complete depletion for each period of interest was

verified on peripheral blood (CD4+, CD8

+, Treg) and lymph node biopsy (Treg).

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2 . 11 .1 . CD4+/CD8

+T cel l deplet ions

CD8+ T cells were depleted using YTS169 mAb (Monoclonal Antibody Facility, Perth

Western Australia). CD4+ cells were depleted with GK1.5 mAb (Monoclonal Antibody

Facility). For both CD4+ and CD8

+ depletions, 200 g of mAb was administered by

intraperitoneal injection, 24 hours before the depletion period. The same dose was also

given on the first day of the depletion period. Depletion was maintained by

intraperitoneal mAb, at a dosage of 150 g every second day. Completeness of CD4+ or

CD8+ depletion was assessed by flow cytometry on peripheral blood. Briefly, glass

capillary tubes were used to collect blood from the retro-orbital sinus. Each blood

sample was diluted with PBS (Invitrogen)/10% FCS (CSL), up to a volume of 1mL.

Diluted samples were placed atop 1mL of Ficoll® (Sigma-Aldrich) and centrifuged at

4°C , 2000rpm, for 20 minutes. After centrifugation, a buffy coat (lymphocytes)

appeared on the Ficoll® surface, which was harvested and transferred to FACS tubes

(Round Bottom 12mm x 75mm with cell strainer cap, BD FalconTM

, BD Biosciences).

2mL of PBS/Turbo was then added to each FACS tube, and the mix was centrifuged at

4 degrees, for 5 minutes.

Pellets were re-suspended in 100 L of PBS (Invitrogen)/10%FCS (CSL) with 1/500

CD4-FITC (PharMingen), CD8-PeCy5 (BioLegend), or isotype controls: rat IgG2a-

FITC (PharMingen) and rat IgG2bK-PECy5 (BioLegend). Antibodies were incubated at

4 degrees Celsius for 30 minutes, shielded from light. After incubation, samples were

washed with PBS/Turbo, and flow cytometry was performed. Calibration of the flow

cytometer (FACScan, Becton Dickinson) was performed with unstained and single stain

samples, and the specificity of staining was verified using the isotype controls.

Depletion was considered successful when >90% of CD4+ cells were removed and/or

>95% of CD8+ cells were absent from treated samples

2 . 11 .2 . T r e g deplet ion

PC61 Anti-CD25 mAb was obtained from the Monoclonal Antibody Facility (Perth,

Western Australia) and used to deplete Treg in vivo, as per standard protocols.335

Briefly,

PC61 was administered intra-peritoneally at a dosage of 500 g, 24 hours prior to the

period of required Treg depletion. Successful Treg depletion was confirmed on the first

day of the period of interest by retro-orbital bleeds and processing for

CD4+CD25

+Foxp3

+ flow cytometry, as described earlier. The period of Treg depletion

was approximately 10-14 days and the gradual re-emergence of Treg at 7-10 days was

- 68 -

verified by lymph node biopsy and flow cytometry. While CD25+ cells were absent on

flow cytometry after anti-CD25 mAb therapy, some preservation of the Foxp3+CD4

+

population was found, consistent with recently published data.336

2 .12 . Ident i f icat ion of sent ine l nodes

Vital dyes are a recognised method of visualising lymphatics and identifying sentinel

lymph nodes.129,130

Methylene blue was used to identify the lymphatics and sentinel

nodes for the BALB/c flank.

2 . 12 .1 . Methylene b lue

10mg/mL methylene blue (Sigma) was formulated into sterile PBS and 100 L was

injected into the subcutaneous flank of anaesthetised BALB/c mice. Animals were

systemically dissected and photographed at 5 minutes, 15 minutes, 30 minutes, 1 hour

and 24 hours after inoculation.

2 .13 . DC tracking

Skin DC migration was tracked in vivo using CFSE, GM-CSF and the standard

cutaneous (Langerhans) DC markers of CD11c and DEC205.337

Animals were

preconditioned at the typical tumour injection site using 10 g of recombinant murine

GM-CSF (ProSpec-Tany Technogene Ltd, Rehovot, Israel). 24 hours after treatment

with GM-CSF, 100 L of 10 M CFSE (Molecular Probes) was injected into the same

site.

A further 24 hours after CFSE inoculation, regional lymph nodes were harvested and

processed for DC isolation as per 2.6.1. Samples were then incubated with 1:50 CD11c-

PE (eBioscience), 1:200 CD11b-APC (0.2mg/mL, BioLegend), and 1:125 DEC-205-

Biotin (CedarLane Labs, Hornby, Ontario Canada). Additional samples were incubated

as single stained, unstained controls, or IgG1-PE (eBioscience) isotype control and rat

IgG2b -APC (BioLegend) isotype controls.

Samples were washed twice with PBS, and fixed with 4% paraformaldehyde (Sigma-

Aldrich) prior to flow cytometry. Flow cytometry was performed using the FACS

- 69 -

Vantage (Becton Dickinson) instrument, with associated CellQuest V3.1 software

(Becton Dickinson). The mononuclear population was selected from the forward scatter

and side scatter plot, and sub-gated for CD11c positivity (x-axis) against DEC-205 (y-

axis). CFSEHI

CD11cHI

DEC-205HI

CD11bHI

cells were quantified as a proportion of total

CD11c positive cells. Different node groups within the animal were compared and

correlated with findings on methylene blue injection.

2 .14 . Histology

Histological sections were undertaken to assess the completeness of resection, the

presence of lymph node invasion, and to document the morphology of RencaHAM in

vivo. Slides were prepared for histology by standard haematoxylin and eosin (H&E)

staining, as below.

2 . 14 .1 . H&E staining

Specimens were fixed in OTC (Sakura Finetek, Tokyo Japan) and frozen at -80C.

Frozen specimens were cut at 10 m sections on L-lysin (Sigma) coated slides, by

cryostat. Slides were fixed with Carnoy‟s fixative. Carnoy‟s fixative was comprised of

10% acetic acid (Sigma), 30% chlorophorm (Sigma), and 60% absolute alcohol

(Pronalys, Biolab, Mulgrave, Victoria Australia).

Samples were then washed with tap water for 1 minute, and stained with Gill‟s

haematoxylin (VWR International Ltd, Poole, England) for 1 minute. Slides were rinsed

in tap water for a further minute, then in Scott‟s water for 30 seconds. Scott‟s water was

formulated from 1L of distilled water, 20g Sodium Bicarbonate (Chem-Supply,

Gillman, South Australia), and 7g of Magnesium Sulphate (Sigma).

After washing with Scott‟s water, samples were rinsed in tap water for another minute,

70% alcohol (Merck Pty Ltd, Kilsyth, Victoria Australia) for 30 seconds, and then 95%

alcohol (Merck) for 30 seconds. Slides were counterstained with Eosin (Gurr

Certistain®, BDH Laboratory Supplies, Pool, England) for 1 minute, and washed with

absolute alcohol (Pronalys, Biolab, Mulgrave, Victoria Australia) for 30 seconds each

time. Slides were cleaned three times with xylene (BDH), for one minute at a time.

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Finally, slides were mounted in DPX (Scot Scientific, Welshpool, Western Australia),

air dried at room temperature, and examined by microscopy.

2 . 14 .2 . Resect ion specimens

To verify complete resection, pairs of animals undergoing surgery were randomly

selected for histology. The presence of a clear resection margin (i.e. healthy tissue),

located on superficial, lateral and deep aspects of the surgical specimen suggested

complete excision.

2 . 14 .3 . Kidneys

Mice with renal RencaHAM tumours were euthanased, and kidney specimens were

collected for histology. Four randomly selected specimens were processed as described

above.

2 . 14 .4 . Lymph nodes

Lymph node dissection was undertaken on two, randomly selected mice for each route

of RencaHAM transplantation (intravenous, intra-cardiac, intra-renal). Lymph nodes

included: cervical, axillary, brachial, inguinal, iliac, caudal, para-aortic, renal, celiac,

mesenteric and mediastinal. Samples were processed for histology, and examined for

the presence of tumour cells by an independent observer.

2 . 14 .5 . Lungs

Whole lungs were teased clear of the thoracic cavity by blunt dissection. Each trachea

was cannulated with a 25 gauge needle (BD PrecisionGlideTM

, BD, Singapore) and

injected with approximately 1mL of 50% OTC (Sakura Finetek)/50%PBS (Invitrogen).

Lung regions of interest (e.g. middle lobe) were incised and rapidly immersed in OTC

(Sakura Finetek) for freezing. H&E sections were subsequently processed, as previously

described.

- 71 -

2 .15 . Cul ture of necropsy spec imens

Lung and lymph nodes from RencaHAM bearing animals were assayed for the presence

of viable tumour cells by tissue culture. Pairs of mice with RencaHAM disease (day 21)

were randomly selected from groups of differently transplanted animals (intravenous,

intra-cardiac, intra-renal).

2 . 15 .1 . Lung

Lungs were collected into chilled R10, mechanically digested by glass slide, and

transferred into 75cm2 BD Falcon culture flasks (BD Biosciences) containing R10

supplemented with 400g/mL G418. Samples were incubated at 37°C and 5% CO2. 48

hours after culture was commenced, the flask culture surface was thoroughly rinsed

with warmed PBS (Invitrogen), and fresh medium was added. Flasks were inspected for

the presence of tumour cells, every 3 days. Medium was changed at the same time, as

per standard cell culture protocols. Once tumour cells were present at microscopy,

photographs were taken and the flasks were discarded. If no cells grew out after 6 weeks

of culture, the result was considered negative.

2 . 15 .2 . Lymph nodes

To determine whether viable tumour cells were present in the lymph nodes of

RencaHAM animals, total lymphadenectomy was performed on randomly selected pairs

of RencaHA bearing mice (intravenous, intra-cardiac, intra-renal). Samples were

collected into chilled R10 and cultured, as for 2.15.1. Once again, if no cells grew out

after 6 weeks of culture, the result was considered negative.

2 .16 . HA-speci f ic rea l t ime PCR

Resection beds and lymph nodes of RencaHAM or AB1HA tumour bearing and/or post-

operative animals were examined for HA positive cells (tumour cells). Genomic DNA

extraction and isolation were undertaken by standard methods, as below. HA specific

primers and real time PCR (RT-PCR) were used, as previously described.20

Preparations

of the relevant tumour cells (AB1HA and RencaHAM) were obtained from tissue

culture and used as positive controls. RNase free water, non-transfected cell lines (AB1,

Renca) and DNA extracted from healthy skin were used as negative controls.

- 72 -

2 . 16 .1 . Extract ion of DNA

Samples for DNA extraction were stored at -20°C in 1.5mL Eppendorf tubes (Sarstedt,

Ingle Farm, South Australia). Lysis buffer was formulated from 50mM Trizma ®

(Sigma-Aldrich), 0.4M NaCl (Sigma-Aldrich), 100mM EDTA (Sigma-Aldrich), 0.5%

SDS (Sigma-Aldrich) and sterile H2O. 600 L of lysis buffer was added to each sample,

along with 350 g of proteinase K (Promega, Annandale, NSW Australia).

Samples were incubated at 55°C and 200 rpm for one hour, or until completely

digested. 167 L of Saturated (6M) NaCl (Sigma-Aldrich) was added to each sample,

followed by a 15 second vortex. Samples were centrifuged, and the pellets were placed

in clean Eppendorfs (Sarstedt). 95% ethanol (Merck Pty Ltd) was added of

approximately equal volume to each pellet. Samples were inverted to precipitate DNA

and centrifuged for 30 minutes at 4°C . Pellets were washed with 70% ethanol (Merck

Pty Ltd) and dried at room temperature. Once the alcohol was gone, samples were re-

suspended in 200 L of RNase free H2O, in preparation for PCR.

2 . 16 .2 . PCR of DNA templates

The presence and absence of HA DNA was detected using HA specific forward and

reverse primers. A 30 L reaction mix was used, containing 10pmol of each primer,

12.5L of 2x QuantiTec SYBER Green PCR Master Mix (Qiagen, Doncaster, Victoria

Australia), 9.25L of RNase free water (Qiagen), 0.25L of 1/1000 Fluoroscein (Bio-

Rad, Hercules, California USA), and 5L of template DNA. HA was detected on the

Real Time PCR Machine (Bio-Rad) using the following protocol: 95°C for 15 minutes,

94°C for 15 seconds, 35 cycles of 58°C for 30 seconds, followed by one cycle of 72°C

for 30 seconds, 80 cycles of 55°C for 10 seconds. Amplified DNA was held at 10°C.

PCR amplification and melt graphs were obtained using the iCycler (Bio-Rad) and

associated software. Subject curves were compared to RNase free water, healthy skin,

non-transfected tumour, and HA transfectant tumour controls.

- 73 -

Chapter 3

- 74 -

3. Surgery and cross presentation

3 .1 . Introduct ion

Numerous immune therapies can be used in combination with surgery. Tumour

vaccines are a major class of such therapies, and as of 2003, there were some 200 – 300

tumour vaccines in phase II or phase III clinical trial.66

By definition, tumour vaccines

provide an exogenous source of tumour antigen and/or a mechanism of enhancing

antigen priming. The notion that antigen priming is limiting to the immune response is

implicit to the principle of tumour vaccination.

There are two possible sources of antigen in a tumour-bearing patient: the primary

tumour and secondary deposits (metastases). Primary tumours are usually the largest

and are examined first, but whether tumours in distal sites present antigen is also

investigated.

In the setting of bulky disease, when tumour antigen is abundant, it remains uncertain

whether antigen presentation is a limiting factor. Rather, it might be the quality of the

interaction between APC and CD8+ that is defective, and/or tumours immune

subversion mechanisms may operate. As evidence for this, tumour vaccination has been

almost universally disappointing in the setting of bulky disease.338

Even if antigen presentation is not a limiting factor with bulky tumour, it may become

limiting after surgery. Despite highly efficient antigen presentation, by removing

tumour mass, surgery may reduce antigen presentation to below stimulation threshold.

The effects of surgery on soluble tumour antigens are well characterised in the literature

(e.g. decline in CEA after colorectal carcinoma excision339

or the fall in PSA after

prostate resection340

) but post-operative tumour antigen presentation has not been

studied in vivo.

Understanding how surgery affects tumour antigen presentation is critical, because

tumour antigen provides the obligatory priming signal to the anti-tumour immune

response (see Figure 1.2) Thus, understanding the impact of surgery on tumour antigen

presentation could be important to planning the optimum type of immune therapy after

- 75 -

surgery. In particular, if tumour antigen presentation becomes limiting post-operatively,

this would indicate a role for tumour vaccines. In contrast, if tumour antigen

presentation remains sufficient after primary resection, it may be more appropriate to

enhance the quality of tumour antigen priming, or to boost the effector component of

the immune response (e.g. by adoptive immunotherapy).

Not only is it important to understand the effects of surgery on tumour antigen levels,

but also the impact of resection on the distribution of that antigen. For instance, surgery

is associated with neutrophil influx, vascular injury, and local protease activity.341

Such

phenomena might lead to the systemic efflux of tumour antigen and might have

implications for planning the mode of delivery for post-operative immune treatment.

More specifically, some immune therapies might work best in the vicinity of tumour

antigen.342

Thus while intra-tumoural or peritumoural treatments may be important

before surgery, intravenous therapy could be more useful if antigen becomes systemic

post-operatively.

In this chapter, the effects of surgery on the presentation of cell associated tumour

antigen are studied, aiming to facilitate a logical approach to planning the optimum

timing, type and mode of delivery for post-operative immune treatments. The effects of

surgery on the presentation of cell associated tumour antigen are examined using the

AB1HA mesothelioma tumour model.148

AB1HA expresses HA as a cell associated

antigen that is constitutively cross presented.343

Thus the emphasis of this investigation

is the effects of surgery on cross presented, cell-associated antigen. Given that CD8+

lymphocytes are the predominant anti-tumour effectors,344-346

and since continual high

level CD8+ lymphocyte effector function is closely related to continued tumour antigen

presentation,347

determining the impact of surgery on CTL function in vivo is logically

the second component to the study.

3 .2 . Resul t s

3 . 2 . 1 . AB1HA in wi ld type and immunodefic ient mice

Initially, the subcutaneous growth of AB1HA tumours was assessed in wild type and

immunodeficient mice. When 1x106

AB1HA cells were inoculated into wild type and

BALB/c nu-/-

, solid and vascularised tumours were the result (Figure 3.1B) Outgrowth

- 76 -

of AB1HA was slightly slower in wild type mice relative to immunodeficient BALB/c

(Figure 3.1A, P < 0.001, two way ANOVA) suggesting that AB1HA could evoke an

immune response. However, that immune response was of insufficient magnitude to be

protective because the tumour take rate was 100%, and all mice succumbed (data not

shown).

3 . 2 . 2 . HA-speci f ic presentat ion during AB1HA growth

The level of HA specific presentation in tumour draining lymph nodes (ipsilateral

axillary and inguinal), non draining nodes (contralateral inguinal, contralateral axillary,

brachial, cervical, iliac, caudal, para-aortic, celiac, mesenteric, mediastinal) and spleens

was measured using the Lyons Parish321

assay for days 4, 10, 16 and 21 of subcutaneous

AB1HA growth (representative data for day 21: Figure 3.2).

When background was defined as CL4 proliferation in the nodes and spleens of normal

(non tumour-bearing) animals, statistically significant proliferation (two tailed P < 0.01,

relative to normal controls) was seen in the draining lymph node at day 4 after tumour

inoculation (Figure 3.3). This corresponded to a mean tumour diameter of 0.85

0.11mm. Cross presentation increased in parallel to tumour size, reaching 51.96

6.93% by day 21 after inoculation (Figure 3.3A). In contrast, cross presentation was

not detectable in the non-draining nodes nor in the spleen, for any time point of tumour

growth (Figure 3.3B,C).

3 . 2 . 3 . Speci f ic i ty of CL4 prol i ferat ion

To ensure CL4 proliferation was specific for HA (i.e. not just due to the presence of a

tumour in the region) presentation was assayed in animals bearing established HA-

negative parental AB1 tumours. Proliferation was assessed on day 16 of AB1 tumour

growth, at which stage tumours were 3.86 0.29mm in diameter. No significant CL4

proliferation was detected in the draining lymph nodes of AB1 bearing animals on day

16 (P > 0.05 relative to background, student‟s t test). The lack of proliferation seen in

non-draining nodes and spleens of AB1HA tumour bearing mice was reinforced by the

statistical equivalence of these tissues to the non draining nodes and spleens of AB1

bearing animals (P = 0.050 and P = 0.080 respectively, student‟s t test).

- 77 -

3 . 2 . 4 . HA presentat ion af ter surg ery

To determine the effect of complete resection (Figure 3.5) on the kinetics of antigen

presentation, HA-specific presentation was assayed in groups of animals at various time

points before and after surgery. As was the case pre-operatively, cross presentation was

not seen in the non-draining lymph nodes nor the spleen for any post-operative

timepoint (data not shown). Within the draining lymph nodes, no immediate effect on

cross presentation was evident (Figure 3.6A). However, HA-specific presentation did

decline steadily after the day of surgery, until day 14 post-operatively, when antigen

presentation was no longer statistically significant (relative to matched sham surgery

controls), Figure 3.6B.

Figure 3.1. Growth kinetics of subcutaneous AB1HA in wild type and nude mice

A. AB1HA was grown subcutaneously in BALB/c and congenic BALB/c nu-/-

. Mean tumour diameter

SEM shown for each cohort. Data are shown from a single experiment (n = 10 for each group). *P <

0.001 (two way ANOVA). B. Subcutaneous tumour in BALB/c mouse at day 16.

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Figure 3.2. Location of HA-specific presentation in AB1HA tumour bearing mice

A. Location of lymphoreticular tissues commonly assayed. B. Representative forward scatter (FSC)/side

scatter (SSC) plot showing inclusion gate (R1) used to detect proliferating lymphocytes. C.

Representative plots (from single experiment) showing region used to measure HA specific proliferation

in the draining (left), non-draining nodes (centre), and spleen (right). D. Representative histograms of

regions R2, showing HA specific proliferation “daughter” peaks in the draining nodes (left) but neither

non-draining nodes (centre) nor spleen (right).

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Figure 3.3. CL4 proliferation during tumour growth

HA specific CD8+ proliferation was assessed in BALB/c mice during AB1HA tumour growth. Single

mice are indicated by points on the graph, mean CL4 proliferation is indicated by the bar for each cohort,

with a minimum of seven animals per cohort. A. CL4 proliferation in the draining nodes over time. B.

HA-specific CD8+ proliferation in the non-draining nodes. C. CL4 proliferation in the spleen. *Denotes

statistically significant proliferation relative to background (two tailed P < 0.05, student‟s t test).

- 80 -

Figure 3.4. Specificity of CL4 proliferation.

CL4 proliferation was assayed in animals bearing day 16 HA-negative parental AB1 tumours and

compared to BALB/c with established AB1HA tumours and naïve controls. Single mice are indicated by

points on the graph, mean proliferation is shown by bars. Data are shown from a single experiment with

at least five animals present per group. *Denotes two-tailed P < 0.05, relative to naïve controls (student‟s

t test). Key: LN = lymph node, SPL = spleen, DLN = draining lymph node, NDLN = non draining lymph

node.

3 . 2 . 5 . Completeness of resect ion

To determine if the persistence of tumour antigen presentation after surgery was due

to incomplete resection, surgical margins were examined by PCR to detect small

amounts of residual tumour cells. As previously described, this technique was

sensitive to as few as ten AB1HA tumour cells.20

Three pairs of animals undergoing

surgery were randomly selected for resection bed biopsy. Resection bed biopsies

were taken from on the day of surgery, 24 hours post-operatively, or 2 weeks after

surgery. Biopsies were examined for the presence of HA transgene by genomic PCR,

as per 2.14. HA transgene positive cells were absent from all biopsies of the surgical

site, on the day of surgery, 24 hours after surgery, and 2 weeks post-operatively

(Figure 3.7A,B). Additionally, surgical cure rates were assessed in congenic

BALB/c nu-/-

, who were athymic and had a markedly reduced capacity to

immunologically reject residual tumour. Tumours were grown in 10 nude BALB/c

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until day 16 after inoculation (when tumour diameter averaged 5.75 0.61 mm) and

resection was undertaken. Animals remained free of recurrence for > 60 days after

surgery, and were thus cured by resection. Complete resection was also checked with

histological sections; clear resection margins were observed in three randomly

selected animals undergoing removal of AB1HA tumour (Figure 3.7C).

Figure 3.5. Surgery for AB1HA tumours

Subcutaneous AB1HA tumours were excised, as detailed in 2.3.9. A. Resection bed after ligature of

vascular pedicles and complete excision of AB1HA lesion plus adherent skin. B. Closure of tissue defect.

C. Appearance at two weeks after surgery.

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Figure 3.6. CL4 proliferation before and after surgery

A. CL4 proliferation in the draining nodes before and after complete AB1HA resection. B. CL4

proliferation in naïve mice before and after sham surgery. Individual mice are shown by points, mean

proliferation indicated by bars. *P value < 0.05 for equivalent means between A and B at indicated time

points (two tailed student‟s t test). Key: Pre-op = pre-operative, DOS = day of surgery.

- 83 -

Figure 3.7. Completeness of resection.

A, B. Soft tissue and skin from resection bed biopsies and healthy mice were digested and subject to RT-

PCR analysis for HA transgene. Representative melt (A) and fluorescence (B) curves are shown.

Fluorescence seen from positive control sample (HA tumour, light blue) - corresponding melt curve peak

at 82°C. No fluorescence seen in negative controls (sham surgery, water, healthy skin) or in test samples:

24h postoperative (dark blue) and 2 weeks postoperative (red). C. Representative histology of AB1HA

tumour on removal. Keratinised epithelium with hair follicles visible at top of slide, subcutaneous

AB1HA tumour towards lower aspect of slide. Occasional vessels were seen in tumour (middle and lower

thirds of slide).

- 84 -

3 . 2 . 6 . HA presentat ion from recurrent AB1HA

The effects of local recurrence or post-operative metastasis on antigen presentation were

then investigated. To simulate local recurrence, BALB/c underwent incomplete

resection of AB1HA tumours by primary resection and immediate re-challenge with

AB1HA into the wound (n = 4). To simulate post-operative metastasis, mice with

AB1HA tumours were completely resected and then re-challenged into the healthy flank

on the day of surgery (n = 4). Locally recurrent tumours were clearly visible by day 16

after surgery in the 3 out of 4 animals in the enucleation group, measuring 8.05

0.75mm. “Metastatic” tumours were also detectable 3 out of 4 animals of the

“metastatic group”, measuring 2.95 0.51 mm at day 16.

In the locally recurrent cohort, antigen presentation was clearly detectable only in the

lymph nodes draining the locally recurrent tumour (Figure 3.8A). Average proliferation

rates of 74.62 5.65% were seen in that group, which was statistically comparable to

BALB/c with primary AB1HA tumours of similar size day 21 (51.96 6.93%) - Figure

3.8C. In animals with “metastatic tumour” after surgery, antigen presentation was seen

in the nodes draining the metastatic deposit, but was no longer detectable in the lymph

nodes of the post-operative site (Figure 3.8B). HA-specific proliferation from the

“metastatic deposit” was 52.98 5.61%, comparable to that normally seen from a

primary AB1HA tumour of similar size (day 10, 44.44 6.38%) - Figure 3.8C.

3 . 2 . 7 . Antigen presentat ion to CD4+ T cel l s pos t -op

HA presentation to the CD8+ T cell compartment was robust in animals with established

tumours, but was absent by two weeks post-operatively. However, CD4+ cells are

crucial to effective CTL generation and maintenance.224,226,230

To determine if tumour

antigen presentation to the CD4+ compartment was deficient compared with antigen

presentation to CD8+ cells, assays were repeated using HA CD4

+ epitope specific

(HNT) congenic T cells pre- and post-operatively (Figure 3.9A) Significant HNT

proliferation was detected on day 16 of tumour growth relative to naïve controls (Figure

3.9B, P < 0.005), although these levels were lower than CL4 proliferation at the same

stage of tumour growth (12.42 2.08% versus 45.96 7.28%, P = 0.006). As with CL4

proliferation, no HNT proliferation was observed two weeks after complete resection

(Figure 3.9A,B).

- 85 -

Figure 3.8. CL4 proliferation with locally recurrent or “metastatic” AB1HA.

BALB/c mice underwent incomplete primary resection, or complete primary resection with re-challenge

into the healthy flank on the day of surgery. Lyons Parish analyses were undertaken 16 days after surgery.

The experiment was performed once, with four animals per group. A. Lyons Parish analysis from local

recurrence group. B. Lyons Parish analysis from animal with metastatic tumour. C, Left: Proliferation

rates in the local recurrent bed compared to its nearest primary AB1HA equivalent (day 21). C, Right:

Proliferation rates of the metastatic nodes c.f. nearest primary AB1HA equivalent (day 10). Key: DLN =

draining lymph node, NDLN = non draining lymph node, Met = metastasis, Local Rec = local recurrence.

- 86 -

3 . 2 . 8 . Post -operat ive DC phenotype

Antigen presentation is governed by a number of factors including DC numbers,348

maturation,349,350

and subtype.350

It was therefore postulated that the decline in tumour

antigen presentation after surgery (3.2.4) may correlate with the disappearance or

emergence of a particular DC subtype. To evaluate this, changes in DC subtypes were

examined. Naïve and sham surgery animals were used as controls. Each sample

(draining and non draining nodes) was pooled from five animals.

DC were identified by CD11c positivity on side scatter/CD11c plots. High background

staining from the rat IgG2K-PE isotype required the use of stringent CD11c gating

(Figure 3.10A). Samples were divided into plasmacytoid (CD11c+CD45RA

+CD8

+/-),

CD8a+ (CD11c+CD45RA

-CD8

+) , and double negative (CD11c

+CD8a

-CD45RA

-)

populations as per accepted practice - Figure 3.10B.326,327

Surgery (sham or tumour) had

no impact on DC phenotypes, at either one week or two weeks post-operativelyA,B).

There was also no difference in DC phenotype between the non draining and draining

lymph nodes themselves, before and after surgery (Figure 3.11A,B)

3 . 2 . 9 . Cross presentat ion and in v ivo CTL funct ion

CD8+ lymphocytes require several signals to become continually activated, proliferate,

and differentiate into effectors.347,351

The first is antigen [peptide-MHC], the second a

co-stimulatory signal [e.g. CD80/86]214

and the third a cytokine signal [e.g. IL-12].352

Although antigen presentation declined after surgery (Figure 3.6), it was not certain that

a similar fall in CTL function would follow. To determine whether in vivo CTL function

decreased in parallel with antigen presentation, in vivo CTL assays were performed.

Assays were taken before surgery (day 16 tumours), 24 hours post-op, 1 week post-op,

or 2 weeks post-op. Inguinal, axillary, non-draining (e.g. contralateral inguinal/axillary)

and spleens were assessed in each animal. In vivo CTL lysis in subject animals was

compared to CL4 (positive) and naive (negative) controls.

HA-specific target lysis was routinely 40% in CL4 TCR transgenic mice (positive

controls), and absent from naïve animals (negative controls) - Figure 3.12B. Modest

endogenous responses (<10% killing) were seen in all tissues of tumour bearing mice

(<10%) except for the axillary node, where 23.77 2.45% killing was observed (Figure

3.12C). When targets were administered 24 hours post-op, killing in the inguinal node

markedly increased (24.00 4.56% post-op versus 6.13 0.35% pre-op, P = 0.0039) and

- 87 -

axillary node target lysis decreased (P = 0.0294). Thereafter, in vivo CTL function

declined gradually in all tissues, similar to the pattern seen in cross presentation (Figure

3.12C), indicating a close association between antigen and continued effector CTL

function.

Figure 3.9. HA-specific CD4+ proliferation before and after surgery.

HNT proliferation was assayed in BALB/c with established (day 16) AB1HA tumours, and two weeks

after surgery. Experiment was performed once, with five animals per group. A. Representative flow

cytometry showing HNT proliferation in the draining nodes of animal with day 16 tumour (left) but

absent two weeks after surgery (right). B. Single mice represented by points on the graph. Mean HNT

proliferation rates shown by bars. P values were two tailed and derived from the student‟s t test at the 5%

level of significance. Key: Pre-op = pre-operative (day 16 tumour), post-op = post-operative, 2w = two

weeks.

- 88 -

Figure 3.10. Flow cytometry for DC cell phenotyping.

Lymph nodes from animals after tumour resection or sham procedures were processed for analysis of

standard DC phenotypes.326,327

Compensation and thresholds were set using unstained, single stained, and

isotype controls. DCs were sub-gated from plots of CD11c and side scatter. The CD11c+ population was

then plotted for CD8 positivity (y-axis) and CD45RA positivity (x-axis). The experiment was performed

once, with five animals pooled from each group. Representative flow cytometry is shown from the

sentinel lymph node of a tumour bearing mouse. A: hamster PE isotype (left) and CD11c-PE mAb

staining (right). B: plasmacytoid (CD11c+CD45RA

+), CD8a-DC (CD11c

+CD8a

+CD45RA

-) and double

negative (CD11c+CD8a

-CD45RA

-) DC populations.

- 89 -

Figure 3.11. DC phenotype before and after surgery.

The proportions of DC that were CD8+, plasmacytoid, or double negative were quantified in the

draining and non-draining nodes. Assays taken pre-operatively and post-operatively. Data presented from

a single experiment, with pooled tissue from five animals in each group. A. DC phenotype in the draining

nodes. B. DC phenotype in the non-draining nodes. Key: pre-op = pre-operative, DOS = day of surgery,

2w = two weeks, post-op = post-operative.

- 90 -

Figure 3.12. Post-operative in vivo CTL

CFSE stained targets (CL4 peptide-pulsed and non-pulsed) were administered to BALB/c mice before

and after surgery. Representative flow cytometry and in vivo CTL readouts are shown for a single

experiment. Results are pooled from two separate experiments. At least 5 animals are present in each

group (range: 5 – 10). A. Gating of target and reference populations for in vivo CTL assay. B. Typical

profiles of in vivo CTL lysis from pre-injection, naïve, and CL4 mice. C. HA-specific in vivo CTL

function in lymphoreticular tissues before and after surgery. Mean SEM percentage killing of HA pulsed

targets shown for each tissue and timepoint, P values derived from student‟s t test at the 95% level of

significance. Key: (+) = pulsed, (-) = non-pulsed, DOS = day of surgery, 1w = 1 week, 2w = two weeks,

post-op = post-operative.

- 91 -

3 . 2 . 10 . Recurrent tumour and sys temic CTL responses

From 3.2.9, in vivo CTL responses correlated with antigen presentation. However, when

tumours emerge after surgery, they also present antigen (3.2.6). Hence they may impact

on existing effector and memory responses. To assess how the post-operative immune

system would function in the presence of metastatic tumour, CL4 peptide pulsed targets

were administered in the setting of post-operative tumour re-challenge. In vivo CTL was

measured for the following time points: day 16 of primary tumour growth, day of

surgery, and two weeks after surgery. CL4 pulsed and non-pulsed targets were

administered intravenously to all animals, four days after tumour re-challenge into the

healthy flank. As seen previously (Figure 3.12), in vivo CTL activity was predominantly

in the draining nodes before surgery. While there was no immediate change in draining

node CTL activity after surgery (P = 0.14), HA-specific killing did increase in the

draining nodes by two weeks post-op (P = 0.0011), Figure 3.13. The most striking

change occurred in the non-draining nodes and spleen, where minimal CTL was seen

pre-operatively (2 – 7%), but 12 – 19% killing was seen at two weeks post-operatively

(P ≤ 0.0162) - Figure 3.13.

3 .3 . Discussion

Tumour antigen presentation is the obligatory priming signal for a tumour-specific

immune response. Without sufficient antigen priming there can be no effector

differentiation, CD8+ proliferation, or memory development.

347,351 Therefore, the effects

of surgery on tumour antigen presentation were investigated using a non metastatic

tumour model20

bearing a membrane-bound model tumour neo antigen (HA) that is

constitutively cross presented.343

In the AB1HA model, HA presentation was previously found to be driven solely by

cross presentation in the tumour-draining lymph nodes.149,351

Consistent with these

previous findings, cross presentation of HA in this study was also found to be confined

to the draining lymph nodes, even after a more exhaustive examination of other

peripheral nodes (e.g. popliteal, brachial, para-aortic). The levels of HA cross

presentation were observed to be both constitutive and efficient, as previously

noted.149,351

Indeed, significant presentation was observed as early as four days post

implantation, when tumours were frequently too small to be visible. Extending on these

- 92 -

previous reports, the kinetics of tumour antigen presentation in vivo were found to be

directly proportional to tumour size, with no sign of plateau, even after 21 days of

tumour growth.

Figure 3.13. Primed in vivo CTL after surgery

In vivo CTL was assayed in animals four days after re-challenge, before and after surgery. Draining

nodes, non-draining nodes, and spleens were processed separately as shown. Experiment was performed

once, with a minimum of ten animals per group. Mean HA-specific lysis SEM shown for each group.

Cohorts compared by two sample student‟s t test at the 95% level of significance. *P = 0.0011, **P =

0.0079, ***P = 0.0162.

As hypothesised, complete surgery ablated antigen cross presentation. However,

intriguingly, tumour antigen presentation was not immediately ablated by surgery, but

persisted for at least a week post-operatively. This phenomenon was not attributable to

incomplete resection, as validated by the PCR, histology, and nude BALB/c data. The

decline in cross presentation was mirrored by changes in MHC II dependent antigen

presentation to CD4+ T cells. With only a single published abstract alluding to week-

- 93 -

long persistence of cross presentation after surgery, no substantive study had previously

defined this phenomenon.236

The finding that complete surgery did eventually ablate antigen presentation appeared at

odds with studies that suggest antigens are permanently presented through follicular

DCs.353,354

It has been postulated that permanent antigen presentation may be critical to

persistence of memory T355

and B356

cells. Indeed, “permanent” cross presentation has

been observed in the AB1HA model on one occasion. Marzo and colleagues found that

cross presentation could persist for 6 months despite the absence of visible tumour.20

Notably, this durable antigen presentation was in a rejection setting, rather than post-

operatively. The mechanism was also unclear, but the retained presentation may have

arisen from a permanent antigen reservoir on an accessory cell,20

or from tumour

dormancy. The latter concept refers to cancers that remain in small volumes, contained

by angiostatins,40

cell mediated immunity,357

or factors attributable to the tumour

microenvironment.40

Although it was not tested, dormant tumour might have been

detectable on PCR or immunohistochemistry in those experiments.40

As such, the

dormant tumour might have provided a continuous source of antigen.20

Numerous factors may have explained the loss of antigen presentation that was seen in

the data presented in this chapter. First, while permanent antigen presentation can occur

via follicular DCs,353,354

membrane bound antigens (like HA) are probably processed

through CD8+

- non-follicular DC.358

A second explanation could be attrition of the

DCs presenting HA in the draining lymph nodes. While the in vivo survival of different

DC subsets has yet to be fully elucidated,359

the half life of murine DC in general may

be in the order of 1.5 – 3 days.360

Accordingly, efforts were made to identify gross

changes in DC phenotype to correlate with the loss of antigen presentation. No change

in DC phenotype was detected after surgery, and so further efforts and time were not

devoted to that project. Other research in our laboratory361

suggests that CD11b+B7DC

+

cells may be important in processing tumour antigen. If those results are borne out in

further research, re-visiting the kinetics of DC phenotype (particularly the CD11bB7

DC population) after surgery might then be fruitful.

The final aspect of the work in this chapter is CD8+ lymphocyte function in vivo. With a

tumour in situ, in vivo CTL function is inextricably linked to antigen presentation, as

has been previously reported for the AB1HA model.155

An interesting pattern emerged

- 94 -

after surgery. Endogenous CTL function declined to background levels 14 days after

surgery, matching the decline in antigen presentation from the original tumour. When

there was recurrent disease after surgery, CTL function was “awakened” in the original

node bed and was also detectable systemically. This suggested that a form of effector

egress might occur after surgery, whereby primary resection releases trapped CD8+

from the draining nodes. Those effector cells may only be active in secondary nodes

when antigen is present, thus they are not visible on un-primed in vivo CTL assays.

If CD8+ effectors do egress from the draining nodes after surgery, it would be

consistent with the systemic appearance of memory CD4+ seen after surgery,48

and also

the egress of tumour-specific CD8+ with activating anti-CD40 mAb treatment.

155 One

concerning aspect was the pre-operative co-localisation of tumour antigen presentation

and tumour-specific in vivo CTL function. This suggested that node removal can

adversely affect the tumour-specific immune response, by removing the sites of priming

and CTL concentration. This hypothesis is tested in Chapter 5.

Also, the decline in cross presentation after surgery suggests patients with minimal

residual disease may become relatively depleted of antigen priming. Thus tumour

vaccines, which augment priming, may be of benefit in the eradication of minimal

residual disease. While the extent to which small recurrences and metastases engage the

immune system remained unclear, the efficacy of cross presentation (robust CL4

proliferation even when tumours were small) suggested local recurrences may interact

with the immune system at an early timepoint. Cross presentation of metastases was

also studied in this Chapter, the “metastasis” being created by inoculation of fresh

AB1HA cells into the healthy flank, rather being a true distant deposit from an

aggressive tumour. Metastases may be clonotypically and immunologically disparate

from the primary deposit, and it would be preferable to study cross presentation from an

orthotopic model that produces metastases ‟naturally‟. This issue is pursued in Chapter

6, where the orthotopic RencaHAM model is used.

3 .4 . Summary

In this Chapter, the kinetics of tumour antigen presentation before and after surgery

were described. This provides a useful principle in the planning of combined

surgery/immune therapy approaches. First, the localisation of tumour antigen to the

draining nodes suggested a place for locally delivered adjuvant therapy into the

- 95 -

resection bed, in the early post-operative phase. Second, patients with complete primary

resection and possible micrometastatic disease might benefit from additional immune

priming (e.g. tumour vaccination). Finally, given the efficiency of cross presentation,

patients who undergo debulking surgery for extensive disease probably have significant

reservoirs of tumour antigen. While they may benefit from strategies to improve the

quality of priming (e.g. DC preparations), it may be more appropriate to augment the

effector arm of the anti-tumour immune response (for example with adoptive immune

therapy).

In addition to the findings on cross presentation, surgery also had a profound impact on

endogenous tumour-specific CTL function. While in vivo CTL declined in parallel with

cross presentation after complete resection, recurrent tumours provided a fresh source of

antigen that evoked a powerful and systemic CTL response. This suggested surgery

enhanced CD8+

function overall, partially validating the practice of combining surgery

with immune therapy. With the effects of tumour resection on antigen presentation and

CTL function in mind, the impact of surgery on tumour resistance are now investigated

(Chapter 4).

- 96 -

Chapter 4

- 97 -

4. Sinecomitant immunity


The effects of surgery on cross presentation and in vivo CTL function were examined in

the previous chapter. Surgery produced a gradual ablation of tumour antigen cross

presentation, but where antigen remained, surgery enhanced systemic CD8+

T cell

function. Based on those findings, it was hypothesised that surgery would enhance anti-

tumour immunity overall. To do so, tumour resistance after primary resection should

exceed the degree of tumour immunity when the primary remains in situ.

When primary tumours remain in situ, experimental animals show some degree of

resistance to re-challenge by the same tumour in a separate site. This phenomenon,

known as concomitant immunity, was first described by Ehrlich in 1906. Concomitant

immunity has previously been shown to be strong when the primary tumour is large362

and when the dosage of re-challenge is low.24

Concomitant immunity may arise from

immune mechanisms,363

but may also occur because of non-specific factors24

(e.g.

angiostatins released from the primary tumour).116,117

Concomitant immunity is

distinguishable from sinecomitant immunity, which is the tumour resistance seen in a

post-operative host. Numerous studies indicate that sinecomitant immunity is tumour-

specific rather than a non-specific (innate) phenomenon,24,25,106

but T cell dependency

has not been established. As with concomitant immunity, the extent of sinecomitant

immunity depends on numerous factors, including the antigenicity of the tumour, the

size of the primary tumour, the timing of re-challenge, and the strength of the re-

challenge inoculum.24,106,107

In this chapter, post-operative tumour resistance (sinecomitant immunity) is compared

to pre-operative tumour resistance (concomitant immunity) to establish the net effect of

tumour resection on tumour immunity in the AB1HA model. Extrapolating from the

data of Chapter 3, a number of immunological pre-requisites for post-operative tumour

resistance are identified, along with a several factors that can antagonise post-operative

tumour immunity. Finally, enhanced responses to immune therapy are seen in the post-

operative setting, reinforcing the hypothesis that surgery boosts tumour-specific

immunity.

- 98 -

4 .2 . Resul ts

4 . 2 . 1 . Concomitant immunity in the AB1HA m od el

To determine the degree of concomitant immunity in the AB1HA model, animals with

established AB1HA tumour (3.90 0.41 mm) were injected with a second inoculum of

AB1HA. Incidence of the secondary tumour was used in proxy of overall survival

(Figure 4.1B), because animals succumbed rapidly to the primary tumour. On average,

animals were culled 15.1 2.85 days after re-challenge (29.1 2.85 days from primary

AB1HA inoculation). Concomitant immunity was present, because a 3 – 6 day delay in

incidence of the secondary tumour was observed relative to naïve controls (P = 0.032,

Figure 4.1C). However, once tumours had emerged, their growth kinetics were

indistinguishable from naïve BALB/c (P = 0.401, Figure 4.1B). This was not surprising,

given that tumour-specific effector CTL responses to cross-presented tumour antigens

were weak and remained localized to the draining lymph node (3.2.9).

4 . 2 . 2 . Sinecomitant immunity in the AB1HA m odel

Post-operative improvements in CTL function (Chapter 3) suggest that surgery may be

beneficial to tumour immunity. To determine the overall effect of surgery on tumour

resistance, BALB/c mice were curatively resected of primary AB1HA tumour, and then

re-challenged with 1x106 cells of AB1HA into the opposite flank. Re-challenge inocula

were administered on the day of surgery, one week post-operatively, or two weeks after

surgery (Figure 4.2A). These time points were chosen because they corresponded,

respectively, to high antigen presentation from the original tumour, fading/low antigen

presentation from the original tumour, and absent cross presentation from the original

tumour.

Significant survival from rechallenge was seen in the healthy flank at all time points

after surgery (P < 0.0001 for equivalence to naïve controls, Figure 4.2B). Overall, cure

rates were 33 19.2% for the day of surgery group, 57.14 18.8% at one week after

surgery, and 66.67 12.2% for the two week post-operative cohort. In the animals

resistant to re-challenge, secondary tumours were neither visible nor palpable at any

time after inoculation.

- 99 -

Figure 4.1. Concomitant immunity in the AB1HA model.

BALB/c with established AB1HA tumours were challenged with 1x106 cells of AB1HA and compared to

naïve controls. All mice were monitored for tumour incidence and growth at the re-challenge site. Data

are pooled from two separate experiments. There is a minimum of ten animals per group. A. Timeline of

tumour inoculation and culls. B. Mean tumour diameter SEM shown for each timepoint after tumour

inoculation, *P = 0.401 (ANOVA). C. Tumour incidence in re-challenged animals and naïve controls,

**P = 0.0318 (Log Ranks).

4 . 2 . 3 . Sinecomitant immunity in the wounded f lank

Sinecomitant immunity was seen when animals were re-challenged into the healthy

flank post-operatively (4.2.2). However, tumours may recur locally after surgery. To

determine whether sinecomitant immunity occurred at the surgical site, BALB/c

underwent curative surgery and were then re-challenge into the wound site. Re-

- 100 -

challenges were administered on the day of surgery, one week after surgery, or two

weeks post-operatively (Figure 4.3A)

Significant resistance to re-challenge was observed in the surgical site (Figure 4.3B) for

all time points (P < 0.001). However, overall survival was <15% for all groups.

Moreover, there was no increase in tumour resistance when rechallenges were delayed

beyond the day of surgery (P > 0.90 for equivalent survival across cohorts).

Consequently, beyond one week post-operatively, a disparity in survival between the

surgical site and the healthy flank emerged (95% C.I. for H.R., 0.02 – 0.25).

Figure 4.2. Sinecomitant immunity in the AB1HA model.

Post-operative tumour resistance was assessed in BALB/c after surgery. Data are pooled from multiple

experiments, with a minimum of ten animals in each group. Kaplan Meier survival are shown for each

cohort. A. Timeline of surgery and rechallenge. B. Resistance to rechallenge over time. P values derived

from Log Ranks analysis.

4 . 2 . 4 . Sinecomitant immunity and re -chal lenge dose

Sinecomitant immunity depended on the location and timing of the re-challenge

(Sections 4.2.2, 4.2.3). To determine the relative strength of the sinecomitant response,

a log fold reduced re-challenge inoculum was used. Specifically, BALB/c underwent

- 101 -

curative resection, followed by rechallenge with 1x105 cells of AB1HA. Re-challenges

were administered into the surgical site, or the opposite (healthy) flank. AB1HA inocula

were given on the day of surgery, one week post-operatively, or two weeks after surgery

(Figure 4.4A). For all time points after surgery, a robust resistance to rechallenge was

seen in the healthy flank when an inoculum of 1x105 cells was used. Moreover, BALB/c

resisted tumour equivalently on the day of surgery and two weeks post-operatively (P =

0.10, Figure 4.4B) with an inoculum of 1x105 cells. When a log fold lower inoculum

was used into the surgical site, a robust resistance to rechallenge was observed. In fact,

survival from surgical site tumours approached equivalence to the healthy flank (95%

C.I. for H.R., 0.005 – 1.99), Figure 4.4C.

4 . 2 . 5 . Surgical t rauma and s inecomitant immunity

From Figure 4.3, sinecomitant immunity was significantly weaker in the surgical site.

To determine whether this was due to the effects of surgical wounding per se, two

different sham experiments were undertaken. In the first experiment, mice underwent

sham surgery alone in the left flank, followed by inoculations of 106 AB1HA cells into

either the wound, or into the opposite flank. Inoculations were administered on the day

of surgery. In this experiment, sham surgery had no effect on tumour growth rates

relative to naïve controls (Figure 4.5A). This was evident for both the site of the surgery

and for the opposite flank. Thus surgery had no effect on primary AB1HA growth, and

wound proximity was similarly unimportant to growth kinetics. Although wounding had

no impact on primary tumour growth, it did not exclude an effect on post-operative

tumour growth. To determine whether surgery caused a difference in tumour resistance

after surgery, a second sham experiment was undertaken (Figure 4.5B). In this case,

BALB/c underwent tumour resection as well as sham operations on the distant flank

(opposite the tumour site). Mice were then re-challenged with AB1HA at two weeks

after surgery, into the site of sham operation. Tumour resistance within the sham

wounded flank was still superior to tumour resistance within the original tumour site

(Figure 4.5C)

4 . 2 . 6 . HA in s inecomitant immunity to AB1HA

AB1HA has been transfected with the nominal neo-antigen, haemagglutinin (HA) of

PR8 influenza H1N1 (A/PR/8/34).148

However, it was not known whether HA was

required or redundant in the sinecomitant immune response. Indeed, there are presumed

to be many tumour neo-antigens in AB1 tumours independent of the model tumour neo-

- 102 -

antigen HA. To determine the relative importance of HA and non-HA antigens to

sinecomitant immunity, primary AB1HA was grown and resected in BALB/c mice.

Those animals were re-challenged into the healthy (non-surgical) flank at two weeks

after surgery, with AB1HA (as before) or AB1 (the parental line, lacking HA).

Alternatively, BALB/c underwent surgery for AB1 primary tumour, followed by re-

challenges with AB1, at two weeks post-operatively. Survival was equivalent in all

cohorts (P > 0.05, Figure 4.6) indicating that HA was redundant in the sinecomitant

immune response (i.e. a true, “spy” antigen). The minor role of HA in the immune

response accorded with the low in vivo CTL activity seen previously (Chapter 3), and

the low frequency of endogenous HA-specific effectors on pentamer staining (0).

However, the minor role for HA might also be observed if sinecomitant immunity was

an innate phenomenon, rather than an antigen dependent phenomenon (see next section,

4.2.7).

Figure 4.3. Sinecomitant immunity in the surgical site.

BALB/c underwent curative primary resection, followed by re-challenge into the surgical site on the day

of surgery, one week post-operatively, or two weeks after surgery. Data are pooled from multiple

experiments, with a minimum of ten animals present per group. A. Timeline of surgery and re-challenge.

B. Kaplan Meier survival shown for each cohort. P values derived from the Log Ranks test.

- 103 -

Figure 4.4. Sinecomitant immunity: significance of re-challenge dosage.

BALB/c underwent curative primary resection, followed by re-challenge with 1x105 AB1HA cells. Re-

challenges were administered into the surgical site or into the opposite (healthy flank). AB1HA was

administered on the day of surgery or two weeks post-operatively. Data are pooled from two experiments,

with a minimum of ten animals per group. A. Timeline of surgery and re-challenge. B. Kaplan Meier

survival from re-challenge into the surgical site. C. Kaplan Meier survival from re-challenge into the

healthy flank. P values derived from Log Ranks analysis.

4 . 2 . 7 . T cel l dependence of s inecomitant immunity

To determine whether sinecomitant immunity was antigen specific, congenic BALB/c

nu-/-

mice were used to assess the T cell dependency of post-operative tumour

resistance. Nude mice were curatively resected of primary AB1HA, and then re-

challenged at two weeks after surgery (when the sinecomitant response was usually

strong in wild type mice). There was no significant difference in survival between post-

- 104 -

operative BALB/c nu-/-

and naïve BALB/c nu-/-

controls (Figure 4.7), suggesting that the

sinecomitant immunity seen in wild type mice was T-cell dependent.

To further characterise the T cell dependency of sinecomitant immunity, YTS 169 mAb

and GK1.5 mAb were used to selectively deplete for CD8+ and CD4

+ cells respectively.

In each instance, (Figure 4.8A) depleting antibody was administered one day prior to

tumour re-challenge (14 days post-operatively) and continued for two weeks.

Depletions were verified by flow cytometry on whole blood, and rat IgG2a isotype was

given to a control group of animals. Depletions were successful (>95% CD4+ or CD8

+

depletion) in all animals treated with depleting mAb, and normal T cell profiles were

seen in the controls (data not shown).

Post-operative mice treated with CD8 depleting antibody had comparable survival to

naïve controls, indicating complete ablation of the sinecomitant immune response

(Figure 4.8B). In contrast, animals treated with isotype had strong capacity to resist

tumour re-challenge (60 20.91% cure), consistent with preserved sinecomitant

immunity. CD4+ depletion significantly reduced survival compared to isotype control

(Figure 4.8B), but this was less marked than depletion for CD8+ cells. Indeed, some

30.77 12.8% of BALB/c were able to resist tumour re-challenge in the absence of

CD4+ cells.

4 . 2 . 8 . Pers is tent tumour and s inecomitant immunity

Complete resection evoked sinecomitant immunity (Section 4.2.2 and Figure 4.2). To

determine if sinecomitant immunity also occurred when resection was incomplete,

sinecomitant immunity was tested in the setting of persistent primary AB1HA tumour.

For this experiment, tumour resistance at two weeks after surgery was tested in cohorts

of BALB/c with or without recurrent primary AB1HA tumours.

Given the progressive growth of the recurrent primary AB1HA tumours, the period of

follow-up for this experiment was short. One mouse of the “recurrent primary” AB1HA

cohort did resist rechallenge, but had to be euthanased at just 10 days of follow-up. This

animal had to be considered a “survivor” despite the short follow-up. Notably, the

failure of tumour to emerge by ten days did lie beyond the usual 7 day timepoint for

AB1HA emergence after surgery (95% C.I., 4.52 - 9.48), n = 18.

- 105 -

Figure 4.5. Effect of surgical wounding on sinecomitant immunity.

Both sham surgery experiments (A and B/C) were performed once, with a minimum of five animals per

group. A. BALB/c underwent sham surgery and re-challenge with AB1HA into the surgical site, or the

opposite flank. Mean tumour diameter SEM shown for each timepoint, P values derived from two way

ANOVA. B. Timing and location of re-challenge in post-operative sham surgery experiment. C. Kaplan

Meier survival of post-operative sham surgery experiment cohorts. P values derived from Log Ranks

analysis.

- 106 -

Figure 4.6. HA specific immunity does not dominate the sinecomitant response

BALB/c underwent surgery for AB1 or AB1HA primary tumours, followed by re-challenge at two weeks

after surgery. Re-challenge inoculums contained AB1 or AB1HA, as indicated above. Data are pooled

from two separate experiments, with a minimum of seven animals per group. Kaplan Meier survival

shown for each cohort, P values derived from Log Rank analysis.

Figure 4.7. Sinecomitant immunity in BALB/c nu-/-

Nude BALB/c underwent resection for AB1HA, followed by re-challenge with AB1HA at two weeks

after surgery. Tumour resistance was compared to naïve BALB/c nu-/-

controls. Kaplan Meier survival

Data are shown for a single experiment, with at least five animals per group. P value was derived from

the Log Ranks test.

- 107 -

Figure 4.8. The effect of T cell depletion on sinecomitant immunity

BALB/c mice were treated with CD4+ or CD8

+ depleting antibody, and re-challenged with AB1HA two

weeks after surgery. Reference cohorts were isotype treated and naïve controls. Data are shown from a

single experiment, with at least five animals per group. A. Time-line of surgery, re-challenge, mAb

treatment, and monitoring of depletion. B. Kaplan Meier survival for each cohort. P values derived from

Log Ranks analysis.

4 . 2 . 9 . Pers is tent ant igen and s inecomitant immunity

Incomplete resection ablated sinecomitant immunity (4.2.8). This could be due to the

persistence of non-specific tumour associated factors (e.g. TGF, VEGF). Alternatively,

incompletely resected tumours might cause antigen persistence (Chapter 3), paralysing

the tumour-specific response.287,292

To dissect the relative importance of tumour factors

and tumour antigens in antagonising sinecomitant immunity, tumour resistance was

tested when there was persistent tumour and/or persistent tumour antigens after surgery.

- 108 -

Firstly, sinecomitant immunity was measured in the presence of persistent HA antigen,

without tumour. HA could be provided as purified peptide, PR8 virus, or RencaHAM

(see Chapter 6). The third option was chosen, as purified protein was unavailable and

PR8 virus contains viral-associated immunostimulatory agents (e.g. viral RNA) that

could confound results. Accordingly, BALB/c mice underwent primary surgery for

AB1HA, followed by re-challenge into the surgical site with RencaHAM. In this

setting, there was no persisting tumour in the surgical bed, but HA antigen persisted

post-operatively (Chapter 6). Tumour resistance to AB1HA was impaired in the

presence of RencaHAM, indicating that persistent tumour antigen alone (even without

persistent tumour) was sufficient to antagonise the sinecomitant immune response.

To investigate whether other mesothelioma associated antigens could similarly

antagonise the sinecomitant response (even without persisting tumour), BALB/c were

curatively resected for AB1HA and inoculated with AB1 into the surgical site, before

re-challenge with AB1HA. AB1 failed to grow in any of the animals and in doing so,

presumably, it provided a source of AB1 antigen that persisted until tumour re-

challenge (similar to RencaHAM). While this could not be verified, because there are

no other “spy antigens” in the AB1 model, reduced sinecomitant immunity was

observed. Sinecomitant immunity was also assessed in the presence of persisting

AB1HA tumour. As such, persistent AB1HA tumour should present the full suite of

AB1HA tumour antigens and tumour associated suppressive factors. In that situation,

sinecomitant immunity was nearly ablated. That impairment in sinecomitant immunity

was attributable (at least in part) to the persistence of antigens rather than other tumour

associated factors, because if a highly aggressive364

but antigenically irrelevant murine

malignancy persisted in the surgical site (RencaWT) there was no impairment of the

sinecomitant immune response.

4 . 2 . 10 . Distr ibut ion of HA speci f ic ef fectors p os t -op

In Figure 4.10 and Figure 4.8, sinecomitant immunity was antigen sensitive and CD8+

dependent. Moreover, in vivo CTL function co-located with antigen presentation before

surgery (Figure 3.12) but became systemic post-operatively (Figure 3.13). This

suggested effector cells were present in the draining nodes pre-operatively, but may

have egressed systemically after surgery. In turn, this phenomenon may have

contributed to the delayed sinecomitant response of the healthy flank.

- 109 -

Endogenous HA-specific effectors should form part of the sinecomitant immune

response to AB1HA, and thus the kinetics of HA-specific CD8+ should be

representative of the tumour-specific repertoire. HA specific CD8+ were examined in

the draining (axillary), non draining (contralateral axillary) and spleens from normal

animals, CL4 mice, tumour bearing BALB/c, and postoperative animals (day of surgery,

one week post-op, two weeks post-op). Nodes were examined by flow cytometry, using

fluorescently labelled IYSTVASSL-MHC pentameric complexes (Pro5® MHC

Pentamer, ProImmune) and CD8a antibody (Figure 4.11, A). Due to the high cost of

pentamer reagent, this experiment was performed only once, with four animals present

in each group. CD8+Pentamer

+ cells were quantified as a proportion of lymph node

CD8+ cells in each tissue, and compared between groups as continuous data. Consistent

with the CTL data shown previously (Chapter 3), HA-specific CD8+ cells were

disproportionately represented in the draining node relative to the non draining node

(0.66 0.17% versus 0.12 0.01%). As previously published,155

the relative numbers of

HA-specific effectors in the AB1HA system was low. Despite the small numbers of

HA-specific CD8+, a decline in Pentamer

+ cells was still detectable in the draining

nodes within 24 hours after surgery (“day of surgery” timepoint), 0.65 0.17% to 0.34

0.09% (P = 0.0026). However, no corresponding increase in HA-specific effectors in

the non draining node or spleen (P >0.88) was detected.

Figure 4.9. Incomplete surgery did not protect against new tumour challenges.

Sinecomitant immunity was assessed in mice after curative and non-curative resection for AB1HA. Data

are shown from a single experiment, with at least 5 animals per group. Kaplan Meier tumour incidence

shown for all cohorts. Continued primary AB1HA growth necessitated the truncated follow-up of the

“persisting primary AB1HA” group. *P = 0.044, **P = 0.029 (Log Ranks).

- 110 -

Figure 4.10. Tumour antigen persistence partially ablated sinecomitant immunity

A. AB1HA tumours were grown and then resected, for each cohort to this experiment. On the day of

surgery, animals were re-challenged into the surgical site with AB1 (parental to AB1HA, lacking the HA

antigen – A), RencaHAM (shares no known antigens with AB1HA except for HA – B), AB1HA (C),

RencaWT (shares no known antigens with AB1HA but is a highly aggressive tumour – D), or saline (E).

Each cohort was rechallenged with 1x106

AB1HA tumour cells into the healthy (non-surgical) flank at

two weeks post AB1HA resection. Experiment was performed once, with five animals present in each

group. B. Kaplan Meier survival shown for each cohort. P values derived from Log Ranks.

4 . 2 . 11 . Suppress ion a nd s inecomitant immunity

Surgery enhanced tumour-specific immunity, but tumour resistance was relatively

impaired on the day of surgery and at the original tumour site. Surgical wounding and

tumour antigen kinetics may affect the topography and timing of tumour-specific

immunity (see 4.2.5). However, cancers have also been shown to hamper tumour-

specific immunity by numerous other mechanisms, including Treg366

and MSCs.27

To determine whether post-operative tumour immunity correlated with the

accumulation and distribution of Treg, regulatory T cells were quantified (as a proportion

of lymph node CD4+ cells) in naïve, tumour bearing, and post-operative animals (Figure

4.15). Treg were compared between the surgical flank (lastingly vulnerable to tumour)

- 111 -

and the healthy flank (resists tumour robustly at two weeks after surgery). There was no

significant accumulation of Treg in the nodes with mice bearing AB1HA tumour, the

percentages of CD4+CD25

HiFoxp3

+ did not change after surgery, and Treg were not

over-represented in the region of the original tumour relative to the healthy flank

(Figure 4.16). While Treg did not accumulate in the draining nodes, this cell type might

still hamper post-operative tumour immunity. To determine the impact of Treg on

sinecomitant immunity, PC61 mAb (Monoclonal Antibody Facility) Treg depletions

were performed. Depletions were undertaken when resistance to re-challenge was

weakest (on the day of surgery, with re-challenge into the surgical site).335

Depletions

were verified on whole blood and lymph node biopsy (Figure 4.18A).

While CD25+ cells were reduced with anti-CD25 mAb, the FoxP3

HICD4

HI population

were not significantly depleted (Figure 4.17). This was consistent with a recent

publication reporting that Treg are not removed by PC61,336

but rather CD25 expression

is down-regulated. Nevertheless, PC61-induced downregulation of Il-2R does impair

Treg function.336

Thus PC61 treatment would still reduce Treg function.

CD25 expression was ablated by PC61 at 24 hours after treatment (Figure 4.17A). At 7

days after PC61 mAb injection, CD25 ablation was maintained in 4 out of 5 animals

(Figure 4.18B,C). At that time point, tumour was emerging in the animal that was

beginning to express CD25 (tumour diameter 1.41mm). A single treatment of PC61

mAb delayed the onset of tumours for approximately seven days, significantly

improving survival relative to saline controls (Figure 4.19).

Thus Treg depletion boosted the sinecomitant immune response but this was not

complete. Another factor was additionally investigated, specifically, the relationship

between MSC and sinecomitant immunity. Spleens were collected from BALB/c in the

absence of tumour, with established (day 16 tumour), on the day of surgery, and two

weeks post-operatively.

Splenic MSC were identified by flow cytometry and the characteristic markers of

CD11b and Gr-1.246

As it is unknown which cell type of the MSC population is

responsible for immune suppression,246

the group was quantified as a whole.

Quantification was derived by dividing the number of Gr-1+CD11b

+ cells by the number

of viable, non red cell splenocytes collected over the same period - Figure 4.20A.281

- 112 -

No statistically significant increase in splenic MSC occurred with tumour growth (4.96

0.48% versus 3.87 0.41% seen in naïve animals), P = 0.097 - Figure 4.20B. There

appeared to be a small decrease in splenic MSC from tumour bearing animals relative to

the two weeks post-op group (4.96 0.48% versus 3.59 0.34%), but this was not

statistically significant (P = 0.0728).

Figure 4.11. Distribution of HA-specific CD8+ T cells.

HA-specific CD8+ cells were identified using IYSTVASSL-MHC pentameric complexes (Pro5

® MHC

Pentamer, ProImmune). Due to the high cost of pentamer reagent and the quantities required to detect

HA-specific CD8+ cells, this experiment size had to be restricted. Of the tumour draining nodes, the

axillary nodes were chosen because they contained higher counts of pentamer positive cells, compared to

inguinal nodes. Only four animals were present in each group, and the experiment was performed once.

A. Representative flow cytometry from CL4 nodes, axillary nodes, non-draining nodes, and spleen. B.

Mean % CD8+ cells expressing IYSTVASSL-specific TCR for each cohort, + SEM for each mean value.

*P < 0.05 (student‟s t test).

- 113 -

Figure 4.12. CD127 and CD44 analysis of CD8+Pentamer

+ cells

Representative plots of CD127 and CD44 on pentamer+ and pentamer

- cell populations. A. isotype

staining for CD44 and CD127 mAb. B. Representative flow cytometry for CD44 and CD127 expression

of Pentamer+ and Pentamer

- populations.

- 114 -

Figure 4.13. Expression of CD44+ in Pentamer+ and Pentamer

- CD8

+

Pentamer+ and Pentamer

- CD8

+ cells were assessed for expression of CD44. Mean values +SEM shown

for each cohort. Experiment was performed once, with four animals per group. P values were two tailed

at the 5% level of significance, derived from the student‟s t test.

- 115 -

Figure 4.14. Expression of CD127 in CD8+CD44

+ populations

CD8+CD44

+Pentamer

+ and CD8

+CD44

+Pentamer

- cells were assessed for expression of CD127. Mean

values +SEM shown for each cohort. Experiment was performed once, with four animals per group. P

values were two tailed at the 5% level of significance, derived from the student‟s t test.

- 116 -

Figure 4.15. Representative flow cytometry for Treg quantification.

Treg were quantified in the draining and non-draining lymph nodes of tumour bearing BALB/c, normal

(naïve mice), and post-operative animals. A, B. CD4+ cells were sub-gated from the lymphocyte

population on the forward scatter and side scatter plots. C. CD4+ lymphocytes were plotted for Foxp3

positivity (y-axis) against CD25 positivity (x-axis). CD4+CD25

HIFoxp3

+ cells were selected as the natural

Treg population and quantified as a proportion of CD4+ cells in each tissue. Percentages of

CD4+CD25

HIFoxp3

+ cells were compared between subject groups, as continuous variables.

- 117 -

Figure 4.16. Treg frequency pre- and post-operatively

CD4+CD25

HiFoxp3

+ cells were quantified proportionately in the draining and non-draining nodes, before

and after surgery. Data are pooled from two separate experiments, with a minimum of four animals per

cohort (range: 4 – 8). Individual mouse lymph nodes shown. Mean %CD4+CD25

HiFoxp3

+ indicated by

bars. All groups compared using the student‟s t test, and no statistically significant difference was found

(two tailed P > 0.05).

- 118 -

Figure 4.17. Treg remained despite PC61 mAb.

The presence of CD4+Foxp3

+ cells was assessed in PC61 and saline treated animals, for the Treg depletion

experiment. Each experiment was performed once, with five animals per group. Flow cytometry was

performed on three separate occasions. Representative flow cytometry shown from the 24 hours post-

treatment timepoint. A,B. Whole blood CD25 and Foxp3 expression for saline and PC61 treated animals.

C. Mean %CD4+Foxp3

+ cells +SEM shown for depleted BALB/c and controls. P value derived from the

student‟s t test.

- 119 -

Figure 4.18. Correlation of Treg depletion with tumour emergence

BALB/c were curatively resected of AB1HA, and then re-challenged into the surgical site on the day of

surgery. Concurrently, these mice were depleted for CD25 using PC61 mAb, and followed for emergence

of the locally recurrent tumours. A. Timeline of surgery, PC61 mAb depletion, and monitoring for Treg

depletion. B. Flow cytometry on lymph node biopsy specimens, one week after PC61 mAb/saline

treatment. Depleted animals shown in the top panel, saline controls in the bottom panel. Mouse with

recovering CD25 expression (highlighted) had emerging locally recurrent tumour at the time of biopsy. C.

CD25 expression levels at the time of biopsy (data from B). P value derived from the student‟s t test.

- 120 -

Figure 4.19. Effect of Treg depletion on local recurrence.

BALB/c underwent Treg depletion and re-challenge into the site of AB1HA resection, as for Figure 4.18.

Experiment was performed once, with five animals per group. Kaplan Meier tumour-free survival for

PC61 and saline treated BALB/c. P value derived from Log Ranks analysis.

4 . 2 . 12 . Sinecomitant immunity and immune therapy

If surgery boosted the anti-tumour immune response (4.2.2), immune therapies could be

more effective in treating post-operative recurrence than primary disease. To determine

whether immune therapy was more effective for post-operative recurrence than primary

disease, BALB/c with primary AB1HA or post-operative recurrent AB1HA were

treated with immune therapy. Post-operative recurrent AB1HA was generated by re-

challenge with fresh tumour cells in the healthy flank, on the day of primary AB1HA

resection. In all animals, treatment was initiated when tumours were first palpable

(1mm in diameter). Antigen-based therapy (tumour vaccines) were avoided, given the

apparent importance of antigen decline (4.2.9) to the sinecomitant immune response.

Response to therapy was assessed by the outgrowth of treated tumours and overall

survival. Therapies were chosen for which dosage regimens were previously optimised.

The intra-tumoural route of administration was chosen because it theoretically offered

the highest drug concentration within the tumour, and a lower systemic toxicity.367

Specific treatments were murine activating anti-CD40 mAb (FGK45, Monoclonal

Antibody Facility), CpG-ODN 1668 (Tib-Molbiol), poly I:C (InvivoGen), and 3M019

(3M Pharmaceuticals).

- 121 -

Figure 4.20. MSC in tumour bearing and post-operative mice.

MSC were quantified proportionate to non-erythrocyte splenocytes in BALB/c spleens before and after

surgery. Data are shown for single experiment. A. Representative flow cytometry from naïve, tumour

bearing and post-operative animals (day of surgery and two weeks post-operative). B. Percentages of

CD11b+Gr-1

+splenocytes shown for each cohort. Individual mice shown, along with mean values (bar).

No statistically significant difference was observed between the groups (student‟s t test).

- 122 -

CpG 1668 was equally able to cure all animals with either primary AB1HA or post-

operative AB1HA disease (data not shown). Each of the remaining therapies (FGK45,

3M019, poly I:C) improved survival in the setting of primary AB1HA disease (P < 0.05

relative to saline controls), but cure rates ranged from just 8.33 7.98% (FGK45) to 40.00

21.90% (poly I:C) - Figure 4.21 and Figure 4.22. For each of those treatments, survival

was more remarkable in the post-operative setting (71.43 12.08% to 100.00%) and

significantly improved relative to primary disease (P < 0.05) - Figure 4.21A,B and

Figure 4.22A,B. Cure rates were less impressive for tumours sited in the surgical flank

relative to the healthy flank, but still superior to treatment for primary disease (data

shown for FGK45 - Figure 4.22A,B).

4 .3 . Discussion

Surgery has been much maligned as an immune suppressive treatment.31-33,95,96

However, if surgery is immune suppressive, this seems at odds with a number of key

clinical and empirical findings. First, patients have residual disease more frequently

than predicted from post-operative recurrence and metastasis rates. Indeed, modern

techniques of PCR and flow cytometry indicate that a significant proportion of patients

with clinically localised epithelial malignancy have circulating tumour cells both before

and after surgery. 35,37,38,40

Thus dissemination of tumour cells might occur when as few

as 106 cells are present, several log scales before the earliest tumours are detected.

40 In

breast cancer for instance, at least a subset of such patients never present with

metastasis or recurrence after surgery.40

Such a phenomenon would seem unlikely if

surgery suppressed immune function.

Second, it has been shown that immune therapies are more effective in combination

with surgery than without surgery in renal cancer.12,14,15,112

Once again, if surgery

depressed anti-tumour immunity rather than boosted it, such a finding would be

improbable. Finally, recent empirical publications suggest that surgery can re-set several

important parameters of the anti-tumour immune response, with beneficial effects on

immune suppressive cytokine levels,140

MSC networks,46,285

CD4+ memory phenotype

and trafficking,48

and general cell mediated immunity.285

- 123 -

Figure 4.21. Response to immune therapy after surgery.

BALB/c with primary or post-operative AB1HA tumours were treated with poly I:C, 3M019, or saline.

Post-operative tumours were sited in the healthy flank. All tumours were treated intra-tumourally, on

emergence (tumour diameter 1 mm). Kaplan Meier survival shown for each cohort, P values derived from

the Log Ranks test. Data are shown for a single experiment, with at least five animals present per group.

A. Poly I:C treated mice received a dosage of 10 g every day, for a maximum of six treatments. B. Mice

undergoing 3M019 therapy received 50 g every second day, for a maximum of six treatments.

- 124 -

Figure 4.22. Response to immune therapy in the healthy flank and surgical site.

BALB/c with primary or post-operative AB1HA tumours were treated with FGK45 or saline. Post-

operative tumours were sited in the healthy flank (A) or the surgical site (B). All tumours were treated

intra-tumourally, on emergence (tumour diameter 1 mm). Mice received a dosage of 40 g, up to every

second day, for a maximum of six treatments. Data are pooled from three separate experiments, with a

minimum of 5 animals per group. Kaplan Meier survival shown for each cohort, P values derived from

the Log Ranks test. A. FGK45 versus saline for the treatment of primary AB1HA tumours, or post-

operative tumours in the healthy flank. B. FGK45 versus saline for the treatment of primary AB1HA

tumours, or post-operative tumours in the healthy flank .

- 125 -

In this chapter, tumour resistance was compared between post-operative animals and

mice with untreated primary AB1HA. Animals bearing primary AB1HA did display

somewhat reduced growth of a second AB1HA challenge (concomitant immunity),23,24

but this was a weak phenomenon. In contrast, animals robustly resisted tumour

rechallenge after surgery. That phenomenon, previously dubbed “operation immunity”

or “sinecomitant immunity”,25

would indicate that surgery could boost tumour

immunity, providing a useful platform for post-operative immune therapy. It might also

explain why immune therapy has been more successful after surgery than

preoperatively, as discussed above.

Sinecomitant immunity was never 100%, suggesting that sinecomitant immunity was

variable across individual mice. Numerous factors have been reported to contribute to

such variation, including: tumour size at surgery, duration of primary tumour growth,

and dosage of inoculum.25

Additional factors that may have been operant included:

blood loss, procedure times, pain, anaesthesia dosages, and so on.25,106

To reduce bias,

every effort was made to ensure comparison groups were matched for tumour size, time

of tumour growth, and mouse age. Other variables were more difficult to control but

assumed to be comparable, given that a single surgeon (the author) administered all

anaesthesia and performed all procedures.

A number of factors seemed to affect the strength of the sinecomitant immune response.

In particular and in accordance with the preceding literature, sinecomitant immunity

was affected by the dosage and timing of the re-challenge.110,111

However, sinecomitant

immunity was also affected by the location of the re-challenge. This had not been seen

previously, possibly because preceding publications have used smaller dosages for re-

challenge (105 cells or less).

106-108 As seen in this chapter, dosages of 10

5 could be

resisted reliably irrespective of site of injection. It was not until dosages of 106 cells

were used that the difference in post-operative tumour resistance between the surgical

site and a distant site was observed.

Having established the extent of sinecomitant immunity in the AB1HA model,

numerous immunological correlates of successful and unsuccessful sinecomitant

immunity were sought. Looking at the healthy flank, successful resistance to

rechallenge occurred only at two weeks after surgery. This pointed to a remarkable

parallel between the wane of tumour antigen cross presentation (seen in Chapter 2), and

- 126 -

the emergence of sinecomitant immunity. Indeed, the reduction in tumour antigen, or

“antigen holiday”, seemed critical to sinecomitant immunity, since antigen persistence,

even in the absence of tumour persistence (e.g. rechallenge with AB1 or RencaHA),

could abrogate sinecomitant immunity. Moreover, sinecomitant immunity against

AB1HA was preserved when aggressive364

but antigenically irrelevant tumour

(RencaWT) persisted in the surgical site.

The inverse relationship between sinecomitant immunity and tumour persistence and/or

tumour antigen persistence had been noted twice previously. Gorelick and colleagues

found that primary resection had to be complete for sinecomitant immunity to

develop,24

suggesting that the removal of tumour suppressive factors and/or removal of

tumour antigen en masse could be important to sinecomitant immunity. Further, using

the methylcholanthrene induced fibrosarcoma model (C3H/HeJ inbred mice), Kahan

and colleagues described a series of surgical experiments in which tumour antigen

vaccines were given in the first two weeks after surgery.106

Under these conditions, the

usual resistance to re-challenge was impaired. This led them to conclude that

sinecomitant immunity and (tumour) antigen therapy are “mutually antagonistic”,106

as

was found to be the case in this Chapter.

Given the significance of tumour antigen in sinecomitant immunity, it was not

surprising that T cells were required: congenic BALB/c nu-/-

did not display post-

operative tumour resistance. Depletion studies using YTS169 confirmed this was the

case, with ablation of sinecomitant immunity occurring when CD8+ cells were absent.

Thus CD8+ function seemed integral to the sinecomitant immune response, as with the

anti-tumour immune response more generally.344-346

CD4+ cells were not essential to the sinecomitant response, at least when they were

depleted after surgery. However, CD4+ cells may be more important in the “APC

licensing” phase of the anti-tumour immune response, when effectors are being primed

for the first time.224,225,227,229

Thus significant reductions in sinecomitant immunity may

ultimately have been observed if CD4+ cells were depleted during tumour growth (the

initial priming and licensing phases). Additionally, Treg express CD4 and these were

probably depleted along with helper T cells when GK1.5 CD4+ depleting antibody was

used.363,368

Since Treg depletion enhanced tumour immunity (4.2.11), a reduction in Treg

- 127 -

may have offset any detrimental effects of reduced helper T cells during CD4+

depletion.

A disparity in tumour resistance was seen between the early post-operative phase and

the late post-operative phase, as well as the healthy flank and the surgical site. One

potential explanation was some immune suppressive effect from surgical trauma, and/or

the inherent vulnerability of surgical wounds to tumour growth.96,369,370

To determine

whether surgical wounding contributed to the topography and timing of sinecomitant

immunity, sham experiments were performed. Sham surgery did not accelerate tumour

growth, and only partially impaired the robust tumour immunity of the healthy flank.

Thus surgical wounding was an improbable factor in the distribution and time

dependency of sinecomitant immunity; other mechanisms were more likely to be

operant.

One such potential mechanism was the selective accumulation of Treg in the draining

lymph nodes of tumour bearing mice. CD4+CD25

++Foxp3

+ could be identified in the

lymph nodes of normal animals, and these accorded with previously published values

for both mouse371,372

and man.372,373

Treg did not accumulate in the draining nodes nor

did they decline significantly after surgery. This was consistent with a recent study by

Needham and colleagues in the AE17 C57BL/6J mesothelioma model, who found that

CD4+CD25

+ cells do not accumulate in the nodes, but rather within the tumour itself.

259

The fact that Treg did not decline after surgery suggested they may be impeding post-

operative tumour immunity. As sinecomitant immunity was most impaired in the site of

the surgery and on the day of surgery, the role of Treg was investigated in that setting.

Accordingly, Treg depletion with PC61 could ameliorate tumour vulnerability in the

operated flank. Not only could local recurrence be prevented during the phase of Treg

depletion, but the return of Treg correlated with the emergence of local recurrence. Thus

Treg may be a critical impediment to sinecomitant immunity, and may be a major factor

in the vulnerability of resection beds to local recurrence.

Enhanced sinecomitant immunity after PC61 depletion suggested that Treg were indeed

dampening sinecomitant immunity. Treg have been previously found to limit both

concomitant immunity363

and sinecomitant immunity.374

Combining Treg depletion with

surgery might therefore have considerable benefit374

and numerous treatment strategies

- 128 -

may be used to boost sinecomitant immunity, including: anti-CTLA4 antibody,375,376

cyclophosphamide (a conventional chemotherapy that reduces CD4+CD25+ cells),377

intra-tumoural Treg depleting antibody,259

denileukin diftitox (Ontak®, Ligand

Pharmaceuticals, San Diego, California USA),372,378,379

cyclosporine/tacrolimus

(transplantation drugs that suppress Il-2 production and signalling and could abrogate

Treg function)372

and anti-glucocorticoid induced tumour necrosis factor receptor related

protein antibody (anti-GITR mAb).372,380

As well as identifying Treg impediments to sinecomitant immunity, the candidate

attempted to investigate the relationship between sinecomitant immunity and MSCs.

MSCs, also known as “inhibitory macrophages” 250,251

and “early myeloid cells”249,250

may accumulate in the lymphoreticular system253

and in tumours of cancer-bearing

mice.27

Those MSCs may deplete arginine in the tumour microenvironment, impairing

CD8+ proliferation and maintenance of the CD3 chain.

256 Moreover, MSC produce

peroxynitrites, which induce CD8+ apoptosis

246 and tumour nutritive polyamines

(through the L-ornithine pathway).282

MSC may also directly produce T cell tolerance,

at least to soluble tumour antigens, by uptake and presentation to T cells in a tolerogenic

manner.254

In this chapter, MSCs accounted for 1.98 0.41% of splenocytes in healthy mice,

consistent with previously published values.381

MSCs appeared to accumulate in the

spleens of tumour bearing animals, but the increase was minimal over naïve mice, and it

was not statistically significant. The increase in MSCs for this system was less than

previously reported in other BALB/c mesothelioma models250

where around 28.5% of

splenocytes had Gr-1 and CD11b positivity during tumour growth.

A number of explanations may underpin that difference, including mouse strain

(C57Bl6 versus BALB/c), tumour model (AB1HA versus TC-1), and technical issues

(antibody choice, isotypes, compensation). However, the most important factor was

possibly tumour size (Albeda and colleagues used tumours 6 – 8 times larger than

studied herein).250

As MSCs are known to accumulate more or less in parallel with

tumour burden,253

a lesser MSC accumulation would be predicted with the smaller

tumour sizes used in this chapter.

- 129 -

Reductions in MSCs after surgery have been reported in previous

publications.46,281,285,382

In this Chapter, no statistically significant decline in MSC levels

was present. If larger AB1HA tumours were grown and/or a large series was

undertaken, a post-operative decline in MSCs might be validated to statistical

significance.

Given the low accumulation of MSCs, it would seem doubtful they were of significance

in impairing sinecomitant immunity. To assess this more rigorously, additional

depletions of MSCs might be fruitful. For example, one could compare sinecomitant

immunity in MSC depleted and non-depleted animals, to determine whether there was a

difference. Techniques that could be used include: all-trans retinoic acid (which induces

MSC differentiation) 383

and gemcitabine (which reduce splenic MSCs).250

In Chapter 3, it was found that systemic CTL activity increased after surgery, and this

correlated with a decline in cross presentation within the sentinel nodes. It was therefore

hypothesised that surgery induced a systemic egress of HA specific CD8+, and that

those cells would have a central memory phenotype; similar to recently published data

for the CD4+ compartment.

48 The robust antigen dependent proliferation of Chapter 3

suggests it would be easy to track the endogenous HA specific CD8+ repertoire in the

AB1HA model. Unfortunately, as has been published previously for this model, only a

few HA specific CD8+ are visible on HA epitope–MHC-fluorochrome flow

cytometry.155

Even with high grade reagent (IYSTVASSL Pro5® MHC Pentamer,

ProImmune), detection of HA specific effectors was extremely difficult.

Nevertheless, it was shown that tumour-specific CD8+, like in vivo CTL function, were

disproportionately represented in the draining lymph nodes of the tumour. After

surgery, there was a reduction in those tumour-specific CD8+ in the regional nodes, as

was recently reported for the CD4+ compartment.

48 This decline in CD8

+ cells was

consistent with an egress of CD8+ from the draining lymph node to the systemic lymph

nodes and spleen. However, again similar to the findings of Benigni and colleagues,48

there was no corresponding increase in CD8+ cells within systemic nodes to correspond

to the reduction in draining node CD8+ cells. Unfortunately, due to the small cell

numbers involved, flow cytometry was unlikely to detect a difference. While apoptosis

would be an alternative explanation for the reduction in tumour-specific CD8+ in the

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sentinel node, more sensitive techniques (e.g. quantitative PCR) might subsequently

support or refute an egress hypothesis.

An attempt was made to determine the kinetics of memory phenotype markers in

tumour-specific effectors after resection. CD44 was used to identify the proportion of

CD8+ lymphocytes that were memory cells. A second memory marker, Il-7 receptor

(C127), was also used. Expression of Il-7R receptor (CD127) is thought to indicate a

true “central memory” cell that is capable of homeostatic proliferation, via Il-7 and the

STAT-5/Bcl-2 mechanism of anti-apoptosis.365

Notably, studies of viral infection have

suggested that with antigen persistence, Il-7R is downregulated and that such cells

display an anergic memory profile.365

In contrast, when infection can be controlled, Il-

7R is upregulated, homeostatic proliferation occurs, and cells are again capable of

producing IFN.365

The persistence of tumour antigen during tumour growth was thought to parallel chronic

viral infection, where antigen burden is high and CD8+ memory cells have an anergic

phenotype (including low Il-7R expression). After surgery, antigen loads are reduced

(Section 3.2.4) and as with acute viral infection, cells might then upregulate Il-7R

expression and the capacity to produce IFN may be recovered.

In this chapter, surgery did not upregulate CD127 expression in the tumour-specific

memory pool. However, assessment of Il-7R expression was extremely difficult in this

system. The problem of low HA specific pentamer+ cell counts was compounded by

high background rat IgG2aK-APC isotype binding and technical difficulties with four

colour FACS compensation. No rigorous Il-7R specific signal was possible to analyse,

and the results obtained (no change in Il-7R) were considered tentative, at best.

Alternative experiments may be helpful to address the effects of surgery on CD127

expression. One method is to adoptively transfer naïve tumour-specific CD8+ cells from

congenic mice, prior to tumour growth. This approach has been used extensively by

other researchers in tumour or viral models298,299,307

and effectively would amplify the

number of tumour-specific CD8+ cells available for antigen encounter and memory

differentiation. This approach was not used by the author, because the required murine

strains were unavailable and because adoptive transfer of CD8+ could fundamentally

modify the dynamics of the immune response. Specifically, the ratio of CD8+ and CD4

+

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cells to the APC could be disrupted, artificially imprinting patterns of memory

development307

that would not otherwise be seen in the endogenous repertoire.

Applying the concepts of this chapter, the finding that surgery enhanced tumour-

specific immunity was encouraging. It was postulated that surgery may induce a decline

in tumour related suppressive factors, including tumour antigen itself. While the role of

immune therapy after surgical resection had yet to be proven,27

the early post-operative

period after surgery was thought to present a “window of opportunity” for effective

immune therapy. In the clinical setting, patients may be optimally responsive to immune

therapy within the early and intermediate post-operative phases: tumour associated

suppressive factors are at their lowest,27

and tumour antigen presentation is declining.

Beyond that period, the growth of locally recurrent tumour or metastases may again

suppress the immune system.27

To delay treatment beyond the early post-operative

phase may therefore miss the optimum time for immunotherapy.384,385

It is not clear how long the “window of opportunity” remains open. In the murine model

of mesothelioma, tumours emerged on 8.14 0.33 days after surgery, i.e. two days

longer than naïve controls. This suggested the immune system of post-operative animals

was competent for 2 more days than a naïve mouse. In humans, the window of

opportunity may be open for longer. Indeed, patients may hold residual cancer cells in

check (tumour dormancy) for years or even decades after surgery.3,26,40,357,386

That

period of immune competence probably varies from patient to patient. Differences may

relate to numerous (tumour histology, disease burden, sites affected, extent of

cytoreduction) and host factors e.g. co-morbidities. With renal cancer, the average

period of immune competence may be several years on average: most patients who

present with recurrence do so within 3 – 5 years post resection.387

For mesothelioma,

the window of opportunity may be shorter – most patients have presented with recurrent

disease by 2 years after surgery.388

In this chapter, the window of opportunity was explored using numerous immune

therapies that target the nexus between CD4+ T cells, DC, and CD8

+ lymphocytes (see

Chapter 1). Given the importance of a decline in tumour antigen to sinecomitant

immunity, exogenous antigen delivery (tumour vaccination) was avoided. The therapies

used were activating anti-CD40 antibody (FGK45, Perth Monoclonal Antibody

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Facility), 3M019 (an imidazoquiniline, 3M), poly I:C (InvivoGen) and 1668 CpG-ODN

(Tib-Molbiol).

CD40 activation immunotherapy has previously been combined with the conventional

therapies of radiotherapy389

and chemotherapy,333

but has not previously been used in

combination with surgery. Similarly, there was no preceding data on the use of locally

delivered poly I:C and 3M019 in the post-operative setting. For each therapy tested

(excepting CpG 1668), responses to immune therapy were much better in animals after

surgery than they were in the setting of de novo (primary) AB1HA disease.

The local recurrence site responded poorly in each instance, which was predicted from

the weak improvements in tumour-specific immunity seen in the surgical flank (4.2.3).

As discussed earlier, the factors underlying that poor response might be the inherent

vulnerability of scar tissue to tumourigenesis369

and/or some form of suppression

imprint from the tumour, specifically sited in the draining nodes (e.g. IDO+

plasmacytoid DC258

or Treg366

). Thus future immune strategies for local recurrence may

have to consider this problem, perhaps by a combined approach of immune stimulation

and Treg depletion.

4 .4 . Summary

Surgery has been postulated to boost tumour-specific immunity by the disruption of

immune suppressive networks, but few publications have addressed this hypothesis. In

this chapter, concomitant immunity (the resistance of a host to a second tumour, by

virtue of a primary lesion being present)25,105

was found to be a weak phenomenon. In

contrast, animals could robustly resist tumour re-challenge after primary resection. This

phenomenon, known as “sinecomitant immunity”,25

supports the concept that surgery

can boost the tumour immune response. As such, surgery could provide a powerful

platform for effective immune therapy. In this chapter, it was demonstrated that primary

resection could synergise with several TLR ligands and activate anti-CD40 antibody to

eradicate locally recurrent and metastatic solid tumours.

Sinecomitant immunity could be hampered by Treg and was absolutely dependent on

cytototoxic lymphocytes. Sinecomitant immunity was also strongest temporally and

spatially disparate from the original tumour site. The cause of the disparity was unclear,

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but there were no significant differences in absolute numbers of MSC, Treg and DC

phenotype to explain that trend. Surprisingly, sinecomitant immunity required the

decline in tumour antigen presentation seen after surgery (reported in Chapter 3) and

tumour antigen persistence (even when the tumour itself was not persistent) could

antagonise post-operative tumour resistance.

Taken together, these findings would support the practice of cytoreduction surgery

(maximal antigen ablation) and suggest tumour vaccines could be contraindicated in the

early post-operative phase. The importance of CD8+ and Treg was also highlighted,

indicating that therapies which target Treg and/or boost CTLs may be beneficial after

surgery.

Despite these insights, the role of sentinel lymph nodes (tumour draining nodes) in the

sinecomitant immune response remained unclear. Certainly the tumour draining nodes

were the local reservoir of antigen presentation after tumour resection, and it seemed

that antigen presentation was inversely correlated with sinecomitant immunity. It was

therefore hypothesised that sentinel node biopsy could accelerate the decline in tumour

antigen presentation after surgery, and enhance tumour resistance in the early post-

operative phase. The next chapter tests this hypothesis, investigating the effects of

sentinel node removal on antigen presentation kinetics and sinecomitant immunity.

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Chapter 5

- 135 -

5. Tumour immunity & sentinel nodes


Sentinel lymph nodes are defined as the first lymph nodes situated along the line of

afferent lymphatics that drain a particular body region.129

The concept of sentinel node

biopsy has become popular because the tumour status of sentinel nodes predicts the

status of the remaining nodes in the regional basin. This obviates the need for extensive

lymphadenectomy for prognostication. 129

The use of sentinel node biopsy has become increasingly frequent in the surgical

management of melanoma,135

squamous cell carcinoma,132,136

and breast cancer.134

The

sentinel node biopsy concept is theoretically translatable to a large number of solid

malignancies,132

and may be increasingly utilised into the future. Despite the increasing

prevalence of sentinel node biopsy, very little is known about how the procedure

impacts on anti-tumour immunity in vivo.

In Chapter 3, the tumour draining lymph nodes (axillary and inguinal nodes) of the

BALB/c flank subcutis were the solitary sites of cross presentation. It had been

postulated that lymph node dissection may dilute antigen delivery from tumours to a

level that falls below immunogenic thresholds (through passage into the vasculature),151

completely ablate antigen presentation,152

or force antigen presentation to secondary

lymph nodes.153

However, these hypotheses had never been confirmed or refuted.

It had also been suggested that numerous factors could affect the contribution of

sentinel nodes to tumour immunity. Several groups have reported that tumour proximity

reduced node function, lowered CD8+ mitogenicity,

171 reduced CD4

+:CD8

+ ratios,

172,173

increased Treg accumulation,174

and reduced tumour reactivity.175

It had additionally

been postulated that tumour invasion of the regional nodes could impact on their

capacity to participate in the immune response against tumour. Previous research had

indicated that nodal metastasis was associated with impaired tumour immune function,

including reduced cytokine production and T lymphocyte anergy.137,145,390

However, the

- 136 -

effects of tumour proximity and nodal invasion on cross presentation and in vivo CTL

function had not previously been assessed.

Also, in Chapter 3, it was identified that surgery improved systemic CTL function, but

that change took time to mature (Chapter 3). This was hypothesised to reflect a systemic

egress of effectors from the draining nodes after surgery, although this was not

demonstrated (Chapter 4). Given the central role of tumour draining lymph nodes in

presenting tumour antigens, and the critical changes occurring within the lymph node

after surgery (e.g. the decline in cross presentation), sentinel node removal seemed

unlikely to be a null event. The effects of sentinel node biopsy on post-operative tumour

immunity (sinecomitant immunity) were therefore investigated.

As the model used (AB1HA) is non-metastatic to the lymph nodes, and given time

constraints, analysis was restricted to the usual scenario – the effect of removing a

tumour negative (healthy) sentinel node on sinecomitant immunity. In future studies

using the RencaHAM model, the impact of removing a tumour-invaded sentinel node

on sinecomitant immunity will be investigated (see also, 7.3).

5 .2 . Resul ts

5 . 2 . 1 . Ident i f icat ion of sent inel nodes

Methylene blue was formulated at a concentration of 10mg/mL and 50 was injected

into a series of BALB/c. Injections were sited in the caudal flank region and serial

photography of the lymph nodes was undertaken at five timepoints after injection (range

5 minutes to 24 hours).

At 5 minutes and 15 minutes after inoculation of methylene blue, afferent lymphatics

were visible, Figure 5.1. Those lymphatics ran parallel to the lateral thoracic and

inferior epigastric veins. In each instance, they led to the axillary and inguinal nodes

respectively. Those nodes invariably appeared blue, with the axillary node tending to

darken more noticeably than the inguinal node (Figure 5.1). The axillary and inguinal

- 137 -

nodes were therefore identified as sentinel nodes for the caudal BALB/c flank subcutis.

Similar results were obtained when a tumour was in situ (data not shown).

At later dissections (1 hour and beyond) the afferent lymphatics were no longer visible,

and the nodes faded. In its place, the kidneys were darkened in appearance (30 minutes

to one hour) and the urine was blue. At 24 hours after inoculation, no dye was visible in

the animals at any location.

Figure 5.1. Transit of methylene blue dye into the sentinel nodes.

50L of 10mg/mL methylene blue was administered into the caudal flank subcutis of 10 BALB/c mice.

Animals were systemically dissected and photographed at 5 minutes, 15 minutes, 30 minutes, 1 hour and

24 hours after inoculation (n = 2 for each timepoint). Afferent lymphatics were visible within 15 minutes

after injection (above left) and the inguinal and axillary nodes took up blue dye (above right).

5 . 2 . 2 . Dendri t ic t racking and the sent inel nodes

Methylene blue showed the transit of soluble factors into sentinel nodes (5.2.1).

However, HA is cross presented by DCs, which requires cellular traffic. To determine

whether the traffic of DC was similar to that of soluble factors, DCs were tracked from

the BALB/c flank using GM-CSF, CFSE, and DC markers (CD11c, CD11b, DEC-205).

- 138 -

The di-acetate form of carboxyfluoroscein succinimidyl ester (CFDASE) is a non-toxic,

non-fluorescent molecule that diffuses passively into cells.322

Once inside the plasma

membrane of live cells, cellular esterases cleave the acetyl groups of CFDASE to form

the active fluorophore carboxyfluoroscein succinimidyl ester (CFSE). CFSE forms dye-

protein adducts that are retained within or at the cell surface. It emits and absorbs light

at wavelengths characteristic of its fluoroscein moiety. Since CFSE is only formed

intracellularly, CFSE fluorescence on flow cytometry can only be present from cells or

debris of cells that encountered CFSE. As such, CFSE can only track cells or cellular

debris from the site where it was inoculated. Thus CFSE is a useful fluorochrome to

mark cellular traffic.

GM-CSF was used in combination with CFSE to amplify signal. GM-CSF is a growth

factor for Langerhans DCs, the major cutaneous antigen presenting cell type.391

Moreover, GM-CSF conditioning of a skin site can increase the trafficking of antigen

presenting cells from the skin site to the regional lymphatics.391

Protocol details were provided in Chapter 2, and outlined in Figure 5.2. By this

approach, CFSE signal was only identified in combination with the DC marker

(CD11c). Langerhans-like DCs, bearing CFSE from the flank, were identified in the

inguinal and axillary nodes (Figure 5.2). No CFSE signal was found in any other node

groups (e.g. popliteal, brachial, cervical, para-aortic, iliac) nor the spleen. Moreover, the

number of CFSE+CD11c

+CD11b

+DEC-205

+ cells, as a proportion of CD11c

+ cells, was

equivalent between the axillary (2.49%) and inguinal (2.25%) nodes.

5 . 2 . 3 . Tumour proximi ty and node funct ion

A transient increase in CTL function and cross presentation was apparent on the day of

surgery (Chapter 3). This suggested a “rebound” phenomenon, whereby nodes were

impaired in cross presentation and CTL function with a tumour in situ, and then

“released” when the tumour was excised. To delineate the “rebound” of cross

presentation and in vivo CTL more precisely, inguinal and axillary nodes were

processed separately in pre-operative and post-operative mice. In each instance, the

AB1HA tumour was located superficial to the inguinal node, and distant to the axillary

node.

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Figure 5.2. Traffic of DC to the sentinel nodes

DC were tracked from the BALB/c caudal flank subcutis. Tracking sites were pre-conditioned with 10g

rGM-CSF (ProSpec-Tany Technogene Ltd), and then inoculated with 100L of 10m CFSE (Molecular

Probes). 24 hours after inoculation, cells from the nearby nodes (cervical, brachial, axillary, inguinal,

popliteal, etc.) were stained with CD45RA-FITC (BD PharMingen), 1:50 CD11c-PE (eBioscience),

CD11b-APC (BioLegend), and CD8-PECy5 (BioLegend). Samples were then examined by flow

cytometry. The experiment was performed once, with three animals studied. Results were similar in each

animal. Representative flow cytometry shown for the axillary node. A. Selection of cells from the

“lymphocyte shoulder” on forward scatter/side scatter. B. Identification of CFSE positive DC. C.

Expression of Langerhans DC markers by the CFSE positive and CFSE negative DC of the axillary node.

- 140 -

In terms of cross presentation, the inguinal node displayed poor CL4 proliferation

(17.90 11.16%) relative to the axillary node (66.16 2.48%) before surgery (P =

0.003). The inguinal node presented HA robustly (39.44 6.24%) after surgery,

significantly enhanced relative to its pre-operative value (P < 0.001). However, cross

presentation was unchanged within the distant (axillary) node (P = 0.283). A similar

pattern was present for in vivo CTL function. With the tumour in situ, inguinal node in

vivo CTL was poor in the inguinal node (6.30 0.54%), although comparable to the

axillary node P = 0.160. After surgery, in vivo CTL improved in the inguinal node

(24.46 4.03%) P = 0.007. By comparison, in vivo CTL was robust in the more distant

sentinel node (axillary node) on the day of surgery (20.28% 8.34%), and remained

similarly robust after surgery (P = 0.381).

Since the inguinal node (close to the tumour) rebounded strongly after surgery but the

axillary (distant) node changed little, a proximity-related suppression phenomenon was

suggested. To test whether tumour proximity affected in vivo CTL and antigen

presentation, these parameters were compared between BALB/c with caudal flank or

rostral flank tumours (Figure 5.5A). The two groups were matched for tumour size,

since this factor affects antigen presentation and in vivo CTL (see 3.2.9) and would

otherwise confound the results.

AB1HA grew faster in the upper flank compared to the lower flank, so for tumour size

equivalence, in vivo CTL and antigen presentation was tested on day 13 for upper flank

tumours and day 16 for lower flank tumours. When tumours were present in the lower

flank, both antigen presentation and in-vivo CTL were strong within the axillary node.

When equivalently sized AB1HA tumours were sited in the rostral flank position

instead (close to the axillary node), axillary nodal antigen presentation (Figure 5.4B)

and in vivo CTL function (Figure 5.4C) were reduced. In vivo CTL and antigen

presentation in the inguinal nodes were close to background levels once tumours were

sited in the rostral flank. This is because the inguinal node did not drain the upper flank,

but instead the brachial node exhibited in vivo CTL killing and HA cross presentation

(data not shown). These differences in antigen presentation and in vivo CTL function

(between inguinal and axillary nodes) were not explained by artefacts in cell viability

(Figure 5.5A) nor inequalities in penetrance of the transferred assay cells (Figure 5.5B).

- 141 -

5 . 2 . 4 . Tumour invasion and nodal funct ion

Tumour proximity was a factor that affected cross presentation and in vivo CTL in

sentinel nodes (5.2.3). These parameters may also be affected by tumour invasion.

Antigen presentation and in vivo CTL function were therefore assayed in BALB/c with

intra-nodal tumours, and compared to mice with extra-nodal tumours of a similar size

and location. Tumours were grown directly within the axillary node (see Chapter 2)

because it was larger than the inguinal node, and therefore easier to inject intra-nodally.

In vivo CTL and antigen presentation for those mice were compared to BALB/c with

similar size tumours, abutting the axillary fossa. When tumours were sited near the

axillary node, but not involving the axillary node, low amounts of in vivo CTL were

seen (4.59 2.09%). In vivo CTL was comparable and poor when the tumour invaded

the axillary node (6.37 2.39%), P = 0.31, Figure 5.6B. For cross presentation, when

tumours were located near the axillary node, proliferation rates of 34.42 9.11% were

seen. As with in vivo CTL, antigen presentation was equivalent when the tumour had

invaded the node itself (25.46 7.71%, P = 0.13) - Figure 5.6C.

5 . 2 . 5 . Surgical d issect ion of the sent inel nodes

The anatomical locations of the two sentinel nodes necessitated distinct surgical

approaches. Steps of the operation were provided in Chapter 2 (see also, Figure 5.7).

Various combinations of sentinel node surgery were explored. Bleeding from the

axillary vein occurred in 2 out of 150 animals and those animals were euthanased.

Another animal developed ischaemia of the forefoot at 24 hours post-operatively, and

was euthanased. No other complications (reduced mobility/contractures, lymphoedema,

infection, etc) occurred.

5 . 2 . 6 . Antigen ablat ion and sent inel node excis ion

Cross presentation from subcutaneous BALB/c flank tumours was confined to the

axillary and inguinal nodes (Sections 3.2.2, 3.2.4). By inference, if the tumour was

removed along with the sentinel nodes, it was hypothesised that cross presentation

would be ablated, i.e. it would not immediately appear in other non-resected nodes or

spleen. To test this, animals underwent primary resection with sentinel node excision,

on day 16 after inoculation. Antigen presentation was assessed on the day of surgery,

and day 5 after sentinel node resection. As before, antigen presentation was quantified

in the spleen and the following nodes: popliteal, brachial, contralateral axillary,

contralateral inguinal, iliac, para-aortic, celiac, mesenteric, renal, mediastinal,

- 142 -

cervical/jugular, and facial. Cross presentation was not detectable in the spleen, or in

any node group after tumour resection and sentinel node biopsy. Thus cross presentation

was immediately and completely ablated by primary resection and sentinel node biopsy

(Figure 5.8).

Figure 5.3. Cross presentation and in vivo CTL function after surgery.

BALB/c mice underwent assays for cross presentation and in vivo CTL function on day 16 of AB1HA

tumour growth, or 24 hours after resection. Inguinal and axillary nodes were processed separately.

Individual mice were depicted by points on the graphs, mean values were shown by bars. P values were

derived from the student‟s t test. A. Antigen presentation before and after surgery, for inguinal and

axillary nodes. B. In vivo CTL function pre- and post-operatively, for inguinal and axillary nodes.

- 143 -

Figure 5.4. Tumour proximity and nodal function.

Antigen presentation and in vivo CTL were quantified in the axillary and inguinal nodes for BALB/c mice

with caudal flank (A, Left) or rostral flank (A, Right) tumours of equivalent size. Individual mice were

shown by points on the graphs, mean values were denoted by bars. B. Antigen presentation in the axillary

and inguinal nodes. C. In vivo CTL for axillary and inguinal nodes. *P = 0.019, **P = 0.013 (student‟s t

test).

- 144 -

Figure 5.5. Viability and assay cell penetrance in the axillary and inguinal nodes.

Lymphocyte viability (% trypan blue exclusion) and CL4 penetrance (% lymphocytes with CFSE signal

at Lyons Parish analysis) were assessed in BALB/c inguinal and axillary nodes. Both experiments were

performed once, although measurements were taken in triplicate. Points on graph were from individual

mice, and a minimum of 5 animals was in each cohort. Mean values were shown by the bars and

compared by student‟s t test. P values were two tailed at the 5% significance level. A. Viability of

lymphocytes from inguinal and axillary node preparations. B. CL4 penetrance into axillary and inguinal

nodes.

- 145 -

Figure 5.6. Nodal invasion: effects on antigen presentation and in vivo CTL.

In vivo CTL and antigen presentation were quantified in the axillary node. Tumours were involving the

node itself (intra-nodal), or located nearby (extra-nodal). All tumours were of similar size. Data was

pooled from two separate experiments with a minimum of four animals per group. Individual mice were

shown as points, mean values were depicted by bars. P values were derived from student‟s t test. A.

Typical appearance of intra-nodal tumour (left). Comparison between nodally tumour-invaded and a

normal axillary node (right). B. In vivo CTL function for axillary nodes with nodal invasion (intra-nodal)

and those with tumour nearby (extra-nodal). C. Antigen presentation for axillary nodes with intra-nodal

tumour (intra-nodal) or tumours nearby the axillary node (extra-nodal).

- 146 -

Figure 5.7. Primary resection with sentinel node excision.

The axillary fossa was exposed via oblique incision (A), and the lower border of pectoralis major was

retracted superiorly (B). The axillary node was dissected clear of the axillary vein (C). Inguinal nodes

were excised en bloc with the tumour and vascular pedicle (D), or via a separate inguinal incision (not

shown). Wounds were closed with interrupted suture (E). Animals healed without complication (F)

5 . 2 . 7 . Tumour ant igen presentat ion af ter node removal

Complete primary resection plus sentinel node biopsy ablated cross presentation (5.2.6).

However, sentinel node biopsy is commonly undertaken for aggressive tumours (e.g.

advanced melanoma) that may not be completely resected. To determine the impact of

sentinel node biopsy on cross presentation from incompletely resected tumours, animals

had sentinel node biopsy in the setting of persistent tumour (i.e. without resection). CL4

were transferred on the day of surgery, or five days after surgery.

When CL4 were transferred on the day of sentinel biopsy, no cross presentation was

visible in any nodal location (Figure 5.9A). This was consistent in all animals. When

CL4 were transferred five days after sentinel biopsy, cross presentation was detected in

distant locations (Figure 5.9B). There was no clear pattern in cross presentation after

sentinel node biopsy, but the ipsilateral brachial node (6/8 animals) and the mediastinal

nodes displayed cross presentation most frequently (5/8 animals) - Figure 5.9C.

Importantly, this systemic cross presentation pattern did not reflect metastases at these

locations, as BALB/c that undergo primary resection remained tumour-free for at least

six months post-operatively.

- 147 -

Figure 5.8. Antigen presentation after resection and sentinel node biopsy.

Antigen presentation was quantified in BALB/c mice after primary resection plus sentinel node biopsy.

Lyons Parish assays were undertaken on the day of surgery and five days post-operatively. Individual

mice were shown as points on the graph, and mean proliferation was indicated by the bar.

5 . 2 . 8 . Sent inel node removal and re -chal lenge

To determine the effect of sentinel node removal on sinecomitant immunity, animals

underwent sentinel node removal and tumour excision, followed by re-challenge with

AB1HA. (Figure 5.10A). As before, when animals were re-challenged with AB1HA on

the day of surgery, there was a modest sinecomitant immune response (approximately

20% survival) seen in the surgical site and the opposite (healthy) flank. Sentinel node

biopsy did not alter this effect (Figure 5.10B,C).

If BALB/c were re-challenged at two weeks after sentinel node biopsy plus tumour

resection instead (Figure 5.10A) a significant decline in survival was seen for re-

challenges into the healthy flank (Figure 5.10E). However, sentinel node removal did

not impact on survival from re-challenge into the surgical site (Figure 5.10D).

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Figure 5.9. Cross presentation from local recurrence, after sentinel node removal

BALB/c mice with AB1HA tumours underwent sentinel lymphadenectomy. Antigen presentation was

assessed on the day of surgery (A) or 5 days post-operatively (B). Representative flow cytometry shown

from node specimens in each instance (clockwise from lower left): ipsilateral popliteal, abdominal

(mesenteric, para-aortic, iliac), ipsilateral brachial, cervical, mediastinal, contralateral axillary, spleen,

contralateral inguinal). C. Proportion of mouse lymph node specimens with >10% CL4 proliferation, five

days post sentinel node excision.

- 149 -

5 . 2 . 9 . Sent inel sampl ing and s taged lymphadenectomy

Two strategies were attempted to reduce the “harm” of sentinel node biopsy on post-

operative tumour resistance, for both the surgical site and the healthy flank. In the first

instance, the sentinel nodes were sampled (axillary or inguinal lymphadenectomy)

rather than both nodes being removed. In the second experiment, both sentinel nodes

were removed, but staged at two weeks after tumour resection. For both experiments,

the readout was survival from re-challenge into the surgical site or the opposite

(healthy) flank, at two weeks post resection.

As before, sentinel node biopsy was a null event for sinecomitant immunity into the

surgical site. Removing one or other of the sentinel nodes, or delaying sentinel node

removal was also a null event (data not shown). Importantly, survival from re-challenge

into the healthy flank was equivalent between animals that had sentinel node sampling,

and those with intact sentinel nodes (Figure 5.11B). Similarly, when sentinel node

removal was staged at two weeks after surgery, survival from re-challenge was similar

to animals that had no sentinel node procedure (Figure 5.11C).

5 .3 . Discussion

The role of lymph node surgery has evolved considerably over the years. In recent times

there has been a waning of radical lymph node dissection206

in favour of incomplete

dissections or targeted biopsy (sentinel node biopsy). Nevertheless, lymph nodes yield

useful prognostic information and are frequently invaded by tumour.21

Therefore

lymphadenectomy will probably remain a component of cancer surgery.

Identification of sentinel nodes for the BALB/c flank subcutis had previously been

reported using vital dyes (e.g. isosulfan blue392

or Evans blue393

), radio-isotope

imaging,394

and even micro-magnetic resonance lymphangiography.395

The first of the

techniques (vital dye) was chosen because it was inexpensive, presented few

occupational hazards, was easily formulated from standard ingredients, and could be

readily transported to sites of experimentation. Using the methylene blue dye, inguinal

and axillary nodes were identified as the sentinel nodes for the BALB/c flank subcutis.

Those nodes were not only the site of first transit for soluble factors, but also for DCs.

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Figure 5.10. Effect of sentinel node removal on survival from re-challenge

Data were pooled from two separate experiments, with a minimum of ten animals present in each group.

All animals were matched for pre-operative tumour size. Surgical impost was approximately equivalent

across cohorts (extra incision when axillary lymphadenectomy performed). Kaplan Meier survival was

shown for each cohort. P values were derived from the Log Ranks test. A. Timeline of tumour resection,

sentinel node removal, and re-challenges. B. Survival from re-challenge into the surgical site on the day

of surgery sentinel node removal. C. Survival from re-challenge into the healthy (opposite) flank on the

day of surgery sentinel node removal. D. Survival from re-challenge into the surgical site at two weeks

after tumour resection sentinel node removal. E. Survival from re-challenge into the healthy (opposite)

flank at two weeks after tumour resection sentinel node removal.

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Figure 5.11. Re-challenge after sentinel node sampling or delayed biopsy.

BALB/c underwent tumour resection on day 16 after AB1HA inoculation, and re-challenge into the

healthy flank at two weeks after tumour removal. Tumour sizes were matched across cohorts, and surgical

impost was similar. Data were pooled from two separate experiments. At least 10 animals were present in

each group. Kaplan Meier survival shown for each cohort. P values were derived from the Log Ranks

test. A. Timeline for sentinel node sampling and delayed sentinel node removal. B. Survival from re-

challenge into the healthy flank, after sentinel node sampling (axillary or inguinal lymphadenectomy) at

the time of tumour resection. C. Survival from re-challenge into the healthy flank, when sentinel nodes

(inguinal and axillary) were removed at two weeks post tumour resection.

- 152 -

In Chapter 1, the importance of the interaction between the APC, the CD8+ and the

CD4+ cells was identified. That interaction probably spans only a few cell diameters,

and to occur efficiently it must take place in an architecturally and functionally

optimised environment.366

That optimised environment is the lymph node.366

In Chapter

3 it was found that cross presentation was confined to the axillary and inguinal lymph

nodes: identified as sentinel nodes in this chapter.

Given the importance of the sentinel nodes to DC tracking and APC-T cell interaction,

lymphadenectomy could have a profound effect on tumour antigen priming. It had been

postulated previously that lymph node dissection may dilute antigen below

immunogenic thresholds (through passage into the vasculature),151

completely ablate

antigen presentation,152

or force antigen presentation to secondary lymph nodes.153

This

chapter indicated that the third hypothesis was correct. Cross presentation was absent

for three days after node removal, and then shifted by five days post lymph node

removal. The mechanism by which cross presentation shifted was unknown, but the

new sites of cross presentation were not from metastases (no tumour outgrowth

occurred in corresponding locations).

It was possible that new lymphatic channels were created to explain the shift in cross

presentation. Little is known about how rapidly lymphangiogenesis occurs, but

lymphatic endothelium migrates in culture at only a few micrometres per day. 396

Moreover, if lymphangiogenesis explained the new sites of cross presentation, it would

be likely that axillary and inguinal nodes would be replaced by the next most proximal

nodes i.e. “second-tier nodes” (brachial, popliteal, para-aortic). However, as cross

presentation was frequently found in distant sites (e.g. spleen and contralateral axillary

node), this suggested lymphangiogenesis was not the mechanism. It has also been

identified that lymphatico-lymphatic and lymphatico-venous connections exist. 123,397

As a corollary, when a main lymphatic vessel has been disrupted by surgery, antigen

bearing presenting cells could be shunted into adjacent lymph channels and away from

the dissected sites.

Thus when a lymph node is removed there may be open vessels left behind in the

resection bed,397

which may uptake the DC traffic and shunt it through to systemic sites.

Indeed, angiogenesis is promoted because of pro-angiogenic signals from the hypoxic

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wound environment.120,369,398

Those new vessels, tend to be highly permeable397

which

may further enhance the uptake and systemic intravascular trafficking of DCs.

As an alternative to shifting cross presentation to systemic sites, sentinel node biopsy

could ablate cross presentation when it was combined with complete primary resection.

This accorded with Chapter 3 and the findings of others,20,148,149

where cross

presentation has been confined to draining nodes. Also, since node dissection and

primary excision only left DC in transit to cross present (i.e. DC between the regional

node and the tumour site). This insinuated that DC had a lifecycle of less than five days

(the usual time taken for cross presentation to appear in new node), consistent with the

work of Kamath et al., who reported the DC life cycle to be between 1.5 and 3 days.360

Despite the effects of sentinel node excision on cross presentation, the overall impact of

lymphadenectomy on tumour immunity was ambiguous. A number of factors may

complicate the contribution of sentinel nodes to immunity, including whether those

nodes are invaded by tumour, and how close the tumour is to the nodes.

The data presented in this chapter, and the findings of numerous others,171,173,175,399,22

hint at a topography of lymph node function, based on tumour proximity. Specifically,

those nodes closest to a tumour may be exposed to the greatest tumour stimulation but

also the strongest tumour derived suppression. Those nodes further away will react

poorly also, because they are too far away to be engaged by the tumour. Thus it is the

intermediate zone nodes which function best – encountering antigen, but relatively

spared form tumour suppression.

In the flank tumour model described herein, the inguinal node was sited very close to

the tumour itself. However, the axillary node, which also drained the tumour, was more

distant. With an established tumour in situ, the inguinal node always performed poorly

in terms CL4 proliferation and in vivo CTL function. That difference in function was

not explained by a difference in viability of cells between the two nodes on assay, nor

was it attributable to preferential DC trafficking to the axillary node. When the tumour

was moved close to the axillary node, in vivo CTL and cross presentation declined. That

was despite an equivalent volume of tumour, similar volumes of CL4, and/or equivalent

proportions of tumour-specific targets.

- 154 -

A mechanism to account for the topography of node function was not identified.

However, the proximity effect indicated a soluble factor may have been involved.

Numerous soluble factors have been identified previously, including: VEGF,400

Il-10,401

and TGF402

Given the importance of Treg in this system (see depletion studies, Chapter

4), tumour chemokine CCL22 (which recruits and expands Treg cells) may be

significant.264

A second complicating factor was that nodes could be invaded by tumour. By replacing

the space normally occupied by APC and T cells, tumours may tip the balance away

from cross presentation and towards direct presentation. In addition, previous groups

have found invaded nodes to have higher “suppressor” T cell counts,174

anergic T

cells,177-179

and ablated concomitant immunity.180

In this chapter, robust cross

presentation and in vivo CTL function were seen in tumour-invaded axillary nodes. That

level of function was comparable to animals with equivalent size tumours, not involving

the node. Nevertheless, while tumour-invaded nodes may contribute to immunity, they

will likely succumb to increasing tumour growth390

and would be a source of

locoregional recurrence. Hence, it is not advocated that tumour invaded nodes should be

left in situ.

Despite the factors complicating the contribution of sentinel nodes to tumour immunity,

sentinel nodes predominantly suppress rather than activate anti-tumour immunity.

Indeed, the tumour draining nodes may be suppressed within 4 – 5 days by tumour

growth.258

Munn and Mellor suggest the tumour draining nodes are a powerful “factory”

for generating local and systemic tolerance to tumour antigens, however that was not a

major factor in our model.366

In Chapter 4, tumour antigen presentation frustrated post-

operative tumour resistance. By extension, removing sentinel lymph nodes might have

assisted immune function – removing the “factory” of suppression and re-setting the

immune response. Accordingly, in one approach for colorectal cancer, dubbed “immune

corrective surgery”, (ICSTM

, Biocrystal, Columbus Ohio USA), investigators have

attempted to resect all lymph nodes where tumour antigen is found.154

The authors

reported an improvement in survival with this technique in a phase 1 study,154

but a

larger study has not been published.

In this thesis, a somewhat different view is supported. While tumour proximity impaired

cross presentation and in vivo CTL function, that impairment could be alleviated by

- 155 -

surgery. Improved nodal function after surgery would correlate with the findings of Lee

and colleagues, who reported that sentinel lymph node IL-10 levels declined after

surgery140

- presumably enhancing T cell activation. In Chapter 4, the importance of a

decline in cross presentation after surgery was reported. That signal was presumably

integrated in the node. It was further seen that tumour-specific effectors and in vivo

CTL cluster in the draining node, correlating with the findings of others.155

Therefore, the combination of improved nodal function after surgery, the importance of

a decline in cross presentation in the draining nodes, and the localisation of effectors

and CTL to the tumour nodes led to a postulate that sentinel node biopsy would

adversely impact on tumour immunity. The impact of sentinel node biopsy was difficult

to assess in the surgical flank, where sinecomitant immunity was weak (see Chapter 4).

However, sentinel node biopsy had a clear impact on sinecomitant immunity in the

healthy flank. Specifically, only 10% of animals survived re-challenge after sentinel

node biopsy (compared to 50% survival with intact nodes).

While sentinel node surgery seemed to be detrimental to post-operative immunity,

sinecomitant immunity could be preserved by altering the extent and timing of sentinel

node excision. Specifically, leaving one or other of the sentinel nodes intact preserved

tumour resistance (Figure 5.11B) – suggesting a degree of redundancy in the system.

Additionally, it was postulated that surgery induces an effector egress of CD8+ from the

sentinel nodes in parallel with the decline in cross presentation (see pentamer staining of

Chapter 4, and primed in vivo CTL of Chapter 3). It was therefore hypothesised that

immediate sentinel lymph node excision could be detrimental (Figure 5.13) but delayed

sentinel node biopsy may leave sinecomitant immunity intact (Figure 5.14). Indeed,

delaying sentinel excision did improve survival relative to sentinel lymphadenectomy

concurrent with surgery, and approached similar levels to BALB/c with intact sentinel

nodes (Figure 5.11C).

Incomplete sentinel node biopsy may not be practical, because sentinel nodes may need

to be removed en bloc as part of the local clearance procedure. Delayed sentinel node

biopsy may similarly be problematic, because this would require re-operation in close

proximity to the previous procedure – where tissue planes have been distorted.

Therefore, an alternate strategy to reduce the “immunological harm” of sentinel node

- 156 -

biopsy may be preferable. Activating anti-CD40 antibody may be one such strategy,

because FGK45 has been shown to “push” effectors out of the tumour draining nodes

and into systemic sites.155,156

In short, pre-operative activating anti-CD40 antibody

might allow the effector cells to refuge in systemic sites by the day of surgery, so that

sentinel node excision does not deplete the patient of effector cells156

(Figure 5.15).

Although there was insufficient time to test the pre-operative CD40 hypothesis during

this thesis, it would be an attractive hypothesis to test in future pre-clinical work.

Importantly, the effect of sentinel node biopsy on sinecomitant immunity was restricted

to the removal of healthy (non tumour-invaded) nodes. Although in this chapter (section

5.2.4) antigen presentation and CTL function were preserved in the tumour-invaded

nodes, numerous publications have reported that node function is impaired when they

are positive for tumour.137,145,390

It is therefore possible that removing tumour-invaded

sentinel nodes is beneficial to tumour immunity overall, rather than detrimental.

Moreover, lymph nodes that are invaded with tumour may produce local recurrence

and/or metastases,403-405

quite independent of their tumour immune effects. Such

metastases may produce considerable morbidity and mortality. Consequently, it is

difficult to argue for the preservation of sentinel nodes (or even regional nodes) that

bear tumour metastasis, and determining the effect of removing tumour-positive sentinel

nodes on sinecomitant immunity is a priority for future work (see 7.3). The RencaHAM

model (described in Chapter 6) may prove to be a good tool for investigating this area.

- 157 -

Figure 5.12. The post-operative CD8+ effector egress postulate.

In Chapter 3, cross presentation waned after surgery and was associated with enhanced, systemic in vivo

CTL function. In Chapter 4, pentamer staining was confined to the sentinel nodes before surgery and

declined after surgery. In this chapter, removing the sentinel nodes on the day of surgery ablated

sinecomitant immunity, but delaying node excision preserved it. Taken together, these data suggested an

egress of tumour effectors from the draining nodes, possibly relating to the loss of cross presentation

signal in those nodes. The following schema was proposed. Kinetics of cross presentation shown in the

top of the figure: increasing until surgery and then decreasing after surgery. The distribution of tumour-

specific effectors (from pentamer and CTL data) shown on the diagram also: initially confined to the

sentinel nodes (dark green) and becoming systemic (light green) as cross presentation wanes.

Sinecomitant immunity was successful in the majority of animals at two weeks after surgery (indicated by

the star).

- 158 -

Figure 5.13. Predicted implications of sentinel biopsy: scenario 1.

From the postulate indicated in Figure 5.12, if sentinel node biopsy was undertaken on the day of surgery,

then cross presentation would be ablated immediately and effectors would be removed. Thus sinecomitant

immunity would be poor at the two week challenge test-point. Indeed, sinecomitant immunity was more

than halved by sentinel node biopsy concurrent with surgery (Figure 5.10). Sinecomitant was not ablated

completely however, possibly because HA specific CD8+ were representative only of anti-tumour CD8

+

directed against cell-associated antigens. The kinetics of CD8+ directed against soluble tumour antigens

may be different – possibly they are located systemically.406

Secondly, there may be some (lower than

detection threshold) egress of tumour-specific CD8+ during tumour growth i.e. before the day of surgery.

- 159 -


Consistent with the postulate of Figure 5.12, if sentinel node biopsy were to be undertaken at two weeks

after surgery, it was predicted that sinecomitant immunity would be preserved. In this instance, effector

egress would be complete (dark green) and antigen presentation would have waned (dark blue).

Empirically, this was the case (Figure 5.11C and Figure 5.10).

- 160 -


As discussed in this section and as shown in Figure 5.14, sentinel node biopsy may be harmful to post-

operative tumour resistance. That harm could be alleviated by sentinel node sampling (Figure 5.11B) or

delaying sentinel node biopsy (Figure 5.11C). Another alternative would be to force tumour-specific

effectors out of the sentinel nodes, prior to surgery. In the diagram above, it is proposed that pre-operative

activating anti-C40 antibody induces systemic spread of effectors from the sentinel nodes, so that when

the sentinel nodes are removed, strong sinecomitant responses are still seen (dark green) with successful

tumour immunity (yellow). This will be investigated in future research.

- 161 -

5 .4 . Summary

Sentinel node biopsy is increasingly undertaken during cancer surgery, but the

immunological impact of this procedure is unknown. The inguinal and axillary nodes

were identified as sentinel nodes for the BALB/c flank subcutis using methylene blue.

A rapidly reproducible technique of sentinel node surgery was then provided. As

predicted from the cross presentation, in vivo CTL and pentamer data of Chapters 3 and

4, sentinel node removal had a profound effect on tumour antigen processing and

overall anti-tumour immunity. While sentinel node removal might reduce antigen load

and potentially boost tumour immunity on the one hand, it ablated the pool of tumour-

specific effectors on the other.

It may therefore be important to consider the impact of sentinel node biopsy when

planning combined immune therapy/surgery strategies. Where practical, consideration

might be given to sparing the sentinel nodes or staging sentinel node biopsy beyond the

early post-operative phase. Alternatively, pre-operative immune therapy may prove

valuable, as a means of “flushing” effector CTL from the sentinel nodes before surgery.

The final experiment of this thesis is now presented. In this experiment, the derivation

and properties of the RencaHAM model are described. It is hoped that this model will

provide a useful tool for studying the effects of surgery on tumour immunity, and for

the development of combined surgery/immune therapy strategies for renal cancer.

- 162 -

Chapter 6

- 163 -

6. RencaHA


As a Urological surgeon scientist, my initial interest was to develop more effective

immune therapy strategies for renal cancer. Renal cell carcinoma accounts for 3% of

cancers in adults and is the sixth most common cause of cancer death overall.7,407,408

Due to the widespread use of abdominal imaging,409

the incidence of renal cancer has

been increasing, due to increased detection. Overall, about 2000 new cases are

diagnosed each year in Australia and 1100 patients die annually of this disease.410

Renal cell carcinoma is probably the prototypical target for immune therapy because of

its proven sensitivity to biological treatments and its poor response to conventional

strategies (chemotherapy411

and radiotherapy7). Numerous renal cell carcinoma antigens

have been identified and substantial lymphocytic infiltrates have been observed in

deposits of kidney cancer.412,413

Strikingly, metastatic disease spontaneously regresses

in up to 1% of cases with primary resection alone.16,42

There is also some evidence that

more immunogenic renal cancers have better prognosis, and conversely that patients

with high B7-H1 expression fare poorer.414

The underlying objective of this thesis was to develop a rationale for combining surgery

and immune therapy more effectively. As such, renal cancer is the first malignancy for

which a benefit for combining surgery and immune therapy has been demonstrated in

randomised clinical trials. Specifically, primary resection plus IFN therapy is more

effective than IFN alone in the setting of advanced disease,14,16,415

and tumour

vaccines improve disease-free survival and overall survival for patients who undergo

clinically complete primary resection.12,13,237,416

To study combined surgery/immune therapy strategies and tumour-specific immune

responses in renal cancer pre-clinically, a murine renal cancer line with a trackable neo-

antigen was required. The RencaHA cell line seemed ideal, having been previously

developed by Dr Linda Sherman and colleagues.318

Accordingly, RencaHA was sought

and kindly donated by Drs Sotomayer and Cheng at the H Lee Moffitt Cancer Centre

(Tampa, Florida USA).

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A number of unforseen difficulties with the RencaHA line were encountered, and thus a

new sub-clone was derived (RencaHAM). While time did not allow extensive work

with the renal cancer model, different routes of administration for RencaHAM were

explored, including the biologically most valid method (intra-renal implantation).

Having described the RencaHAM subclone and its properties, hopefully this will be

useful to future empirical studies of combined surgery/immune therapy in renal cancer.

Already, a Master of Surgery student at The University of Western Australia has begun

a project based on RencaHAM, and several other researchers at the Tumour

Immunology Group (Sir Charles Gairdner Hospital) are using RencaHAM in their

work.

6 .2 . Resul ts

6 . 2 . 1 . Ini t ia l exper ience wi th RencaHA

After a number of failed attempts to obtain RencaHA by freight, the candidate visited

the H Lee Moffitt Cancer centre in person and was kindly provided with a fresh sample

of cells by Dr Cheng. After a transit time of approximately 24 hours, culture was

commenced in the Tumour Immunology Group laboratory. On arrival, the cell line was

lightly contaminated and mycoplasma positive. The culture was treated with

amphotericin, penicillin and gentamicin, followed by ciprofloxacin. After four or five

passages, the culture was viable and negative for mycoplasma on three consecutive PCR

examinations. While initial experiments have suggested that RencaHA is too

immunogenic to grow subcutaneously,318

subcutaneous transplantation was attempted in

20 mice with 1x105

RencaHA cells (n = 5), 1x106

cells of RencaHA (n = 10), and 2x106

RencaHA cells (n = 5). Subcutaneous tumours grew in 4 out of 5 mice with 1x105

RencaHA, 8 out of ten mice with 1x106 RencaHA, and 2 out of 5 mice with 2x10

6

RencaHA, (data not shown). The lowest inoculum concentration (1x105 cells of

RencaHA) grew significantly slower than the stronger inoculums, but there was no

difference in growth rate between injections of 1x106 and 2x10

6 cells of RencaHA.

Five animals with day 31 RencaHA (1x106 group) were euthanased and the tumours

were prepared into five sterile cultures. After two in vitro passages, those cell lines were

stained for HA expression using H18 antibody and examined by flow cytometry using

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FACScan (Becton Dickinson) and associated Cell Quest V3.1 (Becton Dickinson)

software. HA expression was absent from four of the five samples, and positive in the

remaining culture (Figure 6.1A). The broad HA expression of that culture represented

cells that retained RencaHA expression in vivo. This cell culture was passaged in vitro

and FACS sorted thrice for HA expression, using the FACS Vantage Cell Sorter and

with the assistance of Dr Kathy Heel (BIAF, University of Western Australia, Perth,

Western Australia). Three populations resulted: RencaHALOW

, RencaHAMEDIUM

, and

RencaHAHIGH

. These three subpopulations were quite different with respect to in vitro

growth characteristics: both RencaHALOW

and RencaHAHIGH

grew slowly and sparsely.

By comparison, RencaHAMEDIUM

formed confluent monolayers (Figure 6.1C) and grew

prolifically (tripling approximately every 24 hours). After successive sorting, the

RencaHAMEDIUM

sub-clone was denoted “RencaHAM” and selected for experimentation

because of its favourable HA expression profile and stable in vitro growth

characteristics. RencaHAM fluorescence was at least a log shift higher than background

staining (RencaWT) and unstained controls (Figure 6.1B).

6 . 2 . 2 . Subcutaneous RencaHAM

1x106 cells of RencaHAM line were inoculated subcutaneously into 20 BALB/c and

followed for growth kinetics. However, no tumours were present by day 27 after

inoculation. To determine whether RencaHAM failed to transplant because of

immunological rejection, 1x106 RencaHAM was inoculated into congenic BALB/c nu

-/-

(BALB/c background but athymic). Tumours were successfully transplanted in 100% of

nude BALB/c, with lesions reaching 9.58 0.40 mm by day 31 after inoculation (Figure

6.2). Growth of RencaHAM in nude BALB/c was significantly slower than for

RencaWT in wild type BALB/c. To determine whether HA antigen was presented

despite the absence of tumour in wild type mice, antigen presentation assays were

performed on days 4, 10, 16, 21, and 27 after tumour cell inoculation. As with

subcutaneous AB1HA, class I restricted HA presentation was robust and solely

confined to the sentinel lymph nodes (Figure 6.3A). Antigen presentation was strongly

visible at the day four time point, and declined gradually to background by day 27

(Figure 6.3B).

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Figure 6.1. Derivation of RencaHA sub-clone.

A. Subcutaneous RencaHA tumours were harvested and stained for HA expression using H18 mAb. A

single culture retained HA expression after subcutaneous passage, and was subsequently sorted by flow

cytometry as shown. B. Representative flow cytometry from HA staining of RencaHAMEDIUM

(RencaHAM) sub-clone relative to unstained and H18 treated RencaWT controls. C. Appearance of

RencaHAM in vitro.

- 167 -

Figure 6.2. RencaWT and RencaHAM in wild type and congenic BALB/c nu-/-

RencaHAM and RencaWT were inoculated into wild type and congenic BALB/c nu-/-

mice. Mean tumour

diameter SEM was shown for each cohort. Data was shown for a single experiment, with ten animals per

group. Points of statistically significant difference in mean tumour diameter shown by the asterisk (two

tailed P < 0.05 on student‟s t test).

6 . 2 . 3 . Intravenous RencaHAM

Since RencaHAM did not grow subcutaneously (6.2.2), other routes of transplantation

were investigated. Intravenous injection was studied first, as this route of administration

has previously been described.198,318

To verify tumour kinetics of RencaHAM, 1x106

cells of RencaHAM were injected intravenously into 32 mice. The lateral tail vein was

used to inject most of the mice, but due to technical difficulties, eight mice received

tumour cells by direct intra-cardiac injection. While the results of intra-cardiac injection

were reported, the procedure itself was performed as a “last resort” and did not

constitute a formal component of the investigation. Mice were culled serially for

autopsy and Lyons Parish analysis. In the tail vein group, all animals appeared healthy

at day 21, but two culls were necessitated between day 21 and day 34. By day 34,

remaining animals were cachectic and lethargic. At autopsy of mice with day 21

tumour, the thorax was grossly normal - with the exception of enlarged mediastinal

nodes. Macroscopically, the animals appeared free of tumour. At day 28, pleural

plaques appeared and small solid tumours were visible in the lung. By day 34,

pulmonary and pleural tumours were extensive, and occasionally extended onto the

pericardium (Figure 6.4A). On Lyons Parish analysis at day 21 after inoculation, HA

presentation was robust in the mediastinal nodes (approximately 80% proliferation) and

- 168 -

low levels of presentation were found in the spleen and the para-aortic nodes. No other

nodes (renal, cervical, axillary, inguinal, brachial, iliac, popliteal etc.) demonstrated HA

specific presentation (Figure 6.5).

Figure 6.3. Presentation of HA from subcutaneous RencaHAM.

Class I restricted presentation of HA was assayed in BALB/c after subcutaneous inoculation of

RencaHAM. Data was shown from a single experiment with a minimum of four animals per group. A.

representative flow cytometry from BALB/c at day four after inoculation of RencaHAM. B. Antigen

presentation in cohorts of BALB/c culled serially after inoculation of RencaHAM. Individual mice were

shown as points, mean proliferation depicted with bars. Statistically significant proliferation (relative to

background controls on student‟s t test, P < 0.05). were denoted by asterisks.

- 169 -

Disease progression was more rapid in the animals that underwent intra-cardiac

injection of RencaHAM. By day 14, one cull was required and half the mice looked

ruffled and sluggish (unwell). All were mildly cachectic. At day 21, one of four animals

autopsied had adrenal enlargement (probable metastases) and all had mild pleural and

pericardial infiltration. By day 28, all animals that received intra-cardiac RencaHAM

were euthanased. Injection site tumour was visible in each of the three mice with day 28

tumour, and extensive pulmonary and pleural nodules were present. The adrenals were

enlarged in all three, with two mice showing obvious renal lesions. Most lymph nodes

were grossly enlarged, with a chalky to haemorrhagic appearance, suggestive of tumour

invasion. On Lyons Parish analysis at day 17 after injection, cross presentation was

visible in all node groups (Figure 6.6).

At histology, both intra-cardiac and tail vein administered RencaHAM produced

subpleural tumour and intra-parenchymal deposits. The intra-parenchymal deposits

were more frequent with intra-cardiac RencaHAM (Figure 6.4E,F), but the subpleural

tumours seemed more prominent in tail vein injected RencaHAM (Figure 6.4C,D). In

short, whether RencaHAM was given by tail vein injection or intra-cardiac injection, the

main sequelae were subpleural and intraparenchymal tumour deposits. Animals given

intra-cardiac tumour tended to have accelerated disease progression, with euthanasia

required about one week earlier. Also, there was clinical evidence of lymph node

invasion in animals given intravenous RencaHAM via intra-cardiac injection. The issue

of lymph node invasion was formally investigated in 6.2.5.

6 . 2 . 4 . Orthotopic ( intra -renal) RencaHAM

To study post-operative tumour-specific immunity, a biologically valid and surgically

resectable form of RencaHAM transplantation was required. While RencaHAM can be

given by intravenous injection, those deposits are not surgically resectable. Moreover,

Renca is known to behave differently when transplanted orthotopically (i.e. into the

kidney) as opposed to ectopic growth (e.g. subcutaneous inoculation).317

For this reason,

the intra-renal injection of RencaHAM was studied, for which there was no previously

published data. To establish the tumour kinetics of intra-renal RencaHAM, 16 animals

were administered 1x106 RencaHAM cells into the right kidney (Figure 6.7A). Renal

RencaHAM grew in a subcapsular manner on histological section, and this was

irrespective of whether the cells were originally injected subcapsularly or

intraparenchymally (Figure 6.7C,D). By 21 days after surgery, right flank tumours were

- 170 -

palpable in all animals (Figure 6.7B). Euthanasia was obligatory in one in three animals

by this stage, due to cachexia and/or lethargic behaviour. By 28 days after injection, all

remaining animals were euthanased because of the onset of cachexia and indicators of

distress (particularly sluggish behaviour and ruffled fur). At necropsy, very large renal

tumours were present with extensive peritoneal deposits, and significant metastatic

disease (Figure 6.7E,F). Metastatic sites included spleen, liver, contralateral kidney and

the lung. When BALB/c were assayed for class I – restricted HA presentation at 21 days

after intra-renal injection of RencaHAM, multiple sites of HA-specific CD8+

proliferation were seen. In all five animals, HA presentation was seen in the spleen,

renal, para-aortic and mediastinal nodes. Importantly, HA presentation within the

mediastinal nodes correlated with the presence of “micrometastatic disease” in the lung

(Figure 6.9A,B) and the absence of mediastinal node invasion on histology (6.2.5). This

suggested the immune system was not ignorant of orthotopic, micrometastatic disease

and that cross presentation was important to the immune recognition of

micrometastasis.

6 . 2 . 5 . Lymph node m etas tases from RencaHAM

HA was presented robustly after intra-cardiac, intra-renal, and tail vein injection of

RencaHAM. However, it remained unclear whether direct or cross presentation was

important in this system. To determine whether HA presentation could occur

independently of lymph node tumour, the extent of lymph node invasion in the

RencaHAM system was determined. Pairs of mice at day 21 after intravenous or intra-

renal RencaHAM were selected and examined for nodal invasion. Specifically, renal,

para-aortic and mediastinal nodes were sampled separately for histology. Additionally,

lymph nodes from the individual mice were assayed en masse (i.e. total lymph node

dissection) for PCR and culture (Figure 6.10E) Intravenous RencaHAM was found to

metastasise to the lymph nodes on histology, culture, and PCR (Figure 6.10A,B,D).

While tumour cells were only identified histologically in mediastinal nodes, diffuse

lymph node invasion was present clinically at later time points (e.g. day 28, 6.2.3); all

nodes were markedly enlarged, chalky, and haemorrhagic in appearance. Orthotopic

RencaHAM also metastasised to the lymph nodes. On histology, tumour was identified

in the renal (Figure 6.10C) and para-aortic nodes. However, RencaHAM tumour was

not seen in the mediastinal nodes of the orthotopic system. This suggested that

mediastinal node CL4 proliferation in animals with micrometastases (Figure 6.8) was

cross-presentation.

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Figure 6.4 Pulmonary morphology and histology post intravenous RencaHAM

RencaHAM was administered intravenously into BALB/c mice at a dosage of 1x106 cells per mouse.

Tumour was administered via tail vein or intra-cardiac injection. A. Thick subpleural tumour rind in

BALB/c at day 34 after intravenous injection of RencaHAM by tail vein. B. Multiple pleural nodules,

pericardial plaque (with healed right ventricular scar) of BALB/c at day 28 post intra-cardiac injection of

RencaHAM. C. Histological section (x20) of lung surface from BALB/c at day 34 after tail vein injection

of RencaHAM. D. Histological section (x20) of deep lung from BALB/c. day 34 after tail vein injection

of RencaHAM. E. Histological section (x20) of lung surface from BALB/c, day 28 after intra-cardiac

injection of RencaHAM. F. Histological section (x10) of deep lung from BALB/c at day 34 after intra-

cardiac injection of RencaHAM.

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Figure 6.5. Antigen presentation from i.v. RencaHAM.

BALB/c were assayed for class I restricted HA presentation at day 21 after tail vein injection of

RencaHAM. The histograms were representative of CD8+CFSE

high cell populations at flow cytometry.

Data were shown for a single animal, representative of eight BALB/c assayed with similar results. The

proportion of CFSEhigh

cells with lower fluorochrome intensity than parental was shown for each lymph

node group. Clockwise from left: axillary and inguinal nodes, cervical, mediastinal, spleen, renal, and

para-aortic nodes.

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Figure 6.6. Antigen presentation from intra-cardiac RencaHAM.

BALB/c were assayed for class I restricted HA presentation at day 17 after intra-cardiac injection of

RencaHAM. The histograms were representative of CD8+CFSE

high cell populations, at flow cytometry.

Data were shown for a single animal, representative of five animals assayed with similar results. The

proportion of CFSEhigh

cells with lower fluorochrome intensity than parental was shown for each lymph

node group. Clockwise from left: axillary and inguinal nodes, cervical, mediastinal, spleen, renal, and

para-aortic nodes.

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Figure 6.7. Gross morphology and histology of orthotopic RencaHAM.

A. Orthotopic transplantation of RencaHAM cells via operative exposure and subcapsular injection. B.

Flank tumour of typical BALB/c mouse, day 21 after intra-renal inoculation of RencaHAM. C. Kidney at

21 days after inoculation of RencaHAM (above) relative to normal kidney (below). D. Histological

section (H&E, x10) of renal capsule and kidney with RencaHAM at day 21 after injection. E. Kidney at

day 28 after intra-renal injection of RencaHAM. F. left sagittal view of BALB/c coelom, day 28 after

intra-renal injection of RencaHAM to the right kidney.

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Figure 6.8. HA presentation from orthotopic RencaHAM.

BALB/c were assayed for class I restricted HA presentation at day 17 after intra-renal injection of

RencaHAM. Representative histograms of CD8+CFSE

high cell populations at flow cytometry. Data were

shown for a single animal, representative of five animals assayed with similar results. Proportions of

CFSEhigh

cells with sub-parental fluorochrome intensity shown for each lymph node group. Clockwise

from left: axillary and inguinal nodes, cervical, mediastinal, spleen, renal, and para-aortic nodes.

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Figure 6.9. Pulmonary micrometastases from orthotopic RencaHAM.

A. Macroscopic appearance of BALB/c lungs at 21 days after intra-renal injection of RencaHAM.

Puncture site from adoptive transfer seen at cardiac apex. B. Histological section of lung parenchyma

(H&E, x20) from the same mouse. Experiment was repeated on two additional mice, with similar results.

6 .3 . Discussion

In this chapter, the derivation of a new RencaHA clone (RencaHAM) was explained,

together with the properties of that clone. The characteristics of Renca, RencaHA, and

RencaHAM afforded numerous insights into the workings of the immune response

against solid tumour. When RencaHA was grown subcutaneously, 80% of mice lost HA

expression. That finding was consistent with immune escape,245

where CD8+ T cells

effectively target antigenic clones and negatively select against those clones, producing

antigen-negative tumour outgrowth. Moreover, when immune escape could not occur

(i.e. when a stable HA expressing RencaHAM cell line was inoculated), tumours could

not be transplanted. That failure to transplant was not explained by physical (e.g. poor

viability) or nutritional (e.g. poor blood supply) factors because transplantation was

successful in athymic BALB/c mice.

While RencaHAM did not grow subcutaneously, it did cross present HA for somewhere

between 3 and 4 weeks. As such, subcutaneous RencaHAM provided a means of

administering HA antigen without inducing a tumour or requiring the use of virus. In

this regard, in Chapter 4 of this thesis, subcutaneous RencaHAM was used to provide

HA antigen persistence without tumour persistence in studies of sinecomitant immunity.

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Figure 6.10. Evidence of nodal invasion from RencaHAM.

A. mediastinal lymph node from a mouse 21 days after tail vein injection of RencaHAM (H&E section,

x10). B. Culture of total node pool of BALB/c mice, 21 days after tail vein injection of RencaHAM

(phase microscopy, x10). C. Renal lymph node of BALB/c mouse, 21 days after intra-renal injection of

RencaHAM (H&E section, x10). D. RT-PCR for HA transgene in total node pools of BALB/c mice at 21

days after transplantation of RencaHAM via intra-cardiac, intra-renal, or tail vein injection. Fluorescence

shown with reference to positive (RencaHAM and AB1HA culture) and negative (RencaWT, AB1, and

RNase free H2O) controls. Results were similar in both mice processed for each group (intra-cardiac, tail

vein, and intra-renal). E. Summary of PCR, histology and culture evidence of lymph node metastases with

RencaHAM. Culture and PCR was performed on total pooled nodes of each assayed animal (pools

included: mediastinal, renal, para-aortic, inguinal, axillary, cervical), whereas histology was performed on

nodes separately (separate nodes assayed: mediastinal, renal, para-aortic).

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RencaHAM could also be used to model disseminated renal cancer. When RencaHAM

was administered by intravenous injection (tail vein or intra-cardiac), rapidly

progressive, diffusely metastatic disease was apparent. Lymph node invasion was

included in the metastatic profile of intravenous RencaHAM, providing the opportunity

to examine the immune function of lymph nodes when invaded by tumour. However,

intravenous RencaHAM disease was not amenable to surgery – multiple pulmonary

deposits and extensive seeding are not resectable.

For this reason, orthotopic (intra-renal) RencaHAM was probably the real niche of

RencaHAM. In this chapter, RencaHAM could be reliably transplanted intrarenally,

producing contralateral renal, splenic, hepatic and pulmonary metastasis. In future

studies, the tumour-specific response to RencaHAM could be tracked in vivo and

disease burden could be quantified by PCR for HA transgene.

There were differences in RencaHAM growth patterns across the various methods of

transplantation. This different behaviour may have reflected not only the difference in

blood and nutrient supply of each implantation site, but also that the tumour may have

expressed different phenotypes according to its location (e.g. type IV collagenase and

EGFR expression).317

The central question of whether small metastases engage the immune system was also

be addressed in the orthotopic RencaHAM system. As discussed in Chapter 3, tumour

antigen presentation is the obligatory priming signal to the anti-tumour immune

response. Without sufficient antigen priming, there can be no effector differentiation,

CD8+ proliferation, nor effective memory development.

347,351 As a corollary, active

immune therapies, which require an endogenous immune response against the tumour,

will be unsuccessful if priming is inadequate. In renal cancer, one third of patients who

undergo „curative‟ surgery still ultimately progress.12

This suggests a proportion of

patients have microscopic residual tumour after clinically complete resection in renal

cancer. To date, it remained unclear whether those patients had sufficient antigen, or

whether antigen priming was a limiting factor in micrometastatic disease.

In the past, others have proposed that the immune system is not ignorant of small

metastases.198

Indeed, in Chapter 3 of this thesis, cross presentation was identified at

four days after subcutaneous inoculation, where tumours were frequently too small to be

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seen or palpated. However, to determine whether micrometastases cross present antigen

definitively, true micrometastases must be studied. Tumours that metastasise normally

go through a cascade of phenotypic changes that enable them to detach from one

another,417,418

invade the extracellular matrix,419

access blood vessels,419

attach to

vessels at distant sites, extravasate, and acquire necessary properties to establish a new

colony.420

In parallel with those phenotypic changes may be the downregulation of antigen

presentation and other immunological changes. Tumour injections into the skin or

tumours that embolise from intravenous injection do not transit through many of these

steps. Therefore, the immunological characteristics of those tumour models may not

relate to the properties of a true metastasis.

In this chapter, RencaHAM was injected into the kidney, which was the orthotopic

location for this tumour. Left to grow in its natural environment, RencaHAM

metastasised to the renal and para-aortic nodes, the spleen, contralateral kidney and

lungs. Those tumours represented true secondaries, whose biology should parallel

metastases from primary renal cancer in humans. As such, at 21 days after intra-renal

inoculation, micrometastases were present in the lung (proven on histology). In that

setting, micrometastases were associated with robust antigen presentation in the

mediastinal lymph nodes. Whether that antigen was cross presented or directly

presented remained uncertain, although the apparent absence of mediastinal node

invasion suggested the former. To prove cross presentation was the mechanism, it

would be helpful to perform PCR surveys of the mediastinal nodes to exclude

metastases to those sites. Similarly, a bone marrow chimera study would be useful.

There was insufficient time for the candidate to undertake such experiments.

Furthermore, while the data of this chapter suggested antigen presentation was highly

efficient from micrometastatic disease, work was limited to the study of a proliferative

response in tumour-specific CD8+ to cross presentation. This is only one component of

priming. It is still possible those CD8+ cells, while proliferating, were not getting the

necessary second signals and the cytokine milieu to enable them to form capable

effectors. Thus while micrometastases do present antigen adequately, the quality of

priming might be limiting. By extension, while patients may not require additional

quantities of antigen after surgery, they may benefit from strategies that augment the

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quality of that antigen priming (e.g. tumour cell-DC hybridomas,421

gene-modified

tumour vaccines,422

and HSP-antigen complexes etc.).423,424

While micrometastastic disease may insufficiently prime the immune response, efficient

antigen presentation from micrometastases might impair sinecomitant immunity. As

seen in Section 4.2.9, enhanced post-operative tumour resistance requires a fall in

antigen presentation, or “antigen holiday”. If micrometastases present antigen

efficiently, this may lessen the immune benefit of cancer resection. It is still possible

macroscopically complete resection may provide an adequate decline in antigen

presentation, even in the presence of tumour antigen presentation from occult

metastases. To answer this question, the RencaHAM model may prove useful (see also,

7.3).

In future studies, nephrectomies of orthotopic RencaHAM could be undertaken to

render animals with “minimal residual disease” renal cancer. In that setting, the question

as to whether tumour immunity improves despite microscopic disease could be tested.

IYSTVASSL-MHC pentameric staining (Pro5®, ProImmune), Lyons Parish analysis321

and in vivo CTL assays323

could be used to assess tumour-specific immunity in vivo and

metastatic burden could be objectively quantified using RT-PCR for the HA transgene.

6 .4 . Summary

In this chapter, the properties of a stable HA-expressing RencaHAM clone were

expounded. This model may prove useful in future studies of combined surgery/immune

therapy strategies for renal cancer. Using RencaHAM, objective tracking of the tumour-

specific CD8+ response, cross presentation, and metastatic burden would be possible. In

addition, this model has provided three key insights into the workings of the anti-

tumour immune response. Firstly, loss of HA expression with subcutaneous growth of

RencaHAM suggested the immune system can “edit” tumours to select antigenically

negative variants. This issue adds weight to the importance of using whole cell vaccines

rather than single peptide preparations (Chapters 1 and 7), because boosting the

response to one antigen may be associated with loss of that antigen and resistance to

therapy.

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Secondly, antigen presentation was preserved, even when nodes were invaded with

tumour. This was consistent with Chapter 5, where invaded lymph nodes had

comparable antigen presentation and in vivo CTL function to intact nodes. Finally, it

was found that antigen presentation was robust, even with minimal residual

“micrometastatic” disease. Thus patients with micrometastases may not benefit from

additional antigen per se. Rather strategies to improve the quality of priming (e.g. DC-

tumour cell hybridomas421

) or boosting the effector arm of the immune response (e.g.

cytokine therapy) may be more appropriate.

The key findings of this thesis are now summarised. Based on these findings, an

empirical framework for the combination of surgery with immune therapy is

synthesised.

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Chapter 7

- 183 -

7. Thesis Summary

Patients with locally aggressive and metastatic tumours continue to fare poorly,369

and

cancer remains the second most common cause of death in western nations.425

This

thesis advocates and facilitates a new treatment paradigm for malignancy: combined

surgery/immune therapy.3

The concept of combining surgery with immune therapy is not without danger, because

surgery can expose the patient to morbidity and mortality in itself. Indeed, operative

morbidity may preclude that patient from the conventional treatments or immune

therapy they would otherwise receive.112,113,415

A second difficulty with combining

surgery and immune therapy is the prevailing dogma that surgery is immune

suppressive.29,30,73

As such, surgery could impair the response to immune therapy rather

than improve it.

While it is acknowledged that surgery may be generally immune suppressive, in this

thesis it has been argued that surgery can boost the tumour-specific component of

immunity. The concept of sinecomitant immunity was highlighted, a phenomenon that

has been absent from the literature for some thirty years. As such, the benefit of tumour

resection was identified as the excess of sinecomitant immunity (post-operative tumour

resistance)25,106,107

relative to concomitant immunity (tumour resistance without primary

resection).24,25,105

Difficulties aside, a central factor that limits the uptake of combined surgery/immune

therapy strategies is the lack of understanding about how surgery impacts on tumour-

specific immunity. Until these effects can be elucidated, the logic for planning

combined surgery/immune therapy strategies will remain unclear. This thesis has

rectified some of the defects in existing knowledge about surgery and tumour immunity,

by providing insights into the effects of surgery on antigen processing, CTLs, and

overall tumour resistance. It is hoped those insights will aid in the planning of combined

surgery/immune therapy strategies in the future, ultimately improving results for

patients.

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7 .1 . Principal Findings

7 . 1 . 1 . Effects of surgery on ant igen presentat ion

The pre-operative and post-operative kinetics of tumour antigen presentation were

examined in the RencaHAM and/or AB1HA tumour models. It was demonstrated that

HA antigen is presented to CTLs in a highly efficient manner. In particular, HA

presentation was detectable in association with tiny primary lesions and

micrometastases. This implied that the immune system was not ignorant of small

primary tumours and micrometastases.

HA presentation was also confined to the sentinel lymph nodes for all stages of tumour

growth, and increased in parallel with tumour burden. Conversely after surgery, there

was a decline in HA presentation, until it was no longer detectable at two weeks post-

operatively. As was the case pre-operatively, HA presentation was confined to the

tumour draining nodes for all time points after surgery. The localisation of tumour

antigen presentation suggested a role for locally delivered immune strategies, exploiting

the sentinel node reservoir of antigen in the early post-operative phase.

7 . 1 . 2 . Surgery & tumour -speci f ic CTLs

Given the dependence of CTLs on tumour antigen processing, it was unsurprising that

HA specific CTL would co-localise with antigen presentation. HA-specific CTL and

HA-specific in vivo CTL activity were disproportionately represented in the sentinel

lymph glands of tumour bearing hosts. After surgery, there was a decline in antigen

presentation in the tumour draining lymph nodes, and this was associated with enhanced

HA-specific in vivo CTL and systemic egress of that CTL activity. Conceptually, it was

postulated that chemokine release and/or the continuous stream of antigen from the

tumour might “tether” tumour-specific CTL into the draining lymph gland. Surgery

could cut that “tether”, enabling improved and systemic tumour-specific CTL function.

Attempts were made to characterise the phenotype of HA specific CTL before and after

surgery. However, there were no obvious differences in memory characteristics of the

pre-operative and post-operative HA-specific CD8+ repertoire.

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7 . 1 . 3 . Sent inel lymph nodes & ant i - tumour immunity

The sentinel lymph nodes provided a pool of tumour-specific CD8+. They were also the

site where the decline in tumour antigen presentation was seen after surgery (7.1.1).

Accordingly, when the sentinel lymph glands were removed concurrent with the

tumour, tumour antigen presentation was completely and permanently ablated. Given

the localisation of tumour-specific effectors in the sentinel node, it was predicted that

sentinel node biopsy would be detrimental to tumour-specific immunity. Indeed,

removing the sentinel nodes on the day of surgery almost completely ablated the anti-

tumour immune benefit of primary resection. It was envisaged that sentinel node

sampling and/or delaying the sentinel node procedure might preserve the pool of

tumour-specific CTL, as these cells were thought to gradually egress from the sentinel

nodes after surgery. This was the case.

7 . 1 . 4 . Propert ies of s inecomitant immunity

Sinecomitant immunity seemed most successful when the tumour re-challenge was

temporally and spatially distant to the cancer surgery itself. Unfortunately, patients that

have incomplete resection will have tumour “re-challenges” occurring on the same day

as the surgery. Hence strategies that boost sinecomitant immunity in the early post-

operative phase are desirable (including TLR ligands and activating anti-CD40 mAb, as

demonstrated in this thesis).

The inherent susceptibility of the surgical site to recurrence was worrying. Poor tumour

resistance at the surgical site was seen repeatedly, and inferior responses to immune

therapy were also observed in that location. Surgical wounding could partly explain that

phenomenon, and wounds have previously been reported to contain significant

quantities of immune suppressive cytokines.120

However, local tumour suppression

vulnerability may also relate to the accumulation of specific cells. In the AB1HA

model, Treg had an active role in impairing tumour resistance of the surgical site, but

there was no difference in plasmacytoid DC concentrations between tumour draining

and systemic nodes.

Fundamentally, sinecomitant immunity was dependent on the three, logically linked

factors outlined above: the decline in tumour antigen presentation (7.1.1), CTLs (7.1.2)

and the sentinel lymph nodes (7.1.3). If any of these factors was missing, then

sinecomitant immunity was poor. These findings introduce a tone of caution for the

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practice of sentinel node biopsy, and raise the possibility that tumour vaccines (which

boost tumour antigen) may be contraindicated in the early post-operative phase. The

role of CTLs in tumour-specific immunity was also highlighted, indicating that CD8+-

targeted therapy might be fruitful.

7 .2 . Conclus ions

Provided the appropriate requirements are met, surgery may provide a post-operative

“window of opportunity” for effective immune treatments (Figure 7.1). In the early

post-operative phase, CD8+ targeted therapies may be most rewarding – given the

critical role of these cells in tumour resistance. Such therapies might be delivered

locally (i.e. into the tumour bed) where they can be absorbed into the lymphatics and

exert activity on the sentinel nodes (where CTL are initially located).

In the intermediate post-operative period, in vivo CTL function is found systemically.

Thus systemic modes of administration for CD8+ boosting therapies might be best. Such

therapies could be combined with Treg depleting treatments (e.g. cyclophosphamide)372

since Treg actively hamper tumour immunity. In addition, while tumour antigen decline

is initially required for enhancements in CTL function, it might be that tumour antigen

becomes limiting in the adaptive immune response. It may then be necessary to

administer a tumour vaccine, augmenting priming and boosting CD8+ immunity.

At delayed time points after surgery, patients may present with bulky recurrent primary

disease and/or metastases. At that stage, it is likely tumours have re-constituted their

immune suppressive networks,27

and strategies to disrupt those networks (e.g.

cytoreduction surgery) may be paramount. Additional treatments, such as anti-VEGF

antibody (e.g. Bevacizumab, AvastinTM

, Genentech Inc, South San Francisco, California

USA) might be helpful, because these could inhibit tumour associated suppressive

signals at the molecular level. The immune intervention itself may also need to be

complex at this stage.28

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Figure 7.1. The window of opportunity for post-operative immune therapy.

Surgery decreased tumour antigen presentation (see Chapter 3) and presumably, tumour derived soluble factors. In addition, CTL were confined to the sentinel nodes before surgery

but increased and spread systemically post resection (Chapter 3). CTL were integral to tumour immunity and post-operative changes correlated with enhanced tumour resistance

(Chapter 4). These findings suggest a role for locally delivered, CTL boosting therapies in the early post-operative phase (as demonstrated in Chapter 4) and a systemic approach in

the intermediate post-operative phase. In the clinical situation, patients may develop post-operative recurrence and metastases, with re-constitution of tumour antigen burden

suppression networks. In that setting, it may be necessary to again disrupt the immune suppressive network (e.g. by surgery or chemotherapy) and/or use a multi-faceted immune

intervention.

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7 .3 . Future Direc t ions

In this thesis, the in vivo effects of primary resection and sentinel node biopsy upon

tumour immunity were described. It is hoped these findings will provide a logical

framework for the combination of surgery and immune therapy in the treatment of

malignancy. As such, it is anticipated that combined surgery/immune therapy strategies

(examples in Chapter 4) will become standard practice into the future. Indeed, there is

already clinical trial evidence for the combination of surgery and immune therapy in the

treatment of both clinically localised12,237

and advanced renal cancer.14

A major emphasis of this project was to correlate primary resection with certain

endpoints including: tumour resistance, CTL function, and antigen presentation (see

Chapters 3 and 4). In this situation, complete tumour clearance ablated antigen

presentation and correlated with an improvement in CTL function in vivo, and tumour

resistance. Another clinically important question would be whether antigen presentation

is sufficiently ablated after incomplete primary resection (i.e. debulking surgery), and/or

in the setting of residual micrometastases (i.e. “minimal residual disease”) to improve

tumour immunity. The RencaHAM model (Chapter 6) should be a useful tool to address

this question.

A second focus of this thesis was the sentinel lymph nodes and their contribution to

tumour immunity in vivo (Chapter 5). It was suggested that sentinel nodes contribute

positively to tumour immunity in vivo, and that removing the sentinel nodes could be

detrimental to tumour resistance. However, sentinel nodes may be removed because

they have intermediate or high probability of being involved with tumour, thus their

prognostic value. The experiments of Chapter Five would suggest tumour-invaded

nodes can function well in the proliferation of tumour-specific CD8+ and in the

clustering of tumour-specific CTL lysis. However, it remains unknown whether

removing a sentinel node is detrimental to tumour immunity, if that node is positive for

tumour. In future studies, it would be possible to assess the effect of removing a tumour

invaded sentinel node on post-operative tumour resistance, using the intra-nodal

injection method in AB1HA (see 2.3.5, 5.2.4) or with the RencaHAM model (6.2.4),

which metastasises to the nodes.

- 189 -

Additionally, if sentinel lymphadenectomy is detrimental to tumour resistance,

investigating mechanisms of avoiding that harm would be desirable. A previous study

has shown that activating anti-CD40 mAb may flush tumour-specific CD8+ from

tumour draining nodes155

and into systemic lymph nodes (Figure 5.15). Determining

whether anti-CD40 mAb can lessen the “immune harm” of sentinel lymphadenectomy

would therefore be of clinical interest.

Finally, there is clearly a complex relationship between tumour antigen presentation and

CD8+ function (Chapters 3 and 4). Based on findings from virology, it is possible that

tumour antigen persistence leads to the upregulation of PD-1, and CD8+ anergy.

297,300

Certain tumours (e.g. renal cancer)414

may compound this anergy, by expressing B7-H1

and thereby directly ligating CD8+ PD-1. It is postulated that PD-1/PD-1L signalling

will be important in tumour immunology, and that surgery might provide a mechanism

of lessening PD-1 activation. Surgery may also prove synergistic with PD-1/PD-1L

blockade in recovering tumour-specific CD8+ function in vivo. A Master of Surgery

student at The University of Western Australia is now testing this hypothesis, using

RencaHAM (described in this thesis – Chapter 6).

- 190 -

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Appendix B: Abbrevia t ions

Ab Antibody. An immune protein targeting a particular structure e.g.

peptide.

AB1 A murine mesothelioma cell line, developed by immortalising tumour

from the peritoneum of asbestos injected BALB/c mice.

AB1HA AB1 mesothelioma that was transfected with genes for

Haemagglutinin and neomycin resistance.

ANOVA “ANalysis Of VAriance”. A statistical method used to test for

differences between three or more independent groups.

APC Antigen presenting cell. A cell capable of antigen uptake and

presentation to CTLs and helper T cells.

BCG Bacillus Calmette-Guérin. An attenuated form of Mycobacterium

bovis. Used as an active non-specific immunotherapy.

BSA Bovine serum albumin. A protein used in general laboratory work,

including the supplementation of cell culture medium.

CD Cluster determinant. An antigen cluster with which antibodies react.

Characteristic CD patterns assist in identifying cell type.

CD4+ Helper T cells. Secrete cytokines and augment the immune response.

CD8+ Widely used to denote CTLs.

CFSE 5,6-carboxyfluoroscein succinimidyl ester. An intracellular dye used

to stain lymphocytes.

CL4 Clone Four transgenic mouse. Homozygous for CD8+ cells with

specificity for the 1-Ad restricted IYSTVASSL epitope of

haemagglutinin.

CTL CTL. CD8+ T lymphocytes capable of lysing target cells.

95% CI 95% confidence interval. A range of values that, with a probability of

0.95, contains the population mean.

DLN Draining lymph node. Becomes synonymous with “sentinel lymph

node” in this thesis.

DC Dendritic cell. Characteristically CD11c+. Thought to have a critical

role in tumour antigen presentation.

DNA Deoxyribonucleic acid. Contains the genetic code. Certain fragments

of bacterial or viral DNA can be immune stimulatory.

DOS Day of surgery. Occurring within 24 hours of tumour resection.

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DTH Delayed type hypersensitivity. Evoked when macrophages present

antigen to CTLs and helper T cells. Involved in allergic responses,

transplant rejection, and the immune response to mycobacterial

infection.

FasL Fas ligand. Also known as apoptosis antigen ligand 1, or CD95

ligand. Trimerises Fas Receptors of the target cell membrane, leading

to apoptosis of that cell.

FCS Foetal calf serum. A protein used in general laboratory work,

including the supplementation of cell culture medium.

FGK-45 A monoclonal antibody that ligates and activates the CD40 receptor.

Foxp3 A forkhead/winged helix transcription factor, critical to the

development and function of Treg.

GK1.5 Rat monoclonal antibody with specificity for murine CD4+, used to

deplete CD4+ cells in vivo.

GL113 Rat isotype antibody, used as control for depletion and FGK45

therapy experiments.

GM-CSF Granulocyte macrophage – colony stimulating factor. A protein that

stimulates bone marrow to produce myeloid cells. Also attracts DC

and causes proliferation of the same.

HA Haemagglutinin. A characteristic antigen of PR8 influenza that was

transfected into the AB1 and Renca cell lines.

HEPES N-2-hydroxyethylpiperazine-N‟-2-ethanesulfonic acid. A buffer used

in cell culture medium.

HNT Transgenic mouse with high frequency of CD4+ cells with specificity

for the H-2Kd restricted HNTNGVTAACSHE epitope of

haemagglutinin.

HR Hazard ratio. A statistical measure of survival in one group relative to

another.

HSPs Heat shock proteins. A family of chaperone proteins that form

complexes with intracellular peptides in response to cellular stress.

Provide a danger signal.

IFN Interferon alpha. A glycoprotein with immune stimulatory and anti-

angiogenic characteristics. Used in metastatic renal cancer.

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IFN Interferon gamma. A dimeric glycoprotein cytokine that enhances

antigen presentation, activates macrophages, and up-regulates TH1

function.

Il-2 Interleukin two. A T cell growth factor cytokine. Also activates NK

cells. Used in metastatic renal cancer.

Il-3 Interleukin three. Stimulates proliferation of haematopoietic

pluripotent progenitor cells, as well as T cell growth and

differentiation.

Il-4 Interleukin four. A cytokine that activates B cells on numerous levels,

including the promotion of immunoglobulin class switching and

upregulation of MHC class II expression.

Il-5 Interleukin five. A cytokine that enhances eosinophil function,

stimulates B cell growth, and promotes plasma cell immunoglobulin

class switching.

Il-6 Interleukin six. Released by macrophages and T cells in response to

trauma. Involved in fever and the inflammatory response to trauma.

Il-10 Interleukin ten. Sometimes referred to as human cytokine synthesis

inhibitory factor. Impairs production of pro-inflammatory cytokines

and stimulates B cells.

Il-12 Interleukin twelve. Involved in the differentiation of naïve helper T

cells into the TH1 subset. Activates CTLs and natural killer cells and

stimulates IFN production.

iNOS Inducible nitric oxide synthase. Produces nitric oxide, a free radical.

i.p. Intraperitoneal. Used to denote the intra-coelomic route of

administration e.g. for monoclonal antibody.

i.v. Intravenous. Used to denote intravascular administration of a drug or

agent e.g. FGK-45.

LN Lymph node. A highly specialised aggregate of lymphoid tissue that

filters and processes incoming lymph from the tissues.

M1 A subset of macrophages that produce iNOS and are associated with

tumour immunity.

M2 A subset of macrophages that produce arginase, impair tumour

immunity, and foster cancer growth.

mAb Monoclonal antibody. Antibodies identical in specificity, produced by

a single clone of cells.

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2-ME 2-mercaptoethanol. An anti-oxidant used to supplement cell culture

medium.

MHC Major histocompatibility complex. A set of cell surface molecules

involved in self recognition and antigen presentation.

MSC Myeloid-derived Suppressor Cells. A heterogenous population of

immature myeloid cells that accumulate during tumour growth and

suppress anti-tumour immunity.

NDLN Non draining lymph node. Lymph nodes which are not situated on the

afferent lymphatics of the tumour site (e.g. mediastinal nodes).

NK Natural Killer cells. Innate cells with cytotoxic capability, able to

recognize tumors or viral infected cells, plus the absence of MHC

Class I. Characteristically express CD16 and CD56 in humans, or

NK1.1/NK1.2 in mice.

NKT Natural killer T cells. A heterogenous group of cells that have

properties of both NK and T cells. They recognize non-polymorphic

CD1d.

P The probability that the observed or a greater difference in means (t

test) or medians (Mann Whitney test) for two samples could be seen

by chance, if the samples were drawn from the same population.

PBS Phosphate buffered saline. A ubiquitous laboratory solution, helpful

for processing cell samples.

PC61 Monoclonal antibody for the Il-2 receptor alpha (CD25). Can be used

to deplete for Treg in vivo.

PCR Polymerase chain reaction. A technique to amplify DNA enabling the

sensitive detection of minute quantities of a gene of interest (e.g.

haemagglutinin gene).

PgE2 Prostaglandin E2. A lipid derived autocrine and paracrine mediator,

involved in vasodilatation, inflammation, and fever.

Post-op Post-operative. Occurring after surgery.

RCC Renal cell carcinoma. A malignant neoplasm of the renal cortex.

S.C. Subcutaneous. Used to denote the subcuticular route of administration

or location.

SD Standard deviation. Calculated as the square root of the variance.

Measures variation in values about the mean.

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SEM Standard error of measurement. Indicates whether the mean value

observed is a reliable estimate of the population mean. Calculated by

dividing the standard deviation of the sample by the square root of n

(the number of values).

sPS Soluble phosphatidylserine. A tumour derived soluble factor that has

anti-inflammatory properties.

TAM Tumour associated macrophages. Population of tumour infiltrative

macrophages that may develop from MSC and exert immune

suppressive properties within the tumour microenvironment.

TGF Transforming growth factor beta. Affects function, proliferation and

activation of numerous cell types. Sometimes released by tumours.

TH1 T helper cell type one. A subset of CD4+ helper T cells where

cytokines foster CTL function.

TH2 T helper cell type two. A subset of CD4+ helper T cells where

cytokines limit CTL function, but enhance IgE mediated immunity.

TIL Tumour infiltrating lymphocytes. Lymphocytes found within the

tumour, sometimes harvested and cultured for adoptive

immunotherapy.

TLR Toll like receptor. A group of pattern recognition receptors that react

to generic inflammatory signals (e.g. bacterial DNA).

TNF Tumour necrosis factor alpha. A cytokine released by white blood

cells and blood vessels in response to trauma. Chemoattractant to

neutrophils. Pro-inflammatory. Also involved with fever and cachexia

(hence its name).

Treg CD4+CD25

+ regulatory T cells that functionally suppress other cell

types.

TS Suppressor T cells. The former name for CD4+CD25

+ regulatory T

cells.

VEGF Vascular endothelial growth factor. Released in association with

hypoxia. Stimulates vasculogenesis and angiogenesis. Also associated

with the accumulation of MSC.

YTS169 Rat monoclonal antibody with specificity for murine CD8+, used to

deplete CD8+ in vivo.

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