inflammation, immune tolerance and cancer -...

Introduction

Role of hCG in tumor induced inflammation and immune-tolerance 65

Inflammation, Immune Tolerance and Cancer

Inflammation (Latin, inflamers, to set on fire) is part of the biological response of

vascular tissues to pathogens, damaged cells, or irritants (Ferrero-Miliani et al., 2007).

It is a protective mechanism by the organism to remove the stimuli and to initiate the

healing process. In an infection, burn, or other injuries, cells release inflammatory

mediators. Now it is well established that inflammation plays a critical role in

tumorigenesis (Wan Wan and Karin, 2007); an inflammatory environment is believed

to be a necessary component (Mantovani et al., 2008). Recent studies also suggest that

induction of inflammation by viral and bacterial infections increases cancer risks (De

Martel and Franceschi, 2009). Various immune cells, including T and B lymphocytes,

macrophages, dendritic cells (DCs), neutrophils and mast cells are found in tumors

(De Visser, 2006; Johansson, 2007 and De Visser, 2005).

Immunological tolerance refers to the failure to mount an immune response to an

antigen. Such tolerance can be either 'natural' (where the body does not mount an immune response to self antigens), or 'induced‟ (where tolerance to external antigens

can be created). Some tumor cells escape immune detection by decreasing the

expression of antigen-presenting proteins at their surface, which helps them become

invisible to cytotoxic T lymphocytes (Meissner et al., 2005). Also, tumors can secrete

proteins that inhibit effector T cell responses and promote the production of regulatory

T cells that suppress immune responses (Shevach, 2004).

Tregs

An increase in the number of Treg cells in cancer patients has been reported by

numerous investigators. There are two kind of CD4+CD25

+ Tregs - naturally and

adaptive. Natural CD4+CD25

+ Tregs arise in the thymus. Adaptive CD4

+CD25

+ Tregs

arise during inflammatory processes associated with infection and cancer. Tregs

suppress immunity through direct contact or the production of soluble factors such as

TGF-beta and IL-10 (Bluestone and Abbas, 2003). Adaptive Tregs have been

demonstrated in the tumor microenvironment (Liu et al., 2007). Apart from

suppressing the function of T cells, Tregs also inhibit the function of NK cells

(Ghringhelli et al., 2005), B cells (Lim et al., 2005) and other immune cells. Patient

survival inversely correlates with the number of Tregs in the tumor (Curiel et al.,

http://en.wikipedia.org/wiki/Latin

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Introduction


2004); recent reports suggest that the number of CD4+CD25

+FOXP3

+ Tcells

correlates inversely with clinical outcome in epithelial carcinoma, ovarian cancer,

breast cancer and hepatocellular carcinoma (Curiel et al., 2004; Bates et al., 2006 and

Kobayashi et al., 2007). However, in haematological malignancies, the situation is

different, due to unexplained reasons; in follicular lymphoma, increased number of

FOXP3+ cells correlates with increased survival (Carreras et al., 2006).

TGF beta

Transforming growth factor beta (TGF-β) plays an important role in development of

the body plan during embryogenesis and is crucial for tissue homeostasis. The

biological effects of TGF-β are mediated through control of proliferation,

differentiation, apoptosis, adhesion and invasion (Heldin et al., 2009; Yang et al.,

2010; Ikushima and Miyazono, 2010 and Massague, 2008). Malfunctioning of these

pathways can lead to tumorigenesis. TGF-β has been demonstrated to be a major

player in EMT; treatment of normal breast epithelial cells with TGF-β increases

expression of mesenchymal markers (Miettinen et al., 1994). TGF-β mediates

downregulation of the microRNA-200 family that has an inhibitory influence on EMT

(Gregory et al., 2008). Analysis of different tumors suggests a role for TGF- β in

tumor progression; melanomas, breast cancer, colon oesophagus, stomach, liver, lung,

pancreas and prostate cancer secrete high levels of TGF-β (Dong and Blobe, 2006;

Levy and Hill, 2006), and levels appear to be higher in metastasized tumors than in

primary tumors (Dalal et al., 1993). Administration of neutralizing TGF-β antibodies

prevents metastasis (Afrakhte et al., 1998).

It has been demonstrated that TGF-β regulates the activation of angiogenesis (Lebrin

et al., 2005; Pepper, 1997). TGF-β also helps tumors evade mechanisms of immune

surveillance (Torre-Amion et al., 1990; Yang et al., 2010); it inhibits CD8+ mediated

rejection in murine tumors (Torre-Amion et al., 1990). CD4+ CD25

+ regulatory T cells

produce high amount of TGF-β and can inhibit NK cell activity (Ghiringhelli et al.,

2005). Tumors can also promote TGF-β production by the surrounding cells in the

tumor microenviornment (Chang et al., 1993). In turn, TGF-β promotes the generation

of Tregs, further promoting immune privilege (Curiel et al., 2004). Increased TGF-β

levels correspond with poor patient prognosis (Saito et al., 2000).

Introduction


Dendritic cells and IDO

Dendritic cells (DCs) play a central role in immune defence against microbial

invaders. These APCs are found in organs most exposed to foreign antigens.

Conversely, DCs can also help in inducing tolerance and controlling autoimmunity.

Cytokine signalling events determine whether DCs becomes tolerogenic or

stimulatory (Katz et al., 2008). Immature DCs produce cytokines such as IL-10 and

vascular endothelial growth factor. Mature, tolerogenic DCs can promote Th2

polarization and expand the Treg population (Mellor and Munn, 2004; Moser, 2003).

Tumors can play a key role in determining responses and functions of DCs

(Ghiringhelli et al., 2005; Liu et al., 2005). The link between the binding of CTLA-4

on Treg cells to B7 on DCs and Indoleamine 2, 3 dioxygenase (IDO) - induced

tolerance has been demonstrated by several groups (Mellor et al., 2003; Mellor et al.,

2004 and Fallarino et al., 2003) (Figure 4.1). IDO is a tryptophan- catabolising

enzyme. IDO activity is apparently crucial to prevent the rejection of an allogeneic

foetus in mice (Munn et al., 1998). IDO activation is one of the mechanisms by which

tumor cells escape the immune surveillance. Several studies suggested that IDO over-

expression by tumors is associated with poor prognosis; for example, the degree of

expression of IDO in tumors was shown to inversely correlate with the survival of

patients with ovarian cancer (Okamoto, 2005). The relationship between cancer and

enhanced tryptophan catabolism was first demonstrated in the urine of bladder cancer

patients (Boyland and Williams, 1955). CTLA-4 blockade is associated with a

decrease in immunosuppressive molecules like IDO and TGF-β in simian

immunodeficiency virus infected macaques (Hryniewicz et al., 2003). Other studies

have confirmed that systemic administration of CTLA-4-Ig results in IDO up-

regulation in a DCs subset (Mellor and Munn, 2003).

Introduction


Figure 4.1 Katz et al, immunological reviews, 2008.

Figure 4.1: IDO in T-cell tolerance and cancer. IDO is commonly dysregulated in tumors and

in plasmacytoid DCs, in tumor-draining lymph nodes (TDLNs). Through tryptophan (Trp)

depletion and/or production of kynurenine (Kyn) and other catabolites, IDO activity limits

antigen-dependent T-cell activation. In tumor cells, where Bin1 attenuation occurs frequently

during tumor formation or progression, IDO can become dysregulated as a result of Bin1 loss.

Through B7 receptor interactions involving CTLA-4 or other inhibitory ligands, Tregs can

promote IDO expression. In a circular fashion, IDO1 DCs in TDLNs may support further

recruitment of Tregs in a feed forward loop. 1MT is a specific inhibitor of IDO and IDO2 that

has recently moved into human clinical trials for cancer treatment.

IL-10

The relationship between IL-10 and cancer is still unclear. It is an immunosuppressive

and anti-inflammatory cytokine (Schottelius et al., 1999). IL-10 can increase tumor

growth in vitro by stimulating cell proliferation and inhibiting cell apoptosis (Sredeni

et al., 2004; Alas et al, 2001). IL-10-expressing tumor cells and tumor-associated

macrophages (TAMS) can produce BAFF, which promote B cell and lymphoma

survival (Ogden, 2005). In IL-10-/-

mice, B cell lymphoma grow more slowly

(Czarneski et al., 2004). IL-10 fixed cells in B16-melanoma xenograft model shows

more angiogenesis and growth (Gracia-Hernandaze, 2002). IL-10 pre-treatment can

induce CTL-resistance in melanoma and lymphoma cells (Kurte et al, 2004; Peterson

et al., 1998). Lewis lung carcinoma tumors grow faster in IL-10 transgenic mice than

in control mice (Hagenbaugh et al., 1997). IL-10 producing monocytes have been

isolated from the ascites of patients with ovarian carcinoma (Loercher et al., 1999).

Introduction


Anti IL-10/IL-10R blocking antibodies (Jovasevic et al., 2004; Vicari et al., 2002) or

anti-IL-10 antisense oligonucleotides (Kim et al., 2000) improve cancer specific

immune responses in some preclinical cancer models.

Macrophages

TAMS contribute to a significant extent to tumor mass (Pollard et al., 2004). They

provide support to the tumor by degrading the extracellular matrix by the synthesis of

growth and angiogenic factors and by suppression of anti-tumor immune responses

(Condeelis and Pollard, 2006). Recent studies suggest that macrophages-derived

osteoclasts remodel bone in mammary development (Lin et al., 2001). Studies show

that there is a strong correlation between macrophages density and poor patient

prognosis (Ding et al., 2009). Macrophages help in cancer initiation and progression

through intravasion, angiogenesis and immune regulation. A correlation exits between

macrophage numbers and the extent of inflammation. On the basis of their activation

state, receptor expression, cytokine production and function, macrophages have been

classified as either Type 1 or Type 2. Type 1 macrophages (M1) are capable of

producing large amounts of pro-inflammatory cytokines. Type 2 macrophages (M2)

moderate the inflammatory response and promote angiogenesis (Lamanga et al.,

2006). M2 macrophages also produce the immunosuppressive cytokine TGF-β and

have also been implicated in ECM remodelling (Bingle, 2002; Coussens, 1996).

Tumor associated IL-10 drives the differentiation of monocytes into angiogenic M2

macrophages which make VEGF. Pro-inflammatory cytokine like IL-17 and IL-23 are

also released by TAMS (Lamagna et al., 2006). (Fig 4.3)

Introduction


Figure 4.2 Allvena et al., 2009, J of leukocytes Biology

Figure 4.2: Overview of TAM, which originate from blood monocytes recruited at the tumor

site by molecules produced by neoplastic and by stromal cells. Main factors involved in

monocyte recruitment are the chemokine CCL2, M-CSF, and VEGF. When monocytes reach

the tumor mass, they are surrounded by several micro environmental signals such as IL-3 and

M-CSF, able to induce their differentiation toward mature macrophages (now called TAM)

and to shape the “new” cells as needed by the tumor (CSFs, IL-4, IL-10, and TGF-β). Tumor-

molded macrophages resemble M2-polarized cells and play a pivotal role in tumor growth and

progression. TAMS actively work for the tumor: They produce several molecules that sustain

malignant cell survival, modify neoplastic ECM proteins, promote the development of a

newly formed vessel, and assist in tumor progression. Moreover, TAMs significantly affect

adaptive immune responses significantly by recruiting and stimulating Tregs and recruiting

Th2 lymphocytes, which in turn inhibit Th1 cells, and by inducing anergy in naïve T cells.

Versican

Versican is a member of the proteoglycan family (Figure 4.3). It expressed throughout

the body and provides the ECM with hygroscopic properties, creating a loose and

hydrated matrix that is necessary to support key events in development and disease.

Introduction


Many cellular processes including adhesion, proliferation, apoptosis and invasion are

regulated by versican (Wight, 2002; Wight and Merrilees, 2004 and Theocharis,

2008). V0 and V1 are the predominant isoforms of the molecule present in cancer

tissues (Sakko et al., 2001; Ricciardelli et al., 2002; Arslan et al., 2007). Heightened

levels of versican observed in many cancers are associated with relapse and poor

patient outcome (Ricciardelli et al., 1998; Pirinen et al., 2005; Pukkila et al., 2007 and

Kodama et al., 2007). Some observations suggest that versican is mostly secreted by

activated pre-tumoral stromal cells in adenocarcinomas (Kodama et al., 2007). It has

been demonstrated that versican can increase cancer cell motility (Arslan et al., 2007,

Cattaruzza et al., 2004, Zheng et al., 2004 and Ricciardelli et al., 2007), proliferation

(La Pierre et al., 2007) and metastasis (Yee et al., 2007).Versican may also promote

the formation of inflammatory microenviornment in tumor stroma. Interaction

between versican and Toll-like receptor 2 is one of the links between inflammation

and metastasis (Kim et al., 2009). Versican-induced TNF-α and IL-6 production by

macrophages is thought to play a significant role in metastasis (Karin, 2009).

Introduction


Figure 4.3 Yao et al, 2005; Cell

Figure 4.3: Structure of versican. The interaction of versican with other molecules and the

locations of versican motifs that interact with these molecules are shown.

IL-6

IL-6 is a proinflammatory cytokine and is considered a key growth-promoting and

anti-apoptotic factor (Ishihara and Hirano, 2002). It is over expressed in response to

injury, inflammation and infection (Scheller and Rose-john, 2006). IL-6 is produced

by many cells, including osteoblast, monocytes, BMMCs (Bone marrow derived

macrophages) and macrophages (Ara and DeClereck, 2010). Most IL-6 targeted genes

are involved in cell cycle progression and suppression of apoptosis, both of which are

characteristics of tumorigenesis (Haura et al., 2005). Studies suggested an association

between IL-6 and risk of developing Hodgkin‟s lymphoma (Cozen et al., 2004). IL-6

interacts with several pathways (prominent amongst which are Cox2, Wnt, TGF-beta

and NFκB associated signalling events) which contribute to its pro-tumorigenic

activity (Ara and DeClereck, 2010). TGF-β upregulates IL-6 expression in several cell

types and TGF-β and IL-6 act synergistically to potentiate bone degradation

(Yamamoto et al., 2001). The role of IL-6 in cancer is complex and includes autocrine

and paracrine mechanisms. IL-6 has a growth-stimulatory effect on many tumor cells

through the activation of several signalling pathways like Ras, Raf, Mek and Erk1/2

(Ara et al., 2009; Ogata et al., 1997 and Smith et al., 2001). Over-expression of IL-6

in specific organs can attract circulating tumor cells to these organs and promotes

metastasis in tumors (Ara and DeClereck, 2010). In melanoma, IL-6 activates STAT-3

Introduction


which lead the growth of metastatic tumors in brain by inducing the over expression

of bFGF, MMP-2 and VEGF (Xie et al., 2006). Metastasis of Lewis lung carcinoma in

the liver is stimulated by NFκB stimulated IL-6 production (Ara and DeClereck,

2010). IL-6 and IL-8 are thought to create a process referred to as „tumor self-seeding‟

which accelerates tumor growth and angiogenesis (Kim et al., 2009). IL-6 increases

angiogenesis by upregulation of VEGF, HIF-1-α, bFGF and MMP-9 in tumor

associated myeloid cells (Kujawaski et al., 2008; Dankbar et al, 2000; Huang et al,

2004). IL-6 down regulates the activity of NK cells and their anti-tumor function (Yu

et al., 2007). Thus, IL-6 helps in angiogenesis, tumor growth and contributes to an

immune microenvironment more suitable for tumor progression.

TNF-α

TNF-α is a member of the TNF/TNFR family. It is a proinflammatory cytokine and its

role in chronic inflammatory diseases is established (Lin and Yeh, 2005).

Macrophages are the major source of TNF-α, but it can also be produced by

proliferating B cells, NK cells, mast cells and neutrophils (Gemlo et al., 1988;

Kinkhabwala et al., 1990; Stein and Gordon, 1991). TNF-α has been detected in a

number of different tumors including those of the ovary and breast, and also in

haematological malignancies (Naylor et al., 1993; Sati et al., 1999; Warzocha et al.,

2000). TNF-α produced in the tumor microenviornment can promote tumor cell

survival through NFκB dependent anti-apoptotic molecules (Luo et al, 2004). TNF-α

also contributes to tumor initiation by stimulating production of molecules like NO

and ROS (Hussain et al., 2003). TNF-α injected into mice inoculated with a

methylcholanthrene-induced fibrosarcoma increased the number of lung metastasis

(Orosz et al., 1993). Toll like receptor signalling may be involved in production of

TNF-α in cancer (Tsan, 2005). TNF-α is a powerful mitogen for primary hepatocytes

(Maeda et al., 2005). TNF-α can increase the expression of IL-8 and Groα in number

of cell types (Strieter et al., 1995). TNF-α also up regulates MMP-9 and contributes to

metastasis and angiogenesis (Shin et al., 2000). Intestinal cells stimulated with TNF-α

exhibit reduced levels of E-Cadherin and enhanced invasion (Kawai et al., 2002).

TNF-α induces bone marrow cells to produce IL-6 (Hideshima et al., 2001). TNF-α

can cause haemorrhagic necrosis and can, in some instances, generate specific T cell

Introduction


tumor immunity (Lejeune et al., 2002), but when produced in the tumor

microenviornment, it acts as an endogenous tumor promoter (Balkwill, 2002).

hCG, inflammation and immune tolerance

Tolerance towards the foetal allograft during gestation is thought to be achieved

partially due to the presence of CD4+CD25+Foxp3+ regulatory T cells (Tregs)

(Schumacher et al., 2009). hCG induces an increase in the number of these cells in the

periphery during pregnancy (Zenclussen et al., 2006). It is believed that Treg cells

may be attracted to the maternal–fetal interface by hCG (Schumacher et al., 2009). It

has been suggested that hCG also contributes towards local immune tolerance by up-

modulating FasL on uterine cells (Kayisli et al., 2003).

hCG treatment of activated dendritic cells induces up-regulation of MHC class II

expression, and increases IL-10 and IDO expression, inducing an immunosuppressive

environment (Wan et al., 2008). hCG also up-regulates IDO in placenta and

suppresses autoimmune diabetes in NOD mice (Ueno et al., 2007).

hCG can promote functions of macrophages, such as clearance of apoptotic cells and

inflammation, which are necessary for the maintenance of pregnancy (Wan et al.,

2007). While most studies appear to suggest an anti-inflammatory role for hCG, it has

been demonstrated that high doses of hCG induces IL-8 production by monocytes

(Kosaka et al., 2002).

Materials and methods

Role of hCG in tumor induced inflammation and tolerance 75

Assays were carried out to determine whether hCG could induce the

transcription/secretion of anti-inflammatory/immune-suppressive and/or pro-

inflammatory mediators from tumor cells, either directly or in collaboration with non-

transformed cells from peripheral blood. In addition, whether supernatants obtained

upon the incubation of hCG with tumor cells could induce the generation of Treg cells

was determined.

Anti-inflammatory/immune-suppressive effects of hCG

COLO-205, ChaGo and LLC cells were cultured under standard conditions in serum-

free medium, supplemented with hCG (1µg), anti-hCG antiserum, hCG + anti-hCG

antiserum, non-immune serum or hCG + non-immune serum for 48 h.

hCG-induced FOXP3 expression and secretion of IL-10 and TGFβ

from tumor cells

FOXP3 expression in cellular lysates was analyzed by immunoblot. Briefly,

subsequent to 10% SDS-PAGE, separated moieties were transferred to nitrocellulose

membranes. Membranes were “blocked” by incubation with Tris-buffered saline

containing 0.05% Tween 20 and 3% BSA for 2 hours. An incubation was carried out

for 2 hrs at 37oC with rabbit anti-human FOXP3 (Santa Cruz) antibodies. Reactive

moieties were revealed by incubation with an anti-rabbit IgG-HRP conjugate (Jackson

Immuno Research) and subsequent enhanced chemiluminescence (Biological

Industries).

Total RNA was isolated using an RNA isolation kit (Intron) as described in Chapter 2.

RT-PCR for human and murine FOXP3 was performed using a one step RT-PCR kit

(Qiagen). The following primers combinations were employed:

FOXP 3 (human) Forward 5’-CACAACATGCGACCCCCTTTCACC - 3’

Reverse 5’-AGGTTGTGGCGGATGGCGTTCTTC -3’

FOXP 3 (murine) Forward 5’-TCTTGCCAAGCTGGAAGACT-3’

Reverse 5’-AGCTGATGCATGAAGTGTGG-3’

PCR conditions were as follows: Reverse transcription 500C for 30 min followed by a

hold 950C for 15 min. Denaturation at 94

0C for 1 min, annealing at 60

0C for 1 min,



extension at 720C for 1 min, final extension is at 72

0C for 10 min. Number of cycles:

35.

Cells were analysed for the presence of FOXP3 by flow cytometry. 105 cells were

incubated with Permeablization Buffer (Appendix) for 90 secs and then "washed"

three times with FACS Buffer (Appendix) by repeated centrifugation at 400 g at 4°C

for 5 min. Cells were then incubated with 1:1000 diluted rabbit anti-FOXP3 antibody

(Santa Cruz) for 2 hr at 4°C. Excess antibodies were removed by repeated "washes"

with FACS Buffer as before. An incubation was then carried out with 1:200 diluted

anti-rabbit FITC conjugate (Jackson Immuno Research) for 2 hrs 4°C. After further

"washes", cells were resuspended in FACS Buffer. Data was acquired on a BD LSR

flowcytometer.

IL-10 and TGF- levels in culture supernatant were quantified by specific ELISA kits

(E-bioscience), following the manufacturer’s instructions. Briefly, capture antibody

(100µl/well in coating buffer) was dispensed into wells of 96-well flat bottom ELISA

plates and an incubation carried out for 16 hr at 4°C. Wells were "washed" three times

with wash buffer and then "blocked" by incubation for 1 hr at room temperature with

assay diluent (200µl/well). After further "washes", standard or samples (100µl/well,

diluted in assay diluent) were dispensed into appropriate wells, followed by an

incubation at room temperature for 2 h. Subsequent to "washes", biotinlylated

detection antibody (100µl/well, diluted in assay diluent) was added, followed by an

incubation at room temperature for 1 hr. An avidin-HRP conjugate (100µl/well,

diluted in assay diluent) was added, followed by incubation at room temperature for

30 minutes. After further "washes", 100µl/well of substrate solution was added to each

well. 50 µl of stop solution was added at the appropriate time to each well. Optical

densities were determined at 450 nm.

The effect of culture supernatant on allogeneic mixed lymphocyte reactions was

assessed. Human PBMC were isolated from MHC-mismatched donors by density-

gradient centrifugation using Ficoll-Paque, as described in Chapter 4. PBMCs from

two separate donors were dispensed at 2 x 105

cells/well. An incubation with culture

supernatant was carried out for 5 days under standard culture conditions; for the last

16 hrs, [3H] thymidine (1µCi/well) was added to each well. Cell-associated

radioactivity (an index of proliferation) was quantified on a beta scintillation counter.



Antibodies to IL-10 (eBioscience) were employed to assess the contribution of the

cytokine to observed inhibitory effects.

Assessment of the ability of supernatant obtained upon the

incubation of hCG with tumor cells to induce the generation of

Treg cells

CD4+CD25

– and CD4

+CD25

+ T cells were purified from the spleen of adult C57BL/6

mice using a T Regulatory cell isolation kit (Miltenyi Biotec) as per the protocol

supplied by the manufacturer. In the first step, the total CD4+

T population was

purified by negative selection. Briefly, 6 x107 cells were centrifuged at 300 x g for 10

mins and resuspended in 240 µl complete DMEM medium. 60 µl of a biotin-antibody

cocktail was added and the suspension mixed well. An incubation was carried out 10

min at 4°C. Medium (180 µl), anti-biotin micro beads (120 µl) and PE conjugated

anti-CD25 antibody (60 µl) were then added, mixed well and incubated for additional

15 min in dark. Cells were 'washed' with 10 ml medium and resuspended in 500 µl

medium. The separation column was placed in a magnetic field and 'rinsed' with 2 ml

medium. The cell suspension was then 'loaded' onto the column and the unbound cells

(comprising CD4+

T cells) allowed to pass through. Cells were centrifuged at 300 x g

for 10 min, resuspended in 540 µl of medium and 60 µl of anti-PE micro beads were

added, the cell suspension mixed well and an incubation carried out for 15 min at 4°C

in the dark. Cells were 'washed' with 10 ml medium by centrifugation at 300 x g for

10 min and cell pellet resuspended in 500 µl of medium. The cell suspension was

'loaded' onto the column (in the presence of the magnetic field) and unbound cells

(comprising CD4+CD25

- T cells) allowed to pass through. The column was then

'washed' with 1.5 ml medium. The magnet was then removed and the column-bound

cells (comprising CD4+CD25

+ T cells) recovered with the help of a plunger.

Isolated CD4+CD25

– T cells were cultured (10

6/well in a 24-well plate) for 5 days in T

cell medium (1 µg/ml plate-bound anti-CD3 antibody + 2 µg/ml soluble anti-CD28

antibody (eBioscience)), supplemented with supernatant collected

from a 3-day culture

of LL cells with hCG (Figure 4.4). Appropriate negative controls were included. Cell

surface CD4 and CD25 expression levels were assessed by flow cytometry using

specific antibodies (eBioscience).



Figure 4.4

Assessment of the ability of hCG to induce up-modulation of CTLA-

4 on tumor cells and the subsequent generation of indoleamine 2,

3-dioxygenase (IDO) from bone marrow derived dendritic cells

(BMDC)

Cells were incubated with hCG as described above. The presence of cell surface

associated CTLA-4 was determined by flow cytometry. 105 cells were "washed" three

times with FACS Buffer by repeated centrifugation at 400 g at 4°C for 5 min. Cells

were then incubated with 1:1000 diluted rabbit anti-CTLA4 antibody (e-biosciences)

for 2 hr at 4°C. Excess antibodies were removed by repeated "washes" with FACS

Buffer as before. An incubation was then carried out with 1:200 diluted anti-rabbit

FITC conjugate (Jackson Immunoresearch) at for 2 hrs 4°C. After further "washes",

cells were resuspended in FACS Buffer. Data was acquired on a BD LSR

flowcytometer.

C57BL/6 mice (6-8 weeks old) were sacrificed by cervical dislocation and their

femurs and tibiae were removed. The tip of each bone was removed with a scalpel,

and the marrow flushed out with RPMI by inserting a syringe needle (231/2-gauge)

into one end of the bone. A single-cell suspension was achieved by gentle pipetting.

Cells (4 x 106

per well in a 6-well plate) were cultured under standard conditions in

medium containing 50 ng/ml recombinant mouse GM-CSF (Peprotec). At Days 3 and

5, fresh medium containing GM-CSF was added. To induce maturation of BMDCs,

1µg/ml LPS (S.typhosa, Sigma) was added on Day 7; control cultured received only

medium. An incubation was then carried out for an additional 36 hrs. Non-adherent



cells were assessed for the presence of surface CD11c, MHC 1, MHC 2, CD 80 and

CD 86 by flow cytometry as described above. 2 x 106 BMDCs were co-cultured with

2 x 106

LLC cells (either pre-incubated with hCG or not, as described above) for 24

hrs.

IDO activity in cell extracts was determined colorimetrically by a modification of a

procedure described by other investigators. 0.5 ml of the cell extract was added to 0.5

ml of substrate (0.8 mM L-tryptophan (Sigma), 40 mM ascorbic acid (Sigma cat. no

A4403), 20 µM methylene blue (Sigma cat. no. M1940), 200 units/ml catalases and

100 mM potassium phosphate buffer (pH 6.5). Both the enzyme suspension and the

substrate suspension were preincubated independently at 37°C for 5 min before

mixing, followed by further incubation at 37°C for 30 min. The reaction was

terminated by the addition of 0.2 ml 30% trichloroacetic acid; a further incubation was

carried out at 50°C for 30 min to hydrolyze N-formylkynurenine (produced as a result

of IDO activity) to kynurenine. The reactants were then centrifuged at 3000 x g for 20

min to remove sediment. 0.8 ml p-dimethylaminobenzaldehyde (1% (w/v) in acetic

acid) was then added. Absorbance was determined at 480 nm to determine the

presence of kynurenine.

The presence of IDO subsequent to co-culture was also determined in cell lysates by

immunoblot. Briefly, subsequent to 10% SDS-PAGE, separated moieties were

transferred to nitrocellulose membranes. Membranes were “blocked” by incubation

with Tris-buffered saline containing 0.05% Tween 20 and 3% BSA for 2 hours. An

incubation was carried out for 2 hrs at 37oC with rabbit anti-human/mouse IDO

(Millipore) antibodies. Reactive moieties were revealed by an anti-rabbit IgG-HRP

and subsequent enhanced chemiluminescence (Biological Industries).

Proinflammatory effects of hCG

Experiments were carried out to assess whether culture supernatants obtained upon the

co-incubation of hCG and tumor cells contained the pro-inflammatory proteoglycan

Versican and whether such supernatants could induce the secretion of pro-

inflammatory cytokines IL-6 and TNF- from adherent, non transformed cells.



Detection of versican

Versican expression in cell lysates was analyzed by immunoblot. Briefly, subsequent

to 10% SDS-PAGE, separated moieties were transferred to nitrocellulose membranes.

Membranes were “blocked” by incubation with Tris-buffered saline containing 0.05%

Tween 20 and 3% BSA for 2 hours. An incubation was carried out for 2 hrs at 37oC

with rabbit anti-human versican (Santa Cruz) antibodies. Reactive moieties were

revealed by an anti-rabbit IgG-HRP and subsequent enhanced chemiluminescence

(Biological Industries).

Assessment of the ability of culture supernatants induced upon incubation of

tumor cells with hCG to induce the secretion of TNF- and IL-6 from normal

cells

Blood was collected from human volunteers in heparinised tubes, appropriately

diluted with complete medium and gently overlaid over an equal volume of Ficoll.

The suspension was then centrifuged at 400 g, at 25°C for 20 min. The "buffy coat" at

the plasma-Ficoll interface (comprising PBMC) was isolated and transferred to a fresh

tube. Cells were "washed" three times with PBS by centrifugation at 1000 g for 10

min. Similar procedures were followed for murine PBMC and spleen cells, except that

RBCs were first lysed by brief incubation in RBC Lysis Buffer (Appendix).

Human and murine PBMC were individually dispensed into wells of 24-well culture

plates. After 2 hr, non-adherent cells were removed. Supernatants from hCG treated or

control tumor cell cultures were added as appropriate (COLO and ChaGo supernatants

to human adherent cells and LLC supernatants to murine adherent cells) and an

incubation carried out for 16-24 hrs. The concentrations of the inflammatory cytokines

IL-6 and TNF-α in supernatants were determined by specific sandwich kits (E-

bioscience) as per the manufacturer’s instructions. Briefly, the 96-well flat bottom

ELISA plates were coated with 100µl/well of capture antibody in coating buffer. The

plates were sealed and incubated for 16 hrs at 4°C. Plates were washed three times

with wash buffer. Wells were then "blocked" by incubation for 1 hr at room

temperature with 200µl/well assay diluent. Wells were then "washed" six times with

wash buffer. Standards or samples (100µl/well, appropriately diluted in assay diluent)

were then dispensed, followed by an incubation at room temperature for 2 hr. After

further "washes", 100µl/well of diluted detection antibody was dispensed. An

incubation was then carried out at room temperature for 1 h. Subsequent to further



"washes", 100µl/well of diluted avidin-HRP conjugate was dispensed, followed by an

incubation at room temperature for 30 minutes. After further "washes", 100µl/well of

substrate solution was added to each well. Reaction was terminated by the addition of

50 µl of stop solution. Optical densities were determined at 450 nm.

Results

Role of hCG in tumor induced immune tolerance and inflammation 82

Anti-inflammatory/immuno-suppressive effects of hCG on tumor

cells

Effect of hCG on the expression of FOXP3

Though FOXP3 is thought to be mainly expressed in Treg cells, recent studies suggest

that tumor cells can also express the transcription factor, an event which may help

create an immuno-suppressive environment, hence assisting tumor growth. RT-PCR

and immunoblot analysis demonstrated up-regulation of FOXP3 mRNA (Figure 20A)

and protein (Figure 20B) levels upon incubation of COLO 205 and LLC cells with

hCG. Anti-hCG antibodies efficiently prevented these effects, whereas control

antibodies did not exhibit inhibitory effects. hCG did not cause significant up-

modulation of FOXP3 levels in ChaGo cells (Figure 20A, B).

FACS analysis confirmed hCG-mediated up modulation of FOXP3 expression in

COLO 205 and LLC cells and the lack of such an effect in ChaGo cells. Anti-hCG

antibodies caused a significant decrease in hCG-induced expression, while normal

serum had no effect (Figure 20C-E).

Effect of hCG on the expression of immunosuppressive cytokines IL-10 and TGF-

β

Since FOXP3 is known to induce the secretion of the immunosuppressive cytokines

IL-10 and TGF-, the levels of these molecules were estimated in culture supernatants

of hCG-stimulated tumor cells. hCG enhanced TGF-β and IL-10 production by COLO

205 and LLC cells. Anti-hCG antibodies (but normal serum, employed as isotype

(control) displayed significant down-modulation of this effect. ChaGo cells did not

exhibit up-regulation of either TGF-β or IL-10 upon hCG treatment, in consonance

with the lack of effect of hCG on FOXP3 expression in these cells (Figures 21, 22).

Given the immunosuppressive properties of IL-10 and TGF-, supernatants from

hCG-stimulated COLO 205 and ChaGo cells were assessed for the ability to suppress

a two-way mixed lymphocyte reaction. Supernatants from stimulated COLO 205 cells

(but not ChaGo cells) were able to significantly reduce cellular proliferation of human

PBMC, as assessed by a reduction in the incorporation of 3H-Thymidine. Antibodies

against IL-10 were able to reverse these inhibitory effects to significant an extent

(Figure 23).

Results


Effect of hCG on the conversion of CD25-cells to CD25+ cells

Supernatants obtained from hCG-treated LLC cells (which contained both IL-10 and

TGF-, as indicated above) induced the conversion of CD4+CD25

-cells to

CD4+CD25

+; the differentiation was specifically inhibited by anti-hCG antiserum but

not by normal serum (Figure 24A-F). The conversion of T cells to a regulatory

phenotype upon the incubation with supernatants obtained from hCG-treated LLCcells

was further confirmed by RT-PCR analysis for FOXP3 (Figure 24G).

hCG-induced CTLA-4 on tumor cells and the generation of

indoleamine 2, 3-dioxygenase (IDO) from bone marrow derived

dendritic cells (BMDC)

FOXP3 is known to induce up-modulation of CTLA-4 levels. This in turn can bind to

CD80/CD86 on dendritic cells to induce the generation of indoleamine 2, 3-

dioxygenase (IDO) from antigen presenting cells. IDO can have immunosuppressive

effects due to its tryptophan-catabolising action. Whether the action of hCG on tumor

cells could initiate this cascade of reactions was investigated.

Flowcytometric analysis revealed that CTLA-4 was up modulated in COLO 205 and

LLC cells (but not in ChaGo cells, in consonance with a lack of up-modulation of

FOX-P3 in these cells upon incubation with hCG, Figure 1) upon treatment with hCG.

Co-incubation of hCG with anti-hCG antibodies negated this effect (Figure 25).

BMDCs, generated from the cells derived from the bone marrow of C57BL/6 mice as

described in the Materials and Methods section, were first assessed for cell surface

expression of CD11c, MHCI, MHCII, CD80 and CD86. Mature BMDCs (obtained

upon additional incubation of GMCSF-derived BMDCs with LPS) demonstrated

higher levels of MHC-II, CD80 and CD86 (Figures 26A and B).

Mature BMDCs were then co-cultured with LLC cells which had been previously pre-

incubated with hCG, which results in the up-modulation of CTLA-4 levels, as

demonstrated above; controls included LLC cells not pre-incubated with hCG. Cell

extracts were probed for the presence of IDO by Western blot. The incubation of

mature BMDCs with hCG-treated LLC cells (but not with control LLC cells) led to

the production of IDO (Figure 27). To further confirm these findings, cell extracts

Results


were assessed for the presence of kynurenine, the IDO-dependent product of

tryptophan catabolism; a significant increase in kynurenine was observed when hCG-

treated LLC cells were co-cultured with BMDCs (Figure 28).

Inflammatory effects of hCG

Versican is an extracellular glycosaminoglycan, heightened levels of which are

significantly associated with enhanced metastasis. Additionally, versican has been

demonstrated to induce an increase in the secretion of IL-6 and TNF- by

macrophages; these cytokines are associated with tumorogenesis, a link that reinforces

the association between inflammation and cancer. hCG was found to induce an

increase in the secretion of versican from COLO 205, ChaGo and LLC cells as

assessed by Western blot analysis; anti-hCG antiserum reversed this effect (Figure

29).

Further, supernatants of tumor cells treated with hCG were specifically able to induce

the enhanced secretion of TNF- and IL-6 from adherent cells isolated from PBMCs;

the increased secretion of cytokines was effectively neutralized by anti-hCG

antiserum. Supernatants from either tumor cells or adherent cells treated with hCG did

not contain enhanced levels of the cytokines, indicating collaboration between

transformed and non-transformed cells in this phenomenon (Figures 30, 31).

Figure 20: (A) RT-PCR and (B) Western blot analysis depicting hCG-mediated induction of FOXP3 mRNA andprotein levels respectively in COLO 205, ChaGO and LLC cells. (C-E) Flow cytometric analysis depictingexpression of FOXP3 expression in permeabilized (C) COLO 205 (D) ChaGo and (E) LLC cells upon incubationwith hCG. The influence of anti-hCG antiserum and normal serum (NS) on hCG-induced effects is also shown.

- + + + - + +

- - + - - - +

- - - +

hCGAnti-hCGNS

A B

COLO 205

ChaGo

LLC

C D EControl

hCG

hCG + anti-hCG

hCG + NS

NS

Figure 21: The effect of hCG on the production of TGF- from (A) COLO 205, (B) ChaGO and (C) LLCcells. The influence of anti-hCG antiserum and normal serum (NS) on hCG-induced effects is alsoshown.

0

500

1000

1500

2000

2500

3000

0

500

1000

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2000

2500

3000

0

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1500

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3000

Control

hCG

hCG + Anti-hCG

hCG + NS

A B

C

pg

/ml

pg

/ml

pg

/ml

Figure 22: The effect of hCG on the production of IL-10 from (A) COLO 205, (B) ChaGO and (C) LLCcells. The influence of anti-hCG antiserum and normal serum (NS) on hCG-induced effects is alsoshown.

0

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1200

Control

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hCG + Anti-hCG

hCG + NS

A B

C

pg

/ml

pg

/ml

pg

/ml

0

100

200

300

400

500

Figure 23: Human two-way mixed lymphocyte reaction (MLR). The effects of hCG as well as ofsupernatants derived from control and hCG-stimulated (A) COLO 205 and (B) ChaGo cells wereassessed on proliferative responses. Also depicted is the influence of anti-IL-10 antibodies.

3H

-Th

ym

idin

e i

nc

orp

ora

tio

n

0

3000

6000

9000

12000

15000

18000

0

3000

6000

9000

12000

15000

18000

- - + + - - - -

- - - - - - + +

- - - - + + - -

- + - + - + - +3H

-Th

ym

idin

e i

nc

orp

ora

tio

n

ChaGO Sup.

hCG

ChaGO Sup. (hCG stim.) Anti-IL-10 Ab.

- - + + - - - -

- - - - - - + +

- - - - + + - -

- + - + - + - +

COLO205 Sup.

hCG

COLO205 Sup.(hCG stim.) Anti-IL-10 Ab.

A B

Figure 24: (A-F) Conversion of CD4+CD25- T cells into CD4+CD25+ T cells by hCG-treated tumor cells. CD25- cellswere incubated with (A) medium; (B) hCG; (C) supernatant from LLC cells treated with hCG; (D) supernatant fromLLC cells; (E) supernatant from LLC cells treated with hCG + anti-hCG antisera; (F) supernatant from LLC cellstreated with hCG + normal sera. (G) RT-PCR analysis depicting the induction of FOXP3 expression in CD4+CD25- Tcells upon incubated with (Lane 1) hCG; (Lane 2) supernatant from LLC cells treated with hCG; (Lane 3) supernatantfrom LLC cells; (Lane 4) supernatant from LLC cells treated with hCG + anti-hCG antisera; (Lane 5) supernatant fromLLC cells treated with hCG + normal sera. M: Molecular weight in base pairs.

CD25

CD

4

A B C

D E F

1 2 3 4 5 MG

300

200

100

Figure 25: Flow cytometric analysis depicting the expression of CTLA-4 in (A, D) LLC, (B, E) COLO 205and (C, F) ChaGo cells. Cells were either permeabilized (A-C) or not (D-F) before the assay. Blackprofiles: Control cells; Red profiles: Cells + hCG; Green profiles: Cells + hCG + anti-hCG antiserum.

COLO 205LLC ChaGo

A B C

D E F

Figure 26: Phenotypic characterization of (A) immature and (B) mature bone marrow deriveddendritic cells (BMDCs) from C57BL/6 mice.

A

B

CD

11c

CD

11c

CD

11c

CD

11c

CD

11c

CD

11c

CD

11c

CD

11c

MHC-I

MHC-I

MHC-II

MHC-II

CD80

CD80

CD86

CD86

1 2 3 4

5250

130

72

95

28

36

17

11

55

Figure 27: Western blot for IDO in cell lysates. Lane 1: BMDCs; Lane 2: LLC cells; Lane 3: LLC cells+ BMDC co-culture; Lane 4: hCG-treated LLC cells + BMDC co-culture; Lane 5: Negative control(secondary antibody). Molecular weights are indicated in KDa.

Figure 28: Estimation of kynurenine levels in cellular extracts. 1: BMDCs; 2: hCG-treatedBMDCs; 3: LLC cells; 4: hCG-treated LLC cells; 5: BMDCs + LLC cells co-culture; 6: hCG-treated LLCcells + BMDC co-culture. p = .001 LLC cells + BMDC co-culture

1 2 3 4 5 6

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

* *

Ab

so

rban

ce (

280 n

m)

COLO 205

ChaGo

LLC

hCG

Anti-hCG

- + +

- - +

Figure 29: Western blot analysis of the effects of hCG on the secretion of Versican from COLO 205,ChaGO and LLC cells. The influence of anti-hCG antibodies on hCG-induced effects is also shown.

Figure 30: Secretion of TNF-α under the conditions indicated, employing (A) COLO 205, (B) ChaGoand (C) LLC cells. PBAC: peripheral blood adherent cells from human volunteers (A, B) or mice (C).

0

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Serum-free medium

Tumor cell supernatant

PBAC supernatant

hCG-treated tumor cell supernatant

hCG-treated PBAC supernatant

Tumor cell supernatant on PBAC

hCG-treated tumor cell supernatant on PBAC

hCG + anti-hCG antiserum-treated tumor cell supernatant

on PBAC

A B

C

pg

/ml

pg

/ml

pg

/ml

Figure 31: Secretion of IL-6 under the conditions indicated, employing (A) COLO 205, (B) ChaGo and (C) LLC cells. PBAC: peripheral blood adherent cells from human volunteers (A, B) or mice (C).

pg

/ml

0

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2500Serum-free medium

Tumor cell supernatant

PBAC supernatant

hCG-treated tumor cell supernatant

hCG-treated PBAC supernatant

Tumor cell supernatant on PBAC

hCG-treated tumor cell supernatant on PBAC

hCG + anti-hCG antiserum-treated tumor cell supernatant

on PBAC

A B

C

pg

/ml

pg

/ml

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