quezada et al. j. exp. med. vol. 205 no.9 2125-2138 presenters: denise rush szymon rus harleen saini
TRANSCRIPT
Mechanisms for Cancer Immunotherapy Stimulation of the immune system Inhibition of the immunological inhibitors Increased immunogenecity of tumor
cells Stimulating bone marrow (G-CSF)
Improved Cancer Immunotherapy Understanding the effect of T reg cell
depletion on anti-tumor immune responses
Establishing synergy between T reg cell depletion and immunostimulation for effective tumor rejection
Targeting the inhibiting immunotherapy checkpoints▪ Blocking CTLA4▪ CD4+ CD25+ T reg cell depletion
T reg cells or regulatory T cells are CD4+ CD25+ Foxp3+
T reg cell depletion leading to enhanced T-cell response
Studying the prophylactic and therapeutic effect of T reg cell depletion
CD25+ mediated T reg depletionONTAK
▪ recombinant IL-2 fused to diphtheria toxin (DT)▪ IL-2 internalized by IL2 receptor bearing cells▪ Diphtheria toxin leading to apoptosis
Foxp3 directed T reg cell depletion▪ Foxp3-DTR transgenic mice
Anti-CD25 antibodies depleting CD25+ effector T cells
Inefficient depletion of intra-tumoral CD25+ T reg cells
Persistence of CD25-/low Foxp3+ T cells
Conversion of CD4+ Foxp3- to CD4+
Foxp3+ cells
Gvax: GM-CSF-secreting cellular vaccine
CTLA-4 inhibitionProphylactic versus therapeutic
CD25 directed depletion of T reg cells
Mice were injected with anti-CD25 mAb 4 d before (prophylactic) or after (therapeutic) tumor establishment and then treated with Gvax/ αCTLA-4 on days 8, 11, and 14( Fig. 1 A ).
Efficient depletion of CD4 + CD25 + T reg cells has occurred by 4 d after mAb injection ( Fig. 1 B ).
Tumor growth was monitored over time for mice treated with Gvax/ αCTLA-4 (black squares), anti-CD25 d-4 and Gvax/ αCTLA-4 (blue triangles), and anti-CD25 d+4 plus Gvax/ αCTLA-4 (inverted red triangles).
Although prophylactic CD25 depletion and Gvax/ αCTLA-4 synergized to reject established tumors, therapeutic CD25 depletion had no impact on tumor growth and rejection ( Fig. 1 C ).
Prophylactic or therapeutic CD25 depletion induced a significant reduction in the percentage of CD4 + Foxp3 + cells independently of Gvax or Gvax/ αCTLA-4 (Fig. 2 A ).
Gvax and Gvax/ αCTLA-4 resulted in an increase in the absolute number of T reg cells over that of nonvaccinated mice (Fig. 2 B ).
Analyses for expression of the proliferation marker KI-67: Gvax or Gvax/ αCTLA-4 induced a
relatively modest increase in the percentage of KI-67 + CD4 + Foxp3 + cells.
anti-CD25 resulted in a significant increase in the KI-67 + population.
greatest increase was induced by the combination of anti-CD25 and Gvax or Gvax/ αCTLA-4 (Fig. 2 C ).
Therapeutic intervention with Gvax or Gvax/ αCTLA-4 (after tumor implantation) induces accumulation of T reg cells, from surviving T reg cell populations that enter the cell cycle
Analysis of systemic anti-B16/BL6 melanoma responses by assessing T cell proliferation and cytokine production 14 d after tumor challenge.
Gvax/αCTLA-4 treatment caused increased KI-67 expression in all compartments, with the biggest increase (more thanthreefold) in CD4 + Foxp3- T cells ( Fig. 3 A ).
To address issues of specificity or functionality of the proliferating cells, melanoma TCR transgenic CD8 + (pmel) T cells were transferred into mice.
Upon tumor challenge, an increase in KI-67 expression was observed in tumor-reactive pmel cells ( Fig. 3 B ).
CD8 + and CD4 + T cells were purified and tested for IFN-γ and IL-2 production in response to the melanoma cell line. Gvax/αCTLA-4 caused a small but significant increase in
IFN-γ production by both CD8 + and CD4 + T cells (Fig. 3, C and D ).
Prophylactic CD25 depletion further increased IFN-γ secretion (Fig. 3, C and D ).
Therapeutic CD25-depletion caused an additional significant increase (Fig. 3 C ).
A similar trend was observed for IL-2.
Prophylactic and therapeutic CD25 depletion does not result in elimination of effector T cells, but promotes strong systemic T cell responses against B16/BL6 melanoma.
Foxp3-DTR transgenic mice were used as tumor recipients( Fig. 4 A )
In contrast to CD25-directed depletion, this approach depletes CD25-/low Foxp3 + T cells upon DT injection ( Fig. 4 B )
Foxp3 + -directed depletion before challenge with B16/BL6 melanoma resulted in efficient tumor rejection, whereas late depletion failed to synergize with Gvax/αCTLA-4 ( Fig. 4 C ).
Therapeutic Foxp3-directed T reg cell depletion failed to synergize with Gvax/αCTLA-4 in rejection of established tumors.
Failure does not result from the effects of a pool of CD25 - /low Foxp3 + cells escaping CD25-directed depletion.
Intratumor responses 14 d after tumor challenge, and evaluation of expression of KI-67 by the effector T cell (CD4 + Foxp3- and CD8 + Tcells) and T reg cell (CD4 + Foxp3 +) compartments.
More than 70% of CD4 + Foxp3 + T reg cells expressed KI-67 in untreated compartments.
CD25 depletion drove mainly CD8 + T cells into the cell cycle, whereas Gvax/αCTLA-4 without CD25 depletion induced mainly CD4 + Foxp3- T cells to proliferate.
Evidence for independent contributions of Gvax/αCTLA-4 and CD25 depletion to the expansion of the intratumor effector T cell compartment.
BL16/BL6 mice treated with anti-CD25 (-4 d or +4 d) plus Gvax/αCTLA-4
Untreated mice showed minimal T cell infiltration
Prophylactic CD25 depletion resulted in T cell infiltration & increase of Effector/T-reg ratio
Therapeutic CD25 depletion failed to increase number of effectors or switch ratio
Similar results obtained after tumor-specific pmels transferred into mice before treatment
Only Prophylactic CD25 depletion resulted in co-expression of VCAM, ICAM and CD31
Activation of tumor vasculature not linked directly to T reg depletion Analyses of rejecting tumors reveals increased expression of ICAM & VCAM Expression correlates with infiltration & tumor rejection even in absence of T reg depletion
Without Gvax/αCTLA-4, prophylactic depletion resulted in small CD8+ infiltrate & increase in effector/regulator ratio
Therapeutic depletion did not result in CD8+ infiltrate or change in ratio
Changing the ratio of effectors/regulators must allow the combination of vaccination strategy to be effective
Irradiation induced a decrease in # of CD8+ and Foxp3+ cells
DLI resulted in recovery of effector/regulator ratio
Donor CD25 depletion increased effector function (IFN-γ production by CD8+ T cells) in response to B16/BL6
ICAM & VCAM only observed upon irradiation and T cell transfer Infiltrating T cells important
factor to increase vasculature activation enhanced T cell infiltration and rejection
DLI from non-depletd mice resulted in delayed tumor growth & increased survival
Maximal effects from DLI from CD25-depleted donors Correlated with enhanced activity & frequency seen with therapeutic
depletion
Tumor rejection not seen in mice lacking conditioning of the recipients, DLI or Gvax/αCTLA-4 vaccinations for recipients Recipient vaccination after DLI needed to further increase T cell numbers
and reactivity against tumor
Therapeutic CD25-directed T reg depletion controls systemic accumulation of T regs & facilitates activation of systemic and intratumoral cells BUT few cells can access tumor due to abnormal
vasculature and poor ICAM/VCAM expression Restricted infiltration results in low effector/T reg ratio
and inability to induce tumor rejection
Prophylactic T reg depletion allows infiltration of effectors into tumor that synergize with Gvax/αCTLA-4 to increase effector/T reg ratio and induce tumor rejection
Prophylactic vs. Therapeutic Model Therapeutic depletion allows tumor time to generate less
permissive microenvironment for infiltration Prophylactic depletion allows T cells to infiltrate and
contribute from within tumor to enhance vaccination effects
Tumor reactive lymphocytes can be transferred into tumor-bearing hosts (after conditioning) for efficient activation of tumor vascularization, T cell infiltration and tumor rejection Applications for treating melanoma and other cancers