Quezada et al. J. Exp. Med. Vol. 205 No.9 2125-2138 Presenters: Denise Rush Szymon Rus

1. Quezada et al. J. Exp. Med. Vol. 205 No.9 2125-2138 Presenters: Denise Rush Szymon Rus Harleen Saini Limited tumor infiltration by activated T effector cells restricts the therapeutic activity of regulatory T cell depletion against established melanoma

2. Background • Mechanisms for Cancer Immunotherapy • Stimulation of the immune system • Inhibition of the immunological inhibitors • Increased immunogenecity of tumor cells • Stimulating bone marrow (G-CSF)

3. Theme of the paper • 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

4. Regulatory T Cells • 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

5. Establishing T reg depletion • CD25+ mediated T reg depletion • ONTAK • 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

6. Problems with T reg depletion • 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

7. Combinatorial Immunotherapy • Gvax: GM-CSF-secreting cellular vaccine • CTLA-4 inhibition • Prophylactic versus therapeutic CD25 directed depletion of T reg cells

8. Figure 1 • 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 ).

9. Figure 2 • 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

10. Figure 3 • 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.

11. Figure 4 • 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.

12. Figure 5 • 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.

13. Figure 6: Are tumor infiltration & Effector/T Reg ratio dependent on timing of anti-CD25 administration? • 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

14. Figure 6 (cont): Is tumor vasculature different from regular vasculature? • 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

15. Figure 7: What are the effects of Prophylactic CD25 depletion in the absence of Gvax/αCTLA-4? • 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

16. Figure 8: Since therapeutic CD25 depletion results in activation of effector cells, can it induce tumor rejection in vivo? • 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

17. Figure 8 (cont.) • 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

18. Conclusions • 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

19. Conclusions (cont.) • 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