Quezada et al. J. Exp. Med. Vol. 205 No Presenters: Denise Rush Szymon Rus Harleen Saini

 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 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

 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 inhibition  Prophylactic 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