Quezada et al. J. Exp. Med. Vol. 205 No.9 2125-2138

Presenters:Denise RushSzymon 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 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