CD4+ T cells elicit host immune responses to MHC class II-negative ovarian cancer through CCL5 secretion and CD40-mediated licensing of dendritic cells

Abstract

T cell adoptive transfer strategies that have produced clinical remissions against specific tumors have so far produced disappointing results against ovarian cancer. Recent evidence suggests that adoptively transferred CD4(+) T cells can trigger endogenous immune responses in particular patients with ovarian cancer through unknown mechanisms. However, conflicting reports suggest that ovarian cancer-infiltrating CD4(+) T cells are associated with negative outcomes. In this study, we elucidate the phenotypic attributes that enable polyclonal CD4(+) T cells briefly primed against tumor Ags to induce therapeutically relevant endogenous antitumor immune responses. Our results unveil a therapeutic mechanism whereby tumor-primed CD4(+) T cells transferred into ovarian cancer-bearing mice secrete high levels of CCL5, which recruits endogenous CCR5(+) dendritic cells to tumor locations and activate them through CD40-CD40L interactions. These newly matured dendritic cells are then able to prime tumor-specific endogenous CD8(+) T cells, which mediate long-term protection. Correspondingly, administration of tumor-primed CD4(+) T cells significantly delayed progression of MHC class II(-) ovarian cancers, similarly to CD8(+) T cells only, and directly activated wild-type but not CD40-deficient dendritic cells recruited to the tumor microenvironment. Our results unveil a CCL5- and CD40L-dependent mechanism of transferring immunity from exogenously activated CD4(+) T cells to tumor-exposed host cells, resulting in sustained antitumor effects. Our data provide a mechanistic rationale for incorporating tumor-reactive CD4(+) T cells in adoptive cell transfer immunotherapies against ovarian cancer and underscore the importance of optimizing immunotherapeutic strategies for the specific microenvironment of individual tumors.

Figure 1. CCL5 is required within transferred T cells for effective adoptive immunotherapy

A. Survival of ID8-Defb29/Vegf-a-tumor bearing mice treated with wild-type (wt) T cells, CCL5^−/− T cells or left untreated. B. Survival of ID8-Defb29/Vegf-a-tumor bearing mice treated with wild-type (wt) T cells or CCL5^−/− T cells combined with depletion of immunosuppressive DCs with an anti-CD11c immunotoxin (IT). C. Quantification of MHCII⁺CD80⁺CD11c⁺ cells in the peritoneum of mice treated with wild-type (wt) T cells, CCL5^−/− T cells or left untreated. Mice were depleted of immunosuppressive dendritic cells with an anti-CD11c immunotoxin prior to T cell transfer. D. Survival of ID8-Defb29/Vegf-a challenged wild-type or CCL5-deficient mice (host) treated with wild-type (wt) anti-tumor T cells or left untreated (PBS). (n=6 mice per group in three independent experiments). (* means P<0.05; **means P<0.01).

Figure 2. Adoptive (T) Cell Therapy (ACT) induces sustained host immunity that is protective against secondary tumor challenges and is dependent upon the presence of CCL5 in transferred T cells

Mice bearing i.p. ID8-Defb29/Vegf-a tumors were depleted of immunosuppressive dendritic cells with an anti-CD11c immunotoxin (IT) then treated with expanded T cells from wild-type or CCL5-deficient mice on days 7 and 14 of tumor progression. After 28 days of tumor progression host CD3⁺ were sorted from treated mice and transferred into naïve mice that received s.c. ID8-Defb29/Vegf-a tumors 2 days later. Tumor growth of s.c. ID8-Defb29/Vegf-a tumors in mice receiving host CD3⁺ T cells from mice treated with wildtype T cells, CCL5^−/− T cells or left untreated. (n=4 mice per group in three independent experiments).

Figure 3. CD4⁺ T cells are pertinent for the effects of adoptive T cell therapy in enhancing the survival of mice bearing ovarian tumors

IT means anti-CD11c immunotoxin, CD4 represents mice receiving CD4⁺ T cell transfer, CD8 represents mice receiving CD8⁺ T cell transfer and CD3 represents mice receiving a mixed population (3 CD8⁺: 2 CD4⁺) of CD3⁺ T cells. A. Mice bearing 7-day-old ID8-Defb29/Vegf-a tumors were treated on day 7 and 14 of tumor progression with either a mixed population of CD3⁺ T cells or pure populations of CD4⁺ or CD8⁺ T cells combined with the depletion of CD11c⁺ cells. Mice were monitored for survival. B. Secretion of CCL5 as determined by ELISA, for a mixed population of CD3⁺ T cells and individual pure cultures of CD4⁺ and CD8⁺ T cells immediately after expansion. C. Total numbers of host CD80⁺MHCII⁺CD11c⁺ cells accumulated at the tumor site 3 days after treatment. D. Survival of mice bearing ID8-Defb29/Vegf-a treated with wildtype or CCL-5^−/− deficient CD4+ T cells along with depletion of CD11c⁺ cells. E. Total numbers of host CD80⁺MHCII⁺CD11c⁺ cells accumulated at the tumor site 3 days after treatment in D. (n=6 mice per group in four independent experiments). (* means P<0.05; **means P<0.01).

Figure 4. CCL5 specifically produced by transferred tumor-reactive CD4⁺ T cells is required for the elicitation of therapeutic immunity in multiple models of peritoneal carcinomatosis

A. Survival of advanced Lewis lung carcinoma-bearing mice treated with wild-type (wt) T cells (day 7) and regulatory CD11c⁺ cell depletion from tumor locations (day 6, day 8 and day 10), or left untreated. B. Quantification of CCL5 produced by C57BL/6 T cells directly after their in vitro expansion against ID8-Defb29/Vegf-a (BL/6 v ID8) tumor cells or Lewis lung carcinoma cells (BL/6 v LLC) and FVB T cells against MT2 tumor cells (FVB v MT2). C. Survival of advanced Lewis lung carcinoma -bearing mice treated with wildtype (wt) CD4⁺ T cells or CCL5^−/− CD4⁺ T cells combined with depletion of immunosuppressive DCs with an anti-CD11c immunotoxin (IT). D. CD80⁺CD11c⁺ and E. CD70⁺CD11c⁺ cells in the peritoneum of MT2-tumor bearing mice treated with wild-type (wt) T cells (day 7) and DC elimination (day 6, 8, 10, 13 and 15) or left untreated. (n=6 mice per group in three independent experiments). (* means P<0.05; **means P<0.01).

Figure 5. CD40-CD40L interactions are required for the therapeutic effects and induction of host immunity elicited by ACT

A. Expression of CD40L on T cells primed against tumor antigens for 7 days. Shaded gray histogram represents Isotype control; unshaded histogram represents CD40L staining. B. Quantification of MHCII⁺CD80⁺CD11c⁺ cells in the peritoneum of mice treated with wild-type (wt) T cells, or CD40L^−/− T cells. C. Survival of ID8-Defb29/Vegf-a tumor-bearing mice treated with wild-type (wt) T cells, CD40L^−/− T cells, combined with the elimination of regulatory DCs (IT), or left untreated. D. Concentration of CCL5 secreted by wild-type (wt) or CD40L^−/− T cells upon 7-day expansion, as determined by ELISA. (n=6 mice per group in four independent experiments). E. T cells expanded from wild-type (wt) or CD40L-deficient T cells show comparable proportions of CD8 vs CD4 T cells by flow cytometry. F. Activation status of T cells expanded from wild-type (wt) or CD40L-deficient T cells. G. IFN-γ ELISPOT analysis of wild-type (wt) or CD40L-deficient T cells primed against ID8-Defb29/Vegf-a tumor antigens. H. Mice bearing i.p. ID8-Defb29/Vegf-a tumors were depleted of immunosuppressive dendritic cells with an anti-CD11c immunotoxin (IT) then treated with expanded T cells from wild-type or CD40L-deficient mice on days 7 and 14 of tumor progression. After 28 days of tumor progression host CD3⁺ were sorted from treated mice and transferred into naïve mice that received s.c. ID8-Defb29/Vegf-a tumors 2 days later. Mice receiving host CD3⁺ T cells from mice treated with wild-type T cells or CD40L^−/− T cells were challenged with s.c. ID8-Defb29/Vegf-a tumors. Tumor growth was monitored over time. (n=3/4 mice per group in three independent experiments) (* means P<0.05; **means P<0.01).

Figure 6. T cells expanded to tumor antigens induce maturation of tumor-associated DCs

Expression profile of MHCII, CD80, CD70 and CD86 on wild-type or CD40^−/− dendritic cells obtained from tumor-bearing mice and subsequently cultured with tumor-primed wild-type or CD40L^−/− T cells as specified. (n=4 per group in three independent experiments). (* means P<0.05; **means P<0.01).

Figure 7. Wild-type T cells interact with CD40 on wild-type tumor associated DCs to induce their production of IL-12 and license them to prime antitumor CD8⁺ T cells

T cells from wild-type or CD40L-deficient T cells were activated for 7 days against tumor antigen. Wild-type or CD40-deficient DCs sorted from mice bearing ID8-Defb29/Vegf-a tumors for 7 days were co-cultured for 24 hours with activated wild-type or CD40L-deficient T cells and the supernatants thus obtained were tested in ELISA for IL-12 production. A. IL-12 secretion by tumor-associated wild-type (wt) or CD40^−/− DCs that were cultured with tumor-primed wild-type (wt) or CD40L^−/− T cells. B. T cells from wild-type (wt) or CD40L-deficient T cells were activated for 7 days against tumor antigen. Wild-type (wt) or CD40-deficient DCs sorted from tumor-bearing mice were co-cultured for 24 hours with activated wild-type (wt) or CD40L-deficient T cells then admixed with tumor-infiltrating CD8⁺ T cells for 48hours in ELISPOT analyses. C. ELISPOT analyses of the number of CD8⁺ T cells secreting IFN-γ after culture with dendritic cells primed as indicated. C. ELISPOT analyses of the number of CD8⁺ T cells secreting Granzyme-B after culture with dendritic cells primed as indicated. (n=4 per group in four independent experiments). (* means P<0.05; **means P<0.01).