Treatment of Established B16 Murine Melanoma Tumors with Tyrp-1 Specific CD4⁺ Lymphocytes.

T-cell mediated response against solid tumors has been mostly associated with CD8⁺ cytotoxic lymphocytes, which act directly on the MHC class I expressing tumors. In the previously published model, gp-100 melanoma antigen-specific pmel-1 CD8⁺ T cells required co-administration of IL-2 and vaccine to induce significant regression of poorly immunogenic B16 melanoma in mice. MHC class II restricted CD4⁺ T-cells (T helpers) may have multiple direct and indirect effects on the immune response, but their role in adoptive cell transfer (ACT) therapy of solid tumors remains mainly undefined and based on highly manipulated models involving foreign antigens. In order to investigate the function of tumor specific CD4⁺ T-cells we have generated a transgenic mouse expressing a TRP-1 T cell receptor (TCR) directed against class II restricted murine melanocyte differentiation antigen tyrp-1. In vitro expanded TRP1 CD4⁺ cells secreted Th1-like cytokines upon antigen stimulation and caused direct cytotoxic effect against B16 melanoma. In vivo they mediated a highly effective response against large (>1cm²) B16 melanoma tumors after ACT of as few as 2.5×10⁵ cells/mouse into C57B6 animals, which was associated with a massive tumor infiltration with CD11b⁺, MAC3⁺, GR1⁺ cells. TRP-1 T cells caused partial tumor rejection and prolonged survival in MHC class II^−/− hosts implying the ability to directly recognize low level MHC class II on the tumor. This suboptimal effect was significantly enhanced after co-transfer of MHC class II⁺ APCs into MHC class II^−/− hosts allowing for antigen cross- presentation.

Interestingly, Rag1^−/− hosts, deficient in all T and B lymphocytes, demonstrated excellent initial response to treatment, but were not cured and succumbed to late relapse of the melanoma. Long-term responses were even more impaired in Rag1^−/− γc^−/− hosts, while complete and durable cure was observed in TCRα^−/−, CD4^−/− and C57B6 mice, suggesting involvement of other arms of the adaptive immune system. Similarly, co-transfer of 0.1×10⁶ CD4⁺ TRP-1 cells and 1×10⁶ CD8⁺ pmel-1 cells resulted in effective tumor regression, while the same numbers of each cells transferred individually were not sufficient to initiate a rejection. Introduction of tumor-specific CD4⁺ cells therefore eliminates the previously sine qua non need for co-administration of vaccine and IL-2 for effective treatment with CD8⁺ pmel-1 cells. Overall, we show that antigen-specific CD4⁺ T cells are highly effective in mediating the anti-tumor response by causing both the direct anti-tumor effect and by activating innate and adaptive arms of the immune system. These findings suggest that CD4⁺ T helper cells may play a key role in improving efficacy of ACT immunotherapy as central activators of the anti-tumor response.