Tregulatory cell-mediated suppression of tumor elimination by NK cells and CD4 T cells

Cancer immunotherapies have revolutionized the treatment of cancer but immunoediting of tumor cells sculpts tumor cells that become resistant to CD8 T cell responses by selecting for the loss of MHC I antigen presentation. Here we will elucidate how the local ablation of intra-tumoral regulatory T cells (IT-Tregs) leads to the mobilization of NK and CD4 T cells that control CD8 T cell-escaped tumors by investigating the underlying mechanisms that mediate tumor control and then testing the translational potential of using the discovered mechanisms therapeutically. Preliminary data shows that IT-Treg ablation, using tumor-localized Diphtheria Toxin administration in Foxp3DTR mice, can substantially control MHC I-deficient tumors in a manner that is independent of CD8 T cells. Instead, NK and CD4 T cells mediated tumor control. Induction of the NK response was dependent on CD4 T cells and associated with an increased STAT5 signaling signature in NK cells, indicating that CD4 T cells may help NK cell antitumor function by increasing IL-2 or IL-15 signaling in NK cells. Furthermore, CD4 T cell tumor control was partially independent of NK cells and was associated with an increased expression of granzymes and NK-activating receptors on CD4 T cells. Notably, elimination of tumor cells by CD4 T cells did not require MHC II or MHC I expression by tumor cells, suggesting an alternative mode of T cell recognition of tumor cells, potentially via NK receptors. We propose to address these gaps in our understanding, as well as test whether IT-Treg-depletion, cytokine-based therapeutics, or combinations of both approaches can yield similar activities against evolving primary and metastatic cancers with mixed MHC I- deficiency. In Aim 1 we will determine the mechanism(s) of CD4 T cell help of antitumor NK cell response, focused on the role of IL-2 and IL-15 cytokines and dendritic cells in supporting antitumor NK cell mobilization. In Aim 2, we will determine the mechanism(s) of NK-independent, CD4 T cell tumor control by first addressing whether CD4 T cells can directly kill tumor cells. We will then define the mode of killing and the mechanisms of recognition of MHC I- and MHC II-deficient tumor cells, using genetic deficiencies and antibody blockade experiments. Alternatively, we will test whether CD4 T cells control tumors indirectly by activating macrophages or antibody responses. In Aim 3, we will test the hypothesis that clinically translatable cytokine delivery and Treg-depleting approaches enhance the activity of NK and CD4 T cells against primary and metastatic tumors. Importantly, we will test combinations with checkpoint blockade immunotherapies, which select for MHC I-deficiency in tumor cells, in the setting of mixed MHC I+/- heterogenous tumors. This will test the translational potential of the discoveries made in Aims 1 and 2 using clinically applicable approaches.