PROJECT SUMMARY The past twenty years have seen a sea change in the treatment of many types of cancer, with immunotherapy- based approaches, including checkpoint blockade and adoptive cell therapy, yielding remarkable results in some patients. The inability, thus far, to achieve more complete responses in more patients has set off a widespread effort to identify novel targets for improving rates and duration of response, either as single agents or together with first-generation immunotherapies, like PD-1/PD-L1 blockade. One of the second-generation checkpoint targets that has attracted attention from many groups, including our own, is the protein Tim-3. Thus far, mAb’s targeting Tim-3 have under-performed in clinical trials for solid tumors, likely due in part to the fact that while Tim-3 is expressed at high levels on exhausted T cells, it is not expressed on the TpEx cells. Obtaining a better understanding of Tim-3 function in these various cell types may lead to more selective and efficacious Tim3- targeting therapies, either as single agents or, critically, in combination with PD-1 pathway blockade. Work from our groups and others have revealed that Tim-3 appears to be particularly important for the suppressive function of regulatory T cells (Treg) that are present in high numbers in tumors, in particular “effector” Treg (eTreg), which are a particularly suppressive subset of Treg. These cells are enriched within solid tumors, relative to their proportions in normal peripheral tissues, suggesting that they could be attractive targets for more specific augmentation of immune responses within tumors. Since a significant proportion of Treg express Tim- 3, and the function of this molecule in vivo is still being elucidated, we generated a knockout model to study Tim- 3 on these cells. Thus, we found that inducible Treg-specific Tim-3 deletion results in a dramatic decrease in both the growth of syngeneic tumors and the number of Treg infiltrating those tumors. However, Treg-specific Tim-3 deletion did not detectably impact Treg development or immune tolerance under homeostatic conditions. Based on published and preliminary data, we hypothesize that Tim-3 is a critical regulator of effector Treg in the tumor microenvironment. We will test this hypothesis with three Specific Aims. In Aim 1, we will determine why there are fewer eTreg in the tumors of mice with Treg-specific Tim-3 KO. In Aim 2, we will define the effects of loss of Treg Tim-3 on the tumor microenvironment. Finally, in Aim 3, we will determine how the loss of Tim-3 on Treg impacts the response to PD-1 checkpoint blockade, in both mouse models, with an extension toward the role of the interaction in the response to PD-1 blockade therapy in patients with head and neck cancer. Together, these studies will provide the basis for more rational translation of Tim-3 as a target in the treatment of solid tumors, in the context of existing checkpoint targets like PD-1. As such, these studies...