Regulatory T cells (Treg) restrain immune responses against malignant tumors, but their global depletion in cancer patients will likely be limited by systemic autoimmune toxicity. Instead, approaches to "tune" their activities may allow for preferential targeting of tumor-reactive Treg. Although Ag recognition regulates Treg function, the roles of individual TCR-dependent signaling pathways in enabling Treg to promote tumor tolerance are not well characterized. In this study, we examined in mouse tumor models the role of calcineurin, a key mediator of TCR signaling, and the role of the costimulatory receptor CD28 in the differentiation of resting central Treg into effector Treg endowed with tumor tropism. We find that calcineurin, although largely dispensable for suppressive activity in vitro, is essential for upregulation of ICOS and CTLA-4 in Treg, as well as for expression of chemokine receptors driving their accumulation in tumors. In contrast, CD28 is not critical, but optimizes the formation of tumor-homing Treg and their fitness in tumor tissue. Accordingly, although deletion of either CnB or CD28 strongly impairs Treg-mediated tumor tolerance, lack of CnB has an even more pronounced impact than lack of CD28. Hence, our studies reveal distinct roles for what has classically been defined as signal 1 and signal 2 of conventional T cell activation in the context of Treg-mediated tumor tolerance.

During persistent antigen stimulation, CD8(+) T cells show a gradual decrease in effector function, referred to as exhaustion, which impairs responses in the setting of tumors and infections. Here we demonstrate that the transcription factor NFAT controls the program of T cell exhaustion. When expressed in cells, an engineered form of NFAT1 unable to interact with AP-1 transcription factors diminished T cell receptor (TCR) signaling, increased the expression of inhibitory cell surface receptors, and interfered with the ability of CD8(+) T cells to protect against Listeria infection and attenuate tumor growth in vivo. We defined the genomic regions occupied by endogenous and engineered NFAT1 in primary CD8(+) T cells and showed that genes directly induced by the engineered NFAT1 overlapped with genes expressed in exhausted CD8(+) T cells in vivo. Our data show that NFAT promotes T cell anergy and exhaustion by binding at sites that do not require cooperation with AP-1.