PROJECT SUMMARY CD8 T cell responses to viral infections and tumors contribute significantly to the immune responses that dictate the clinical outcomes of such pathologies. The functional integrity of CD8 T cell responses depends on the characteristic properties of CD8 effector (Teff) and memory (Tmem) populations. However, during chronic viral infections and cancer, antigen persistence without clearance precludes effective Teff and Tmem development, instead biasing CD8 T cell differentiation towards an epigenetically distinct “exhausted” lineage (Tex). Tex exhibit progressive dysfunction and loss of effector properties, proliferation capacity, and memory potential, as well as a sustained increase in co-expression of PD1 and multiple other inhibitory receptors (IRs). Interrogating the fundamental mechanisms that initiate and maintain the Tex epigenetic state is of central importance to understanding Tex biology and identifying strategies to selectively target or modulate Tex. However, the field generally lacks a detailed mechanistic understanding of Tex-specific epigenetic processes. In models of exhaustion during chronic infection and of dysfunctional tumor-specific T cells, the transcription factor TOX is essential for the initiation of Tex development, repressing terminal Teff differentiation and potentiating epigenetic commitment to the Tex lineage. This proposal seeks to identify and interrogate the mechanistic details of Tex regulation by TOX that would be required to begin developing immunotherapy approaches to epigenetically reprogram Tex and improve immunotherapy clinical outcomes. The molecular transactions TOX employs to exert its effects remain largely unknown. Understanding the details of TOX activity remains limited by a lack of functional characterization of its N- and C-terminal domains (“NTD” and “CTD”) in relation to its HMG-box DNA binding domain. My preliminary data demonstrate in vitro that loss of either the TOX NTD or CTD is sufficient to abrogate the increase in surface PD1 expression that is characteristically driven by full-length (“FL”) TOX, suggesting important, as yet unknown roles for these domains. The central hypothesis of this proposal is that distinct features of TOX activity are attributable to its N- vs. C-terminal domains and that NTD- or CTD-specific perturbations will enable selective modulation of Tex responses to chronic viral infection. This proposal tests this hypothesis by interrogating features of TOX’s interactions and domain-level function at the Pdcd1 locus (encoding PD1), by defining the extent to which the NTD and CTD exhibit global differences in their Tex-specific roles, by defining how the NTD and CTD program the Tex epigenetic state, and by determining which NTD- and CTD-mediated protein interactions TOX uses to regulate Tex transcription. This proposal will thus advance fundamental knowledge of how the molecular processes regulating exhaustion may be manipulated to improve CD8 T cell respons...