CD4 “help” via soluble factors and cell-surface ligands is critical to sustain CD8 T cell responses and humoral immunity during chronic viral infection. Recently, our lab has further identified a necessity for CD4-derived IL-21 in the formation of a previously unrecognized CX3CR1+CD8 T cell subset that exhibits potent cytolytic function. However, despite their essential role, our understanding of how persistent exposure to viral load and inflammation shapes helper T cell differentiation remains incompletely understood. Using scRNA-seq, our lab has generated substantial preliminary data demonstrating that CD4 T cells responding to chronic LCMV infection are more heterogenous than previously appreciated, with three transcriptionally distinct subsets dominating the antiviral response: Cxcr6+Th1, Cxcr5+Tfh, and Slamf6+ memory-like cells. Notably, our data further indicate that CD4 T cells responding to chronic, but not acute viral infection, preferentially redirect their differentiation towards this memory-like subset. Taken together, we hypothesize that prolonged exposure to antigenic and inflammatory signals (TGF-β, IL-10 etc.) modulate the transcriptional diversity of virus-specific CD4 T cells, and that CD4 differentiation during chronic viral infection is driven towards this memory-like subset to coordinate the following: 1) attenuate Th1-mediated immunopathology, 2) maintain a progenitor pool that can give rise to Th1 and Tfh effector cells, and 3) facilitate a niche wherein memory-like CD4 T cells co-localize with progenitor CD8 T cells to redirect their differentiation towards protective CX3CR1+CD8 T cells. In Aim 1, we will perform adoptive transfer (AT) experiments to examine the proliferative potential, protective capacity, and lineage relationship between the three major subsets of virus-specific CD4 T cells. Additionally, based on the gene regulatory network formulated by SCENIC (an R package), we will use RNA interference models to test the contribution of key transcription factors predicted to play a role in regulating CD4 differentiation during chronic infection. In Aim 2, we will use genetic approaches and AT experiments to manipulate antigen-driven TCR-signaling in order to assess how antigenic signals regulate CD4 differentiation during chronic infection. Moreover, we will block the activity of immunoregulatory cytokines known to be overexpressed during chronic infection to determine their impact on the fate commitment of virus-specific CD4 T cells. Lastly, in Aim 3 we will determine whether AT of IL- 21-producing memory-like CD4 T cells, either alone or in conjunction with PD-L1 blockade, can augment progenitorCX3CR1hi CD8 T cell transition to improve T cell-mediated immunity. Knowledge gained from this research will provide mechanistic insights into the functional adaption process CD4 T cells display in the face of persistent infection and help identify novel strategies aimed at optimizing cellular or humoral-mediated antiviral ...