Longitudinal Proteomic Profiling of T Cell Differentiation In Vivo Reveals Biochemical Remodeling Underlying Exhaustion

CD8 T cell exhaustion impedes immune responses to cancer and chronic infections, and a biochemical understanding of exhaustion is essential to improving immunotherapy. Here, we present the first longitudinal protein abundance and phosphoproteomic analysis of antigen-specific CD8 T cells undergoing differentiation in vivo during acute (LCMV-Armstrong) and chronic (LCMV-Clone 13) infection. Comparing protein abundance across the two infection conditions identified over 180 known and novel exhaustion-associated proteins, including proteins missed by transcriptional analyses. Phosphoproteomic analysis identified >900 differentially regulated phosphosites on >400 proteins, including known inhibitory phosphosites on PD1, PAG1, SHP-1/PTPN6, SLAMF1/CD150. We also calculated phosphosite conservation across mammals, to direct follow-up studies towards sites with likely essential function. Lastly, our analysis uncovers exhaustion-associated kinases with clinical-stage inhibitors, underscoring the translational utility of our dataset to guide immunotherapy development. Together, our datasets define a biochemical atlas of T cell exhaustion in vivo, shedding light on the molecular mechanisms of T cell dysfunction.