Strength of CD28 costimulation directs self-renewal or differentiation of TCF-1+ PD-1+ CD8 T cells through metabolic regulation

During persistent antigen stimulation, such as in chronic infections and cancer, T cells differentiate into a hypofunctional exhausted state to survive. Exhausted PD-1+ CD8 T cell responses are sustained by TCF-1+ precursors (Tpex) that self-renew or differentiate into exhausted T cells (Tex) that retain some effector function. Tpex have high expression of the costimulatory molecule CD28 and are the cells that respond to PD-1 targeted immunotherapy. Here, we demonstrate that abrogation of CD28 signaling impairs maintenance of anti-viral T cell responses during chronic infection. Low-level CD28 signaling was required to preserve mitochondrial fitness and self-renewal of Tpex, whereas stronger CD28 signaling enhanced glycolysis and promoted Tpex differentiation. Our findings show that CD28 signaling is essential for long-term maintenance of antigen-specific T cells during chronic stimulation, and suggest that the activation status of antigen presenting cells determines Tpex differentiation into more functional effector-like Tex cells. Gene expression analysis of bulk sorted Tpex (CD73+) or Tex (Tim3+) P14 CD28flox CREert2+ (CD28neg) or creERT2neg (CD28+) from mice chronically infected with LCMV cl13 and 2 weeks post tamoxifen treatment to understand the molecular impact of CD28 loss in maintenance and differentiation of virus-specific T cells.