Sustained CD28 costimulation is required for self-renewal and differentiation of TCF-1 + PD-1 + CD8 T cells.

During persistent antigen stimulation, such as in chronic infections and cancer, CD8 T cells differentiate into a hypofunctional programmed death protein 1-positive (PD-1 + ) exhausted state. Exhausted CD8 T cell responses are maintained by precursors (Tpex) that express the transcription factor T cell factor 1 (TCF-1) and high levels of the costimulatory molecule CD28. Here, we demonstrate that sustained CD28 costimulation is required for maintenance of antiviral T cells during chronic infection. Low-level CD28 engagement preserved mitochondrial fitness and self-renewal of Tpex, whereas stronger CD28 signaling enhanced glycolysis and promoted Tpex differentiation into TCF-1 neg exhausted CD8 T cells (Tex). Furthermore, enhanced differentiation by CD28 engagement did not reduce the Tpex pool. Together, these findings demonstrate that continuous CD28 engagement is needed to sustain PD-1 + CD8 T cells and suggest that increasing CD28 signaling promotes Tpex differentiation into more functional effector-like Tex, possibly without compromising long-term responses.