PD-1 protects expanding human T cells from premature restimulation-induced cell death by modulating TCR and CD28 signaling

Programmed cell death-1 (PD-1) is a co-inhibitory receptor expressed on T cells that dampens TCR and CD28 signaling in the immunological synapse. PD-1 is significantly upregulated on T cells in the tumor microenvironment, where it promotes exhaustion in the context of chronic antigen restimulation. Exhaustion renders T cells hyporesponsive and ineffectual, but potentially resistant to restimulation-induced cell death (RICD). Restimulation-induced cell death (RICD) is a critical propriocidal apoptosis program triggered in activated T cells upon robust TCR re-engagement, which serves to constrain effector T cell expansion and longevity to prevent collateral tissue damage. While the checkpoint function of PD-1 has profound implications for cancer immunotherapy, the role of PD-1 in regulating newly activated T cells remains unclear. We hypothesized that PD-1 attenuates RICD sensitivity in human effector T cells by modulating TCR signal strength. Here we show that transient upregulation of PD-1 helps to protect clonally expanding human CD4+ and CD8 + T cells from premature RICD, with only moderate protection noted in terminally-differentiated, PD-1^lo effector CD8 + T cells. Restimulation of T cells with beads containing PD-L1 results in significant apoptosis resistance, dependent on PD-L1 dosage and the proximity of PD-L1 to the TCR and CD28. Interestingly, PD-L1 demonstrated a more significant RICD rescue with CD28 co-ligation as opposed to TCR engagement alone, suggesting PD-1 signaling targets both signaling pathways in this context. Furthermore, biochemical/proteomic data suggest PD-1 modulates proximal signaling downstream of both TCR and CD28 and influences the expression of specific pro/anti-apoptotic proteins that govern RICD sensitivity. Despite the original assumption of PD-1 as a programmed death-inducing protein, our research reveals that homeostatic expression of PD-1 in clonally expanding T cells confers RICD resistance that promotes T cell survival and persistence. These findings present significant implications for understanding how blocking or engaging the PD-L1:PD-1 signaling axis may influence apoptosis sensitivity in both normal and exhausted T cells to alter adaptive immune responses.