PD-1 restrains thymic IL-2 production and regulatory T cell development

Inhibitory proteins, such as programmed cell death protein 1 (PD-1), have been extensively studied in peripheral T cell responses to foreign, self, and neoantigens. Notably, these proteins are first expressed during T cell development in the thymus. Reports suggest that PD-1 limits regulatory T cell (Treg) development, but the mechanism by which PD-1 exerts this function remains unknown. The present study expands the evaluation of PD-1 and its ligands in the thymus, demonstrating that some of the highest expressers of PD-1 and PD-L1 are agonist selected cells. Surprisingly, we reveal a selective role for PD-1 in regulating the developmental niche only for Tregs as other agonist selected cell populations, such as natural killer T cells, remain unchanged. We also ruled out PD-1 as a regulator of proliferation or cell death of agonist selected Tregs and further demonstrated that PD-1 deficient Tregs have reduced TCR signaling. Unexpectedly, the data suggests that PD-1 deficient thymocytes produce elevated levels of IL-2, a Treg niche limiting cytokine. Collectively, these data suggest a novel role for PD-1 in regulating IL-2 production and the concurrent agonist selection of thymic Tregs. This observation has implications for the use of checkpoint blockade in the context of cancer and infection. Overall design: Thymus from 3-week-old WT or PD-1 knockout animals (C57BL/6J) were processed for flourescence-activated cell sorting (FACS) to sort CD4+ HSA+ CD73- CD25- (CD4 single positive thymocytes) and CD4+ HSA+ CD73- CD25+ (regulatory T cells and CD25+ regulatory T cell progenitors). We sought to examine differences between the WT and PD-1 knockout cells using scRNAseq.