Background PD-1 pathway blockade has led to improved clinical outcomes in diverse cancer types, and has been approved by the FDA for use in over 25 different cancers. Although PD-1 pathway inhibitors have shown great promise, the mechanisms contributing to protective anti-tumor immunity following loss of PD-1 signals remain incompletely understood. In this study, we interrogated how many of the protective effects observed following PD-1 loss were due to direct cell intrinsic changes to the cell that lost PD-1 signaling vs. larger changes to the tumor microenvironment driving changes that were not due to the cell losing PD-1.

Methods We developed an inducible PD-1 knockout (KO) model whereby PD-1 could be deleted on about half of the cells. Using this method, we utilized single cell RNA seq and TCR seq to interrogate how loss of PD-1 directly modulated CD8 T cells in the tumor, as well as how the CD8 T cells that did not lose PD-1 changed. We developed a computational method to reliably distinguish PD-1 KO from PD-1 non-KO cells, visualizing a physical loss of accumulation of RNA seq reads at the exons of the Pdcd1 gene that were targeted for deletion. Validation of transcriptional findings were performed using flow cytometry.

Results Inducible deletion of PD-1 beginning at day 7 post-implantation of MC38 tumor cells subcutaneously was sufficient to induce robust anti-tumor immunity and tumor control. Remarkably, PD-1-expressing CD8⁺ T cells in the tumor had increased functionality similar to the T cells lacking PD-1. Using paired single cell RNA seq and TCR seq, we found that many of the transcriptional changes following PD-1 deletion were not restricted to CD8⁺ T cells in the tumor that lost PD-1, but instead shared by both PD-1-deleted and PD-1-expressing CD8⁺ T cells. While a cell-intrinsic loss of PD-1 resulted in increased clonal expansion on a clone-by-clone basis, major transcriptional and functional changes were not restricted to CD8⁺ TILs that had lost PD-1.

Conclusions These data provide additional mechanistic insight into how loss of PD-1 signals promotes protective anti-tumor immunity, indicating that PD-1 inhibitors can act beyond each individual cell that they contact to promote increased T cell function. These findings have important implications for design of PD-1-based therapeutic strategies in cancer patients, suggesting that not every T cell needs to be physically bound by the PD-1 inhibitor to experience a functional benefit from receiving these inhibitors.

Ethics Approval All animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) at Harvard Medical School. The approval number was: IS00000041-6.