Proteomic Analyses Identify Novel Regulatory Mechanisms of Coinhibitory Receptor PD-1

Coinhibitory immunoreceptor programmed cell death-1 (PD-1, CD279) plays a critical role in mediating T cell exhaustion and blockade of this pathway can promote antitumor immunity. While PD-1 pathway inhibitors are revolutionizing cancer therapy, only a subset of patients respond and show durable remission, highlighting the need to better understand the basic mechanisms by which the PD-1 pathway inhibits T cell functions. Studies assessing PD-1 signaling mechanisms have the potential to identify novel targets for therapeutic intervention in cancer to improve patient responses. To identify novel mechanisms of PD-1 function, we utilized proximity labeling as an unbiased approach to survey proteins proximal to PD-1 following ligation and identified the mechanosensitive cation channel PIEZO1 as a primary target of PD-1-mediated inhibition. Stimulation of CD8+ T cells through the T cell receptor (TCR) and costimulatory receptor CD28 triggered the activation of PIEZO1 at the immune synapse, while simultaneous ligation of PD-1 countered this activation. We found that mice lacking PIEZO1 selectively on CD8+ T cells exhibited significantly increased tumor growth marked by impaired CD8+ T cell function and this could not be rescued by PD-1 blockade. Conversely, CD8+ tumor infiltrating lymphocytes (TILs) from wild-type (WT) mice treated with PIEZO1 agonist showed greater functionality compared to controls. Coadministration of PIEZO1 agonist and PD-1 blockade significantly reduced tumor burden and improved survival in a PD-1 blockade-unresponsive tumor model. These findings identify PIEZO1 inhibition as an important mechanism by which PD-1 signaling regulates CD8+ T cell functions and suggest that modulating PIEZO1 activity on CD8+ T cells may be a novel approach to augment cancer immunotherapy. To determine how PD-1 tyrosine-phosphorylation motifs differentially impact downstream signaling, we relied on PD-1 mutant mouse models and proteomic methodology to uncouple the functions of PD-1 immune tyrosine-based inhibition motif (ITIM) and immune tyrosine-based switch motif (ITSM), as well as other potential signaling motifs. We found that ITSM-mutant mice controlled tumor growth similar to PD-1 knockout (KO) mice, but did not full recapitulate PD-1 KO phenotypes. Furthermore, ITIM-mutant mice showed no significant improvement in tumor control, similar to WT mice. To further characterize these differences, we performed proximity phospho-proteomic analysis and PD-1 phospho-peptide pulldowns to assess PD-1 binding interactions and phosphorylation alterations. We confirmed that the ITSM recruits inhibitory phosphatases and kinases to dampen TCR-mediated activation and discovered that the ITIM associates with proteins involved in ubiquitin-mediated degradation, suggesting that the ITIM regulates the degradation of TCR effector proteins. Novel phospho-serine sites identified on the cytoplasmic tail of PD-1 also exhibited unique protein binding behavior, implying that PD-1 retains residual signaling function beyond the ITIM and ITSM. While additional experimentation is necessary to confirm these findings, our results significantly advance our mechanistic understanding of PD-1-mediated signaling. Together, our studies have discovered a novel regulatory mechanism of PD-1-mediated inhibition and have substantially built upon our understanding of the function of PD-1 signaling motifs. Specifically, our findings demonstrate that PD-1 inhibition of PIEZO1 is therapeutically relevant in cancer and that novel PD-1 phosphorylation sites and binding partners may participate in uncharacterized functions of PD-1.