Immune checkpoint inhibitors have demonstrated significant efficacy in the treatment of a variety of cancers, however their therapeutic potential is limited by abstruse immune related adverse events. Currently, no robust animal model exists of checkpoint inhibitor-induced adverse events. Establishing such a model will improve our mechanistic understanding of this process, which in turn will inform design of improved therapies. We developed a mouse model to determine inflammatory toxicities in response to dual checkpoint blockade in the presence of syngeneic tumors. Mice from susceptible genetic backgrounds received intraperitoneal injections of anti-mouse PD-1 and CTLA-4 antibodies. The mice were monitored for weight loss and histologic evidence of inflammation. Blood was collected for basic metabolic panels and titers of anti-nuclear antibodies. In parallel, mice were also treated with prednisolone, which is commonly used to treat immune related adverse events among cancer patients. Among all the genetic backgrounds, B6/lpr mice treated with anti-CTLA-4 and anti-PD-1 antibodies developed more substantial hepatitis, pancreatitis, colitis, and pneumonitis characterized by organ infiltration of immune cells. Mice that developed tissue infiltration demonstrated high serum levels of glucose and high titers of anti-nuclear antibodies. Finally, while administration of prednisolone prevented the development of the inflammatory adverse events, it also abrogated the protective anti-tumor effect of the checkout inhibitors. Genetic background and treatment modalities jointly modified the inflammatory adverse events in tumor bearing mice, suggesting a complex mechanism for checkpoint inhibitor-related inflammation. Future studies will assess additional genetic susceptibility factors and will examine possible contributions from the administration of other anti-inflammatory drugs.

Programmed cell death protein 1 (PD-1) is an inhibitory receptor on T lymphocytes that is critical for modulating adaptive immunity. As such, it has been successfully exploited for cancer immunotherapy. Programmed death ligand 1 (PD-L1) and PD-L2 are ligands for PD-1; the former is ubiquitously expressed in inflamed tissues, whereas the latter is restricted to antigen-presenting cells. PD-L2 binds to PD-1 with 3-fold stronger affinity compared with PD-L1. To date, this affinity discrepancy has been attributed to a tryptophan (W110PD-L2) that is unique to PD-L2 and has been assumed to fit snuggly into a pocket on the PD-1 surface. Contrary to this model, using surface plasmon resonance to monitor real-time binding of recombinantly-expressed and -purified proteins, we found that W110PD-L2 acts as an “elbow” that helps shorten PD-L2 engagement with PD-1 and therefore lower affinity. Furthermore, we identified a “latch” between the C and D β-strands of the binding face as the source of the PD-L2 affinity advantage. We show that the 3-fold affinity advantage of PD-L2 is the consequence of these two opposing features, the W110PD-L2 “elbow” and a C–D region “latch.” Interestingly, using phylogenetic analysis, we found that these features evolved simultaneously upon the emergence of placental mammals, suggesting that PD-L2–affinity tuning was part of the alterations to the adaptive immune system required for placental gestation.

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Programmed cell death protein 1 (PD-1) is a critical inhibitory receptor that limits excessive T cell responses. Cancer cells have evolved to evade these immunoregulatory mechanisms by upregulating PD-1 ligands and preventing T cell mediated anti-tumor responses. Consequently, therapeutic blockade of PD-1 enhances T cell mediated anti-tumor immunity but many patients do not respond and a significant proportion develops inflammatory toxicities. To improve anti-cancer therapy, it is critical to reveal the mechanisms by which PD-1 regulates T cell responses. We performed global quantitative phosphoproteomic interrogation of PD-1 signaling in T cells. By complementing our analysis with functional validation assays, we show that PD-1 targets tyrosine phosphosites that mediate proximal T cell receptor signaling, cytoskeletal organization and immune synapse formation. PD-1 ligation also led to differential phosphorylation of serine and threonine sites within proteins regulating T cell activation, gene expression, and protein translation. In silico predictions revealed kinase/substrate relationships engaged downstream of PD-1 ligation. These insights uncover the phosphoproteomic landscape of PD-1 triggered pathways and reveal novel PD-1 substrates that modulate diverse T cell functions and may serve as future therapeutic targets. These data are a useful resource in the design of future PD-1-targeting therapeutic approaches.

The inhibitory receptor PD-1 is expressed on T cells to inhibit select functions when ligated. The complete signaling mechanism downstream of PD-1 has yet to be uncovered. Here, we discovered phosphoprotein associated with glycosphingolipid-enriched microdomains 1 (PAG) is phosphorylated following PD-1 ligation and associate this with inhibitory T cell function. Clinical cohort analysis correlates low PAG expression with increased survival from numerous tumor types. PAG knockdown in T cells prevents PD-1-mediated inhibition of cytokine secretion, cell adhesion, CD69 expression, and ERK204/187 phosphorylation, and enhances phosphorylation of SRC527 following PD-1 ligation. PAG overexpression rescues these effects. In vivo, PAG contributes greatly to the growth of two murine tumors, MC38 and B16, and limits T cell presence within the tumor. Moreover, PAG deletion sensitizes tumors to PD-1 blockade. Here PAG is established as a critical mediator of PD-1 signaling and as a potential target to enhance T cell activation in tumors.