Despite the success of monotherapies based on blockade of programmed cell death 1 (PD-1) in human melanoma, most patients do not experience durable clinical benefit. Pre-existing T-cell infiltration and/or the presence of PD-L1 in tumours may be used as indicators of clinical response; however, blood-based profiling to understand the mechanisms of PD-1 blockade has not been widely explored. Here we use immune profiling of peripheral blood from patients with stage IV melanoma before and after treatment with the PD-1-targeting antibody pembrolizumab and identify pharmacodynamic changes in circulating exhausted-phenotype CD8 T cells (Tex cells). Most of the patients demonstrated an immunological response to pembrolizumab. Clinical failure in many patients was not solely due to an inability to induce immune reinvigoration, but rather resulted from an imbalance between T-cell reinvigoration and tumour burden. The magnitude of reinvigoration of circulating Tex cells determined in relation to pretreatment tumour burden correlated with clinical response. By focused profiling of a mechanistically relevant circulating T-cell subpopulation calibrated to pretreatment disease burden, we identify a clinically accessible potential on-treatment predictor of response to PD-1 blockade.

Immune checkpoint inhibitors1 result in impressive clinical responses2–5 but optimal results will require combination with each other6 and other therapies. This raises fundamental questions about mechanisms of non-redundancy and resistance. Here, we report major tumor regressions in a subset of patients with metastatic melanoma treated with an anti-CTLA4 antibody (anti-CTLA4) and radiation (RT) and reproduced this effect in mouse models. Although combined treatment improved responses in irradiated and unirradiated tumors, resistance was common. Unbiased analyses of mice revealed that resistance was due to upregulation of PD-L1 on melanoma cells and associated with T cell exhaustion. Accordingly, optimal response in melanoma and other cancer types requires RT, anti-CTLA4, and anti-PD-L1/PD-1. Anti-CTLA4 predominantly inhibits T regulatory cells (Tregs) to increase the CD8 T cell to Treg (CD8/Treg) ratio. RT enhances the diversity of the T cell receptor (TCR) repertoire of intratumoral T cells. Together, anti-CTLA4 promotes expansion of T cells, while RT shapes the TCR repertoire of the expanded peripheral clones. Addition of PD-L1 blockade reverses T cell exhaustion to mitigate depression in the CD8/Treg ratio and further encourages oligo-clonal T cell expansion. Similar to results from mice, patients on our clinical trial with melanoma showing high PD-L1 did not respond to RT + anti-CTLA4, demonstrated persistent T cell exhaustion, and rapidly progressed. Thus, PD-L1 on melanoma cells allows tumors to escape anti-CTLA4-based therapy, and the combination of RT, anti-CTLA4, and anti-PD-L1 promotes response and immunity through distinct mechanisms.

Blocking Programmed Death–1 (PD-1) can reinvigorate exhausted CD8 Tcells (TEX) and improve control of chronic infections and cancer. However, whether blocking PD-1 can reprogram TEX into durable memory Tcells (TMEM) is unclear. We found that reinvigoration of TEX in mice by PD-L1 blockade caused minimal memory development. After blockade, reinvigorated TEX became reexhausted if antigen concentration remained high and failed to become TMEM upon antigen clearance. TEX acquired an epigenetic profile distinct from that of effector Tcells (TEFF) and TMEM cells that was minimally remodeled after PD-L1 blockade. This finding suggests that TEX are a distinct lineage of CD8 Tcells. Nevertheless, PD-1 pathway blockade resulted in transcriptional rewiring and reengagement of effector circuitry in the TEX epigenetic landscape. These data indicate that epigenetic fate inflexibility may limit current immunotherapies.

T cell activation is a highly regulated process involving peptide-MHC engagement of the T cell receptor and positive costimulatory signals. Upon activation, coinhibitory 'checkpoints', including programmed cell death protein 1 (PD1), become induced to regulate T cells. PD1 has an essential role in balancing protective immunity and immunopathology, homeostasis and tolerance. However, during responses to chronic pathogens and tumours, PD1 expression can limit protective immunity. Recently developed PD1 pathway inhibitors have revolutionized cancer treatment for some patients, but the majority of patients do not show complete responses, and adverse events have been noted. This Review discusses the diverse roles of the PD1 pathway in regulating immune responses and how this knowledge can improve cancer immunotherapy as well as restore and/or maintain tolerance during autoimmunity and transplantation.

Inhibitors of the PD-1:PD-L1 pathway, a central regulator of T cell exhaustion, have been recently shown to be effective for treatment of different cancers. However, clinical responses are mixed, highlighting the need to better understand the mechanisms of action of PD-1:PD-L1, the role of this pathway in immunity to different tumors, and the molecular and cellular effects of PD-1 blockade. Here we review the molecular regulation of T cell exhaustion, placing recent findings on PD-1 blockade therapies in cancer in the context of the broader understanding of the roles of the PD-1:PD-L1 pathway in T cell exhaustion during chronic infection. We discuss the current understanding of the mechanisms involved in reversal T cell exhaustion, and outline critical areas of focus for future research, both basic and clinical.

SUMMARY Therapeutic blocking of the PD1 pathway results in significant tumor responses but resistance is common. We demonstrate that prolonged interferon signaling orchestrates PDL1-dependent and PDL1-independent resistance to immune checkpoint blockade (ICB), and to combinations such as radiation plus anti-CTLA4. Persistent type II interferon signaling allows tumors to acquire STAT1-related epigenomic changes and augments expression of interferon-stimulated genes and ligands for multiple T cell inhibitory receptors. Both type I and II interferons maintain this resistance program. Crippling the program genetically or pharmacologically interferes with multiple inhibitory pathways, and expands distinct T cell populations with improved function despite expressing markers of severe exhaustion. Consequently, tumors resistant to multi-agent ICB are rendered responsive to ICB monotherapy. Finally, we observe that biomarkers for interferon-driven resistance associate with clinical progression after anti-PD1 therapy. Thus, the duration of tumor interferon signaling augments adaptive resistance and inhibition of the interferon response bypasses requirements for combinatorial ICB therapies.

Programmed death-1 (PD-1) is an inhibitory molecule expressed on activated T cells, however, the biological context in which PD-1 controls T cell tolerance remains unclear. Using two-photon laser-scanning microscopy, we showed that unlike naïve or activated islet antigen-specific T cells, tolerized islet antigen-specific T cells moved freely and did not swarm around antigen-bearing dendritic cells (DC) in pancreatic lymph nodes. Inhibition of T cell receptor (TCR)-driven stop signals depended on continued PD-1-PD-L1 interactions, as antibody blockade of PD-1 or PD-L1 decreased T cell motility, enhanced T cell-DC contacts, and caused autoimmune diabetes. CTLA-4 blockade did not alter T cell motility or abrogate tolerance. Thus, PD-1-PD-L1 interactions maintain peripheral tolerance by mechanisms fundamentally distinct from those of CTLA-4.

Dynamic reprogramming of metabolism is essential for T cell effector function and memory formation. However, the regulation of metabolism in exhausted CD8+ T (Tex) cells is poorly understood. We found that during the first week of chronic lymphocytic choriomeningitis virus (LCMV) infection, before severe dysfunction develops, virus-specific CD8+ T cells were already unable to match the bioenergetics of effector T cells generated during acute infection. Suppression of T cell bioenergetics involved restricted glucose uptake and use, despite persisting mechanistic target of rapamycin (mTOR) signaling and up-regulation of many anabolic pathways. PD-1 regulated early glycolytic and mitochondrial alterations and repressed transcriptional coactivator PGC-1α. Improving bioenergetics by overexpression of PGC-1α enhanced function in developing Tex cells. Therapeutic reinvigoration by anti-PD-L1 reprogrammed metabolism in a subset of Tex cells. These data highlight a key metabolic control event early in exhaustion and suggest that manipulating glycolytic and mitochondrial metabolism may enhance checkpoint blockade outcomes.