Kristen E. Pauken

Radiation and dual checkpoint blockade activate non-redundant immune mechanisms in cancer

Immune checkpoint inhibitors result in impressive clinical responses, but optimal results will require combination with each other and other therapies. This raises fundamental questions about mechanisms of non-redundancy and resistance. Here we report major tumour regressions in a subset of patients with metastatic melanoma treated with an anti-CTLA4 antibody (anti-CTLA4) and radiation, and reproduced this effect in mouse models. Although combined treatment improved responses in irradiated and unirradiated tumours, 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 radiation, anti-CTLA4 and anti-PD-L1/PD-1. Anti-CTLA4 predominantly inhibits T-regulatory cells (Treg cells), thereby increasing the CD8 T-cell to Treg (CD8/Treg) ratio. Radiation enhances the diversity of the T-cell receptor (TCR) repertoire of intratumoral T cells. Together, anti-CTLA4 promotes expansion of T cells, while radiation 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 oligoclonal T-cell expansion. Similarly to results from mice, patients on our clinical trial with melanoma showing high PD-L1 did not respond to radiation plus anti-CTLA4, demonstrated persistent T-cell exhaustion, and rapidly progressed. Thus, PD-L1 on melanoma cells allows tumours to escape anti-CTLA4-based therapy, and the combination of radiation, anti-CTLA4 and anti-PD-L1 promotes response and immunity through distinct mechanisms.

Interactions between PD-1 and PD-L1 promote tolerance by blocking the TCR–induced stop signal

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 show here that unlike naive or activated islet antigen–specific T cells, tolerized islet antigen–specific T cells moved freely and did not swarm around antigen-bearing dendritic cells (DCs) in pancreatic lymph nodes. Inhibition of T cell antigen receptor (TCR)-driven stop signals depended on continued interactions between PD-1 and its ligand, PD-L1, as antibody blockade of PD-1 or PD-L1 resulted in lower T cell motility, enhanced T cell–DC contacts and caused autoimmune diabetes. Blockade of the immunomodulatory receptor CTLA-4 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.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.

Overcoming T cell exhaustion in infection and cancer

Inhibitors of the Programmed Cell Death 1: Programmed Cell Death 1 ligand 1 (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 reversing T cell exhaustion, and outline critical areas of focus for future research, both basic and clinical.

Resident memory CD8 T cells trigger protective innate and adaptive immune responses

Resident memory T cells sound the alarm Immunological memory protects against reinfection. Resident memory T cells (TRM) are long-lived and remain in the tissues where they first encountered a pathogen (see the Perspective by Carbone and Gebhardt). Schenkel et al. and Ariotti et al. found that CD8+ TRM cells act like first responders in the female reproductive tissue or the skin of mice upon antigen reencounter. By secreting inflammatory proteins, TRM cells rapidly activated local immune cells to respond, so much so that they protected against infection with an unrelated pathogen. Iijima and Iwasaki found that CD4+ TRM cells protected mice against reinfection with intravaginal herpes simplex virus 2. Science, this issue p. 98, p. 101, p. 93; see also p. 40 Resident memory CD8+ T cells orchestrate a broad immune response in response to reinfection. [Also see Perspective by Carbone and Gebhardt] The pathogen recognition theory dictates that, upon viral infection, the innate immune system first detects microbial products and then responds by providing instructions to adaptive CD8 T cells. Here, we show in mice that tissue resident memory CD8 T cells (TRM cells), non-recirculating cells located at common sites of infection, can achieve near-sterilizing immunity against viral infections by reversing this flow of information. Upon antigen resensitization within the mouse female reproductive mucosae, CD8+ TRM cells secrete cytokines that trigger rapid adaptive and innate immune responses, including local humoral responses, maturation of local dendritic cells, and activation of natural killer cells. This provided near-sterilizing immunity against an antigenically unrelated viral infection. Thus, CD8+ TRM cells rapidly trigger an antiviral state by amplifying receptor-derived signals from previously encountered pathogens.

T-cell invigoration to tumour burden ratio associated with anti-PD-1 response

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.

Epigenetic stability of exhausted T cells limits durability of reinvigoration by PD-1 blockade

Epigenetic profiling suggests that exhausted T cells are a distinct cell linage. The epigenetics of exhaustion During cancer or chronic infection, T cells become dysfunctional, eventually acquiring an “exhausted” phenotype. Immunotherapies aim to reverse this state. Using a mouse model of chronic infection, two studies now show that the epigenetic profile of exhausted T cells differs substantially from those of effector and memory T cells, suggesting that exhausted T cells are a distinct lineage (see the Perspective by Turner and Russ). Sen et al. defined specific functional modules of enhancers that are also conserved in exhausted human T cells. Pauken et al. examined the epigenetic profile of exhausted T cells after immunotherapy. Although there was transcriptional rewiring, the cells never acquired a memory T cell phenotype. Thus, epigenetic regulation may limit the success of immunotherapies. Science, this issue p. 1104, p. 1165; see also p. 1160 Blocking Programmed Death–1 (PD-1) can reinvigorate exhausted CD8 T cells (TEX) and improve control of chronic infections and cancer. However, whether blocking PD-1 can reprogram TEX into durable memory T cells (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 T cells (TEFF) and TMEM cells that was minimally remodeled after PD-L1 blockade. This finding suggests that TEX are a distinct lineage of CD8 T cells. 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.

The role of the PD-1 pathway in autoimmunity and peripheral tolerance

Programmed death‐1 (PD‐1) is a surface receptor critical for the regulation of T cell function during immunity and tolerance. PD‐1 interactions with its ligands PD‐L1 and PD‐L2 inhibit T cell effector functions in an antigen‐specific manner. This paper examines the role of PD‐1 in limiting autoreactivity and establishing self‐tolerance and discusses the hypothesis that PD‐1 ligand (PD‐L) expression both spatially and temporally dictates the fate of self‐reactive T cells during the breakdown of peripheral tolerance and development of autoimmunity. We focus our discussion on the role of PD‐1/PD‐L interactions during peripheral tolerance, the differential role for PD‐L1 and PD‐L2 in response to environmental or self‐antigens, and the impact of PD‐1 signaling on dynamic T cell motility and the T cell receptor (TCR) stop signal. Finally, we discuss the potential to selectively target the PD‐1 pathway therapeutically to alter T cell function during autoimmunity.

Genetic absence of PD-1 promotes accumulation of terminally differentiated exhausted CD8+ T cells

Using PD-1–deficient mice and co-adoptive transfer approaches, Odorizzi et al. demonstrate that PD-1 is not required for the induction of CD8+ T cell exhaustion (TEX) in chronic LCMV infection. The absence of PD-1 leads to more cytotoxic, but terminally differentiated TEX, with compromised long-term durability. PD-1 may well serve to protect TEX from excessive overstimulation, proliferation, and terminal differentiation.

CD39 Expression Identifies Terminally Exhausted CD8+ T Cells.

Exhausted T cells express multiple co-inhibitory molecules that impair their function and limit immunity to chronic viral infection. Defining novel markers of exhaustion is important both for identifying and potentially reversing T cell exhaustion. Herein, we show that the ectonucleotidse CD39 is a marker of exhausted CD8+ T cells. CD8+ T cells specific for HCV or HIV express high levels of CD39, but those specific for EBV and CMV do not. CD39 expressed by CD8+ T cells in chronic infection is enzymatically active, co-expressed with PD-1, marks cells with a transcriptional signature of T cell exhaustion and correlates with viral load in HIV and HCV. In the mouse model of chronic Lymphocytic Choriomeningitis Virus infection, virus-specific CD8+ T cells contain a population of CD39high CD8+ T cells that is absent in functional memory cells elicited by acute infection. This CD39high CD8+ T cell population is enriched for cells with the phenotypic and functional profile of terminal exhaustion. These findings provide a new marker of T cell exhaustion, and implicate the purinergic pathway in the regulation of T cell exhaustion.

CD4 + T cell anergy prevents autoimmunity and generates regulatory T cell precursors

The role of anergy, an acquired state of T cell functional unresponsiveness, in natural peripheral tolerance remains unclear. In this study, we found that anergy was selectively induced in fetal antigen–specific maternal CD4+ T cells during pregnancy. A naturally occurring subpopulation of anergic polyclonal CD4+ T cells, enriched for self antigen–specific T cell antigen receptors, was also present in healthy hosts. Neuropilin-1 expression in anergic conventional CD4+ T cells was associated with hypomethylation of genes related to thymic regulatory T cells (Treg cells), and this correlated with their ability to differentiate into Foxp3+ Treg cells that suppressed immunopathology. Thus, our data suggest that not only is anergy induction important in preventing autoimmunity but also it generates the precursors for peripheral Treg cell differentiation.