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CD8+ T cells are critical mediators of cytotoxic effector function in infection, cancer and autoimmunity. In cancer and chronic viral infection, CD8+ T cells undergo a progressive loss of cytokine production and cytotoxicity, a state termed T cell exhaustion. In autoimmunity, autoreactive CD8+ T cells retain the capacity to effectively mediate the destruction of host tissues. Although the clinical outcome differs in each context, CD8+ T cells are chronically exposed to antigen in all three. These chronically stimulated CD8+ T cells share some common phenotypic features, as well as transcriptional and epigenetic programming, across disease contexts. A better understanding of these CD8+ T cell states may reveal novel strategies to augment clearance of chronic viral infection and cancer and to mitigate self-reactivity leading to tissue damage in autoimmunity.

Melanoma-derived brain metastases (MBM) represent an unmet clinical need due to central nervous system (CNS) progression as a frequent, end-stage site of disease. Immune checkpoint inhibition (ICI) represents a clinical opportunity against MBM; however, the MBM tumor microenvironment (TME) has not been fully elucidated in the context of ICI. To dissect unique MBM-TME elements and correlates of MBM-ICI response, we collected 32 fresh MBM and performed single cell RNA sequencing of the MBM-TME and T cell receptor clonotyping on T cells from MBM and matched blood and extracranial lesions. We observed myeloid phenotypic heterogeneity, most notably multiple distinct neutrophil states including an IL-8 expressing population that correlated with malignant cell epithelial-to-mesenchymal transition. Additionally, we observe significant relationships between intracranial T cell phenotypes and the distribution of T cell clonotypes intracranially and peripherally. We found that the phenotype, clonotype, and overall number of MBM-infiltrating T cells were associated with response to ICI, suggesting that ICI-responsive MBMs interact with peripheral blood in a manner similar to extracranial lesions. These data demonstrate unique features of the MBM-TME, which may represent potential targets to improve clinical outcomes for patients with MBM.

CD8+ T cell exhaustion is a state of dysfunction acquired in chronic viral infection and cancer, characterized by the formation of Slamf6+ progenitor exhausted and Tim-3+ terminally exhausted subpopulations through unknown mechanisms. Here we establish the phosphatase PTPN2 as a new regulator of the differentiation of the terminally exhausted subpopulation that functions by attenuating type 1 interferon signaling. Deletion of Ptpn2 in CD8+ T cells increased the generation, proliferative capacity and cytotoxicity of Tim-3+ cells without altering Slamf6+ numbers during lymphocytic choriomeningitis virus clone 13 infection. Likewise, Ptpn2 deletion in CD8+ T cells enhanced Tim-3+ anti-tumor responses and improved tumor control. Deletion of Ptpn2 throughout the immune system resulted in MC38 tumor clearance and improved programmed cell death-1 checkpoint blockade responses to B16 tumors. Our results indicate that increasing the number of cytotoxic Tim-3+CD8+ T cells can promote effective anti-tumor immunity and implicate PTPN2 in immune cells as an attractive cancer immunotherapy target.

T cell dysfunction is a hallmark of many cancers, but the basis for T cell dysfunction and the mechanisms by which antibody blockade of the inhibitory receptor PD-1 (anti-PD-1) reinvigorates T cells are not fully understood. Here we show that such therapy acts on a specific subpopulation of exhausted CD8(+) tumor-infiltrating lymphocytes (TILs). Dysfunctional CD8(+) TILs possess canonical epigenetic and transcriptional features of exhaustion that mirror those seen in chronic viral infection. Exhausted CD8(+) TILs include a subpopulation of ‘progenitor exhausted’ cells that retain polyfunctionality, persist long term and differentiate into ‘terminally exhausted’ TILs. Consequently, progenitor exhausted CD8(+) TILs are better able to control tumor growth than are terminally exhausted T cells. Progenitor exhausted TILs can respond to anti-PD-1 therapy, but terminally exhausted TILs cannot. Patients with melanoma who have a higher percentage of progenitor exhausted cells experience a longer duration of response to checkpoint-blockade therapy. Thus, approaches to expand the population of progenitor exhausted CD8(+) T cells might be an important component of improving the response to checkpoint blockade.

Patient outcomes for steroid-dependent or -refractory chronic graft-versus-host diesease (cGVHD) are poor, and only ibrutinib has been US Food and Drug Administration (FDA) approved for this indication. cGVHD is often driven by the germinal center (GC) reaction, in which T follicular helper cells interact with GC B cells to produce antibodies that are associated with disease pathogenesis. The transcriptional corepressor B-cell lymphoma 6 (BCL6) is a member of the Broad-complex, Tramtrack, and Bric-abrac/poxvirus and zinc finger (BTB/POZ) transcription factor family and master regulator of the immune cells in the GC reaction. We demonstrate that BCL6 expression in both donor T cells and B cells is necessary for cGVHD development, pointing to BCL6 as a therapeutic cGVHD target. A small-molecule BCL6 inhibitor reversed active cGVHD in a mouse model of multiorgan system injury with bronchiolitis obliterans associated with a robust GC reaction, but not in cGVHD mice with scleroderma as the prominent manifestation. For cGVHD patients with antibody-driven cGVHD, targeting of BCL6 represents a new approach with specificity for a master GC regulator that would extend the currently available second-line agents.

Tfr cells regulate Tfh-mediated antibody responses. Hou et al. demonstrate that FoxP3 and Ezh2 control the Tfr transcriptional program, and loss of this program results in dysfunctional ex-Tfr cells., Follicular regulatory T (Tfr) cells are a regulatory T cell subset that controls antibody production by inhibiting T follicular helper (Tfh)–mediated help to B cells. Tfh and Tfr cells possess opposing functions suggesting unique programming. Here we elucidated the transcriptional program controlling Tfr suppressive function. We found that Tfr cells have a program for suppressive function fine-tuned by tissue microenvironment. The transcription factor FoxP3 and chromatin-modifying enzyme EZH2 are essential for this transcriptional program but regulate the program in distinct ways. FoxP3 modifies the Tfh program to induce a Tfr-like functional state, demonstrating that Tfr cells coopt the Tfh program for suppression. Importantly, we identified a Tfr cell population that loses the Tfr program to become “ex-Tfr” cells with altered functionality. These dysfunctional ex-Tfr cells may have roles in modulating pathogenic antibody responses. Taken together, our studies reveal mechanisms controlling the Tfr transcriptional program and how failure of these mechanisms leads to dysfunctional Tfr cells.

Significance T cell-mediated immune responses are compromised in aged individuals, leading to increased morbidity and reduced response to vaccination. Finding new ways to boost T cell immunity in the elderly is key for enhancing their immune competence. In this work, we performed a systematic analysis of proteins and metabolites in young versus aged T cells. Metabolic rewiring occurs in young T cells following stimulation but is dampened in aged T cells. Moreover, we show that aged T cell functions can be enhanced by metabolite addition., T cell-mediated immune responses are compromised in aged individuals, leading to increased morbidity and reduced response to vaccination. While cellular metabolism tightly regulates T cell activation and function, metabolic reprogramming in aged T cells has not been thoroughly studied. Here, we report a systematic analysis of metabolism during young versus aged naïve T cell activation. We observed a decrease in the number and activation of naïve T cells isolated from aged mice. While young T cells demonstrated robust mitochondrial biogenesis and respiration upon activation, aged T cells generated smaller mitochondria with lower respiratory capacity. Using quantitative proteomics, we defined the aged T cell proteome and discovered a specific deficit in the induction of enzymes of one-carbon metabolism. The activation of aged naïve T cells was enhanced by addition of products of one-carbon metabolism (formate and glycine). These studies define mechanisms of skewed metabolic remodeling in aged T cells and provide evidence that modulation of metabolism has the potential to promote immune function in aged individuals.

Using single-cell RNA sequencing and TCR sequencing, Pauken et al. detect CD8+ T cell clones shared between blood and tumor in mice or melanoma patients, characterize matching clones in blood and tumor, and identify potential biomarkers for their isolation in blood., The ability to monitor anti-tumor CD8+ T cell responses in the blood has tremendous therapeutic potential. Here, we used paired single-cell RNA and TCR sequencing to detect and characterize “tumor-matching” (TM) CD8+ T cells in the blood of mice with MC38 tumors or melanoma patients using the TCR as a molecular barcode. TM cells showed increased activation compared with nonmatching T cells in blood and were less exhausted than matching cells in tumors. Importantly, PD-1, which has been used to identify putative circulating anti-tumor CD8+ T cells, showed poor sensitivity for identifying TM cells. By leveraging the transcriptome, we identified candidate cell surface markers for TM cells in mice and patients and validated NKG2D, CD39, and CX3CR1 in mice. These data show that the TCR can be used to identify tumor-relevant cells for characterization, reveal unique transcriptional properties of TM cells, and develop marker panels for tracking and analysis of these cells., Graphical Abstract

While much is known about the factors that promote the development of diverse Treg cell responses, less is known about the pathways that constrain Treg cell activities. The studies presented here reveal that at homeostasis there is a population of effector Treg cells that express PD-1, and that blockade of PD-L1 or loss of PD-1 results in increased Treg cell activity. In response to infection with the parasite T. gondii, the early production of IFN-γ results in widespread upregulation of PD-L1. Moreover, blockade of PD-L1, whole body deletion of PD-1, or lineage-specific deletion of PD-1 in Foxp3+ cells prevented the loss of the effector Treg cells but resulted in reduced pathogen specific CD4+ T cell responses during infection. Thus, at homeostasis basal PD-L1 expression constrains and tunes the pool of Treg cells, but during infection the upregulation of PD-L1 provides a mechanism to contract the Treg cell population required to maximize the development of pathogen specific CD4+ T cell responses.

The population is aging at a rate never seen before in human history. As the number of elderly adults grows, it is imperative we expand our understanding of the underpinnings of aging biology. Human lungs are composed of a unique panoply of cell types that face ongoing chemical, mechanical, biological, immunological, and xenobiotic stress over a lifetime. Yet, we do not fully appreciate the mechanistic drivers of lung aging and why age increases the risk of parenchymal lung disease, fatal respiratory infection, and primary lung cancer. Here, we review the molecular and cellular aspects of lung aging, local stress response pathways, and how the aging process predisposes to the pathogenesis of pulmonary disease. We place these insights into context of the COVID-19 pandemic and discuss how innate and adaptive immunity within the lung is altered with age.

The process of pyroptosis is mediated by inflammasomes and a downstream effector known as gasdermin D (GSDMD). Upon cleavage by inflammasome-associated caspases, the N-terminal domain of GSDMD forms membrane pores that promote cytolysis. Numerous proteins promote GSDMD cleavage, but none are known to be required for pore formation after GSDMD cleavage. Herein, we report a forward genetic screen that identified the Ragulator-Rag complex as being necessary for GSDMD pore formation and pyroptosis in macrophages. Mechanistic analysis revealed that Ragulator-Rag is not required for GSDMD cleavage upon inflammasome activation, but rather promotes GSDMD oligomerization in the plasma membrane. Defects in GSDMD oligomerization and pore formation can be rescued by mitochondrial poisons that stimulate reactive oxygen species (ROS) production, and ROS modulation impacts the ability of inflammasome pathways to promote pore formation downstream of GSDMD cleavage. These findings reveal an unexpected link between key regulators of immunity (inflammasome-GSDMD) and metabolism (Ragulator-Rag).

The immune system is capable of generating robust antibody responses to foreign antigens during infection and vaccination, while simultaneously limiting antibodies to self-antigens. T follicular regulatory (Tfr) cells are a subset of follicular T cell with specialized roles in regulating humoral immunity. Although Tfr cells have been studied for the past 10 years, their roles have remained elusive. In this review we discuss the current understanding of Tfr cell functions in autoimmunity and how Tfr cells simultaneously control foreign and autoantigen specific antibody responses. We highlight new tools that enable in-depth study of Tfr cells in vivo and recent data suggesting an important role for Tfr cells in limiting participation of autoreactive B cells in germinal centers.

PD-1 (programmed cell death-1) is the central inhibitory receptor regulating CD8 T cell exhaustion during chronic viral infection and cancer. Interestingly, PD-1 is also expressed transiently by activated CD8 T cells during acute viral infection, but the role of PD-1 in modulating T cell effector differentiation and function is not well defined. To address this question, we examined the expression kinetics and role of PD-1 during acute lymphocytic choriomeningitis virus (LCMV) infection of mice. PD-1 was rapidly up-regulated in vivo upon activation of naive virus-specific CD8 T cells within 24 h after LCMV infection and in less than 4 h after peptide injection, well before any cell division had occurred. This rapid PD-1 expression by CD8 T cells was driven predominantly by antigen receptor signaling since infection with a LCMV strain with a mutation in the CD8 T cell epitope did not result in the increase of PD-1 on antigen-specific CD8 T cells. Blockade of the PD-1 pathway using anti-PD-L1 or anti-PD-1 antibodies during the early phase of acute LCMV infection increased mTOR signaling and granzyme B expression in virus-specific CD8 T cells and resulted in faster clearance of the infection. These results show that PD-1 plays an inhibitory role during the naive-to-effector CD8 T cell transition and that the PD-1 pathway can also be modulated at this stage of T cell differentiation. These findings have implications for developing therapeutic vaccination strategies in combination with PD-1 blockade.

Immune responses to cancer are highly variable, with mismatch repair-deficient (MMRd) tumors exhibiting more anti-tumor immunity than mismatch repair-proficient (MMRp) tumors. To understand the rules governing these varied responses, we transcriptionally profiled 371,223 cells from colorectal tumors and adjacent normal tissues of 28 MMRp and 34 MMRd individuals. Analysis of 88 cell subsets and their 204 associated gene expression programs revealed extensive transcriptional and spatial remodeling across tumors. To discover hubs of interacting malignant and immune cells, we identified expression programs in different cell types that co-varied across tumors from affected individuals and used spatial profiling to localize coordinated programs. We discovered a myeloid cell-attracting hub at the tumor-luminal interface associated with tissue damage and an MMRd-enriched immune hub within the tumor, with activated T cells together with malignant and myeloid cells expressing T cell-attracting chemokines. By identifying interacting cellular programs, we reveal the logic underlying spatially organized immune-malignant cell networks.

During infection, antigen-specific T cells undergo tightly regulated developmental transitions controlled by transcriptional and post-transcriptional regulation of gene expression. We found that the microRNA miR-31 was strongly induced by activation of the T cell antigen receptor (TCR) in a pathway involving calcium and activation of the transcription factor NFAT. During chronic infection with lymphocytic choriomeningitis virus (LCMV) clone 13, miR-31-deficent mice recovered from clinical disease, while wild-type mice continued to show signs of disease. This disease phenotype was explained by the presence of larger numbers of cytokine-secreting LCMV-specific CD8+ T cells in miR-31-deficent mice than in wild-type mice. Mechanistically, miR-31 increased the sensitivity of T cells to type I interferons, which interfered with effector T cell function and increased the expression of several proteins related to T cell dysfunction during chronic infection. These studies identify miR-31 as an important regulator of T cell exhaustion in chronic infection.

Both tumor- and host-derived PD-L1 can play critical roles in immunosuppression; differences in tumor immunogenicity appear to underlie their relative contributions. Juneja et al. show that in immunogenic MC38 tumors, PD-L1 on tumor cells dominates in suppressing tumor immunity by inhibiting CD8 T cell cytotoxicity., It is unclear whether PD-L1 on tumor cells is sufficient for tumor immune evasion or simply correlates with an inflamed tumor microenvironment. We used three mouse tumor models sensitive to PD-1 blockade to evaluate the significance of PD-L1 on tumor versus nontumor cells. PD-L1 on nontumor cells is critical for inhibiting antitumor immunity in B16 melanoma and a genetically engineered melanoma. In contrast, PD-L1 on MC38 colorectal adenocarcinoma cells is sufficient to suppress antitumor immunity, as deletion of PD-L1 on highly immunogenic MC38 tumor cells allows effective antitumor immunity. MC38-derived PD-L1 potently inhibited CD8+ T cell cytotoxicity. Wild-type MC38 cells outcompeted PD-L1–deleted MC38 cells in vivo, demonstrating tumor PD-L1 confers a selective advantage. Thus, both tumor- and host-derived PD-L1 can play critical roles in immunosuppression. Differences in tumor immunogenicity appear to underlie their relative importance. Our findings establish reduced cytotoxicity as a key mechanism by which tumor PD-L1 suppresses antitumor immunity and demonstrate that tumor PD-L1 is not just a marker of suppressed antitumor immunity.

Obesity is a major cancer risk factor, but how differences in systemic metabolism change the tumor microenvironment (TME) and impact anti-tumor immunity is not understood. Here, we demonstrate that high-fat diet (HFD)-induced obesity impairs CD8(+) T cell function in the murine TME, accelerating tumor growth. We generate a single-cell resolution atlas of cellular metabolism in the TME, detailing how it changes with diet-induced obesity. We find that tumor and CD8(+) T cells display distinct metabolic adaptations to obesity. Tumor cells increase fat uptake with HFD, whereas tumor-infiltrating CD8(+) T cells do not. These differential adaptations lead to altered fatty acid partitioning in HFD tumors, impairing CD8(+) T cell infiltration and function. Blocking metabolic reprogramming by tumor cells in obese mice improves anti-tumor immunity. Analysis of human cancers reveals similar transcriptional changes in CD8(+) T cell markers, suggesting interventions that exploit metabolism to improve cancer immunotherapy.

'T cell exhaustion' is a broad term that has been used to describe the response of T cells to chronic antigen stimulation, first in the setting of chronic viral infection but more recently in response to tumours. Understanding the features of and pathways to exhaustion has crucial implications for the success of checkpoint blockade and adoptive T cell transfer therapies. In this Viewpoint article, 18 experts in the field tell us what exhaustion means to them, ranging from complete lack of effector function to altered functionality to prevent immunopathology, with potential differences between cancer and chronic infection. Their responses highlight the dichotomy between terminally differentiated exhausted T cells that are TCF1 - and the self-renewing TCF1 + population from which they derive. These TCF1 + cells are considered by some to have stem cell-like properties akin to memory T cell populations, but the developmental relationships are unclear at present. Recent studies have also highlighted an important role for the transcriptional regulator TOX in driving the epigenetic enforcement of exhaustion, but key questions remain about the potential to reverse the epigenetic programme of exhaustion and how this might affect the persistence of T cell populations.

Immunotherapy using checkpoint-blocking antibodies against PD-1 has produced impressive results in a wide range of cancers. However, the response remains heterogeneous among patients. We used noninvasive immuno-positron emission tomography (PET), using (89)Zr-labeled PEGylated single-domain antibody fragments (nanobodies or VHHs), to explore the dynamics and distribution of intratumoral CD8(+) T cells and CD11b(+) myeloid cells in response to anti–PD-1 treatment in the MC38 colorectal mouse adenocarcinoma model. Responding and nonresponding tumors showed consistent differences in the distribution of CD8(+) and CD11b(+) cells. Anti–PD-1 treatment mobilized CD8(+) T cells from the tumor periphery to a more central location. Only those tumors fully infiltrated by CD8(+) T cells went on to complete resolution. All tumors contained CD11b(+) myeloid cells from the outset of treatment, with later recruitment of additional CD11b(+) cells. As tumors grew, the distribution of intratumoral CD11b(+) cells became more heterogeneous. Shrinkage of tumors in responders correlated with an increase in the CD11b(+) population in the center of the tumors. The changes in distribution of CD8(+) and CD11b(+) cells, as assessed by PET, served as biomarkers to gauge the efficacy of anti–PD-1 treatment. Single-cell RNA sequencing of RNA from intratumoral CD45(+) cells showed that CD11b(+) cells in responders and nonresponders were markedly different. The responders exhibited a dominant population of macrophages with an M1-like signature, while the CD45(+) population in the nonresponders displayed an M2-like transcriptional signature. Thus, by using immuno-PET and single-cell RNA sequencing, we show that anti–PD-1 treatment not only affects interactions of CD8(+) T cells with the tumor but also impacts the intratumoral myeloid compartment.

Therapies that target the function of immune cells have significant clinical efficacy in diseases such as cancer and autoimmunity. Although functional genomics has accelerated therapeutic target discovery in cancer, its use in primary immune cells is limited because vector delivery is inefficient and can perturb cell states. Here we describe CHIME: CHimeric IMmune Editing, a CRISPR-Cas9 bone marrow delivery system to rapidly evaluate gene function in innate and adaptive immune cells in vivo without ex vivo manipulation of these mature lineages. This approach enables efficient deletion of genes of interest in major immune lineages without altering their development or function. We use this approach to perform an in vivo pooled genetic screen and identify Ptpn2 as a negative regulator of CD8+ T cell-mediated responses to LCMV Clone 13 viral infection. These findings indicate that this genetic platform can enable rapid target discovery through pooled screening in immune cells in vivo., The use of functional genomics in primary immune cells has been limited by inefficient vector delivery and risk of perturbing cell states. Here the authors present CHimeric IMmune Editing (CHIME) for in vivo evaluation of gene function and pooled screening approaches.

This study investigates the association of PD-L1 expression and immune cell infiltrates and their impact on clinical outcome, in addition to their overlap with microsatellite instability (MSI), HER2 and ATM molecular subgroups of gastric cancer (GC). PD-L1 membrane expression on tumour cells (TC) and infiltrating immune cells (IC), CD3 + T-lymphocytes, CD8+ cytotoxic T-cells, ATM and HER2 were assessed by immunohistochemistry (IHC) in the ACRG (Asian Cancer Research Group) GC cohort (N = 380). EBV status was determined using in situ hybridization and MSI status was performed using PCR and MLH1 IHC. The PD-L1 segment was associated with increased T-cell infiltrates, while the MSI-high segment was enriched for PD-L1, CD3, and CD8. Multivariate analysis confirmed PD-L1 positivity, high CD3 and high CD8 as independent prognostic factors for both disease-free survival and overall survival (all p

Follicular regulatory T (TFR) cells have specialized roles in modulating follicular helper T (TFH) cell activation of B cells. However, the precise role of TFR cells in controlling antibody responses to foreign antigens and autoantigens in vivo is still unclear due to a lack of specific tools. A TFR cell-deleter mouse was developed that selectively deletes TFR cells, facilitating temporal studies. TFR cells were found to regulate early, but not late, germinal center (GC) responses to control antigen-specific antibody and B cell memory. Deletion of TFR cells also resulted in increased self-reactive immunoglobulin (Ig) G and IgE. The increased IgE levels led us to interrogate the role of TFR cells in house dust mite models. TFR cells were found to control TFH13 cell-induced IgE. In vivo, loss of TFR cells increased house-dust-mite-specific IgE and lung inflammation. Thus, TFR cells control IgG and IgE responses to vaccines, allergens and autoantigens, and exert critical immunoregulatory functions before GC formation.

Tan et al. provide novel insights into understanding how PD-1 regulates regulatory T (T reg) cells. More specifically, this study demonstrates that loss of PD-1 on T reg cells leads to enhanced T reg cell suppressor function in vitro and in vivo. These findings have clinical relevance for understanding the efficacy of PD-1– and PD-L1–mediated checkpoint blockade., Inhibitory signals through the PD-1 pathway regulate T cell activation, T cell tolerance, and T cell exhaustion. Studies of PD-1 function have focused primarily on effector T cells. Far less is known about PD-1 function in regulatory T (T reg) cells. To study the role of PD-1 in T reg cells, we generated mice that selectively lack PD-1 in T reg cells. PD-1–deficient T reg cells exhibit an activated phenotype and enhanced immunosuppressive function. The in vivo significance of the potent suppressive capacity of PD-1–deficient T reg cells is illustrated by ameliorated experimental autoimmune encephalomyelitis (EAE) and protection from diabetes in nonobese diabetic (NOD) mice lacking PD-1 selectively in T reg cells. We identified reduced signaling through the PI3K–AKT pathway as a mechanism underlying the enhanced suppressive capacity of PD-1–deficient T reg cells. Our findings demonstrate that cell-intrinsic PD-1 restraint of T reg cells is a significant mechanism by which PD-1 inhibitory signals regulate T cell tolerance and autoimmunity., Graphical Abstract

The ability to monitor anti-tumor CD8+ T cell responses in the blood has tremendous therapeutic potential. Here, we used paired single-cell RNA sequencing and T cell receptor (TCR) sequencing to detect and characterize “tumor matching” (TM) CD8+ T cells in the blood of mice with MC38 tumors and melanoma patients using the TCR as a molecular barcode. TM cells showed increased activation compared to non-matching T cells in blood, and appeared less exhausted than matching counterparts in tumor. Importantly, PD-1, which has been used to identify putative circulating anti-tumor CD8+ T cells, showed poor sensitivity for identifying TM cells. By leveraging the transcriptome we identified candidate cell surface marker panels for TM cells in mice and melanoma patients, and validated markers in mice. Here, using combinations of markers provided better performance than single markers in identifying TM cells from non-TM cells in the blood. These data demonstrate that the TCR can be used to identify tumor-relevant populations for comprehensive characterization, reveal unique transcriptional properties of TM cells, and develop marker panels for tracking and analysis of these cells. Citation Format: Kristen E. Pauken, Osmaan Shahid, Kaitlyn A. Lagattuta, Kelly M. Mahuron, Jacob M. Luber, Margaret M. Lowe, Linglin Huang, Conor Delaney, Jaclyn M. Long, Megan E. Fung, Kathleen Newcomer, Katy K. Tsai, Melissa Chow, Samantha Guinn, Juhi R. Kuchroo, Kelly P. Burke, Jason M. Schenkel, Michael D. Rosenblum, Adil I. Daud, Arlene H. Sharpe, Meromit Singer. Single-cell analyses characterize circulating anti-tumor CD8 T cells and identify markers for their isolation [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO016.

The PD-1 pathway is a cornerstone in immune regulation. While the PD-1 pathway has received considerable attention for its role in contributing to the maintenance of T cell exhaustion in chronic infection and cancer, the PD-1 pathway plays diverse roles in regulating host immunity beyond T cell exhaustion. Here, we discuss emerging concepts in the PD-1 pathway, including (1) the impact of PD-1 inhibitors on diverse T cell differentiation states including effector and memory T cell development during acute infection, as well as T cell exhaustion during chronic infection and cancer, (2) the role of PD-1 in regulating Treg cells, NK cells, and ILCs, and (3) the functions of PD-L1/B7-1 and PD-L2/RGMb/neogenin interactions. We then discuss the emerging use of neoadjuvant PD-1 blockade in the treatment of early-stage cancers and how the timing of PD-1 blockade may improve clinical outcomes. The diverse binding partners of PD-1 and its associated ligands, broad expression patterns of the receptors and ligands, differential impact of PD-1 modulation on cells depending on location and state of differentiation, and timing of PD-1 blockade add additional layers of complexity to the PD-1 pathway, and are important considerations for improving the efficacy and safety of PD-1 pathway therapeutics.

CD48 (SLAMF2) is an adhesion and costimulatory molecule constitutively expressed on hematopoietic cells. Polymorphisms in CD48 have been linked to susceptibility to multiple sclerosis (MS), and altered expression of the structurally related protein CD58 (LFA-3) is associated with disease remission in MS. We examined CD48 expression and function in experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. We found that a subpopulation of CD4+ T cells highly upregulated CD48 expression during EAE and were enriched for pathogenic CD4+ T cells. These CD48++CD4+ T cells were predominantly CD44+ and Ki67+, included producers of IL-17A, GM-CSF, and IFN-γ, and were most of the CD4+ T cells in the CNS. Administration of anti-CD48 mAb during EAE attenuated clinical disease, limited accumulation of lymphocytes in the CNS, and reduced the number of pathogenic cytokine-secreting CD4+ T cells in the spleen at early time points. These therapeutic effects required CD48 expression on CD4+ T cells but not on APCs. Additionally, the effects of anti-CD48 were partially dependent on FcγRs, as anti-CD48 did not ameliorate EAE or reduce the number of cytokine-producing effector CD4+ T cells in Fcεr1γ−/− mice or in wild-type mice receiving anti-CD16/CD32 mAb. Our data suggest that anti-CD48 mAb exerts its therapeutic effects by both limiting CD4+ T cell proliferation and preferentially eliminating pathogenic CD48++CD4+ T cells during EAE. Our findings indicate that high CD48 expression is a feature of pathogenic CD4+ T cells during EAE and point to CD48 as a potential target for immunotherapy.

Follicular regulatory T cells (TFR cells) inhibit follicular helper T cell (TFH cell)-mediated antibody production. Sharpe and colleagues show that TFR cells induce a distinct suppressive state in TFH cells and B cells that can be reversed by the cytokine IL-21. Follicular regulatory T cells (TFR cells) inhibit follicular helper T cell (TFH cell)–mediated antibody production. The mechanisms by which TFR cells exert their key immunoregulatory functions are largely unknown. Here we found that TFR cells induced a distinct suppressive state in TFH cells and B cells, in which effector transcriptional signatures were maintained but key effector molecules and metabolic pathways were suppressed. The suppression of B cell antibody production and metabolism by TFR cells was durable and persisted even in the absence of TFR cells. This durable suppression was due in part to epigenetic changes. The cytokine IL-21 was able to overcome TFR cell–mediated suppression and inhibited TFR cells and stimulated B cells. By determining mechanisms of TFR cell-mediated suppression, we have identified methods for modulating the function of TFR cells and antibody production.

Chronic viral infections are characterized by a state of CD8+ T-cell dysfunction that is associated with expression of the programmed cell death 1 (PD-1) inhibitory receptor. A better understanding of the mechanisms that regulate CD8+ T-cell responses during chronic infection is required to improve immunotherapies that restore function in exhausted CD8+ T cells. Here we identify a population of virus-specific CD8+ T cells that proliferate after blockade of the PD-1 inhibitory pathway in mice chronically infected with lymphocytic choriomeningitis virus (LCMV). These LCMV-specific CD8+ T cells expressed the PD-1 inhibitory receptor, but also expressed several costimulatory molecules such as ICOS and CD28. This CD8+ T-cell subset was characterized by a unique gene signature that was related to that of CD4+ T follicular helper (TFH) cells, CD8+ T cell memory precursors and haematopoietic stem cell progenitors, but that was distinct from that of CD4+ TH1 cells and CD8+ terminal effectors. This CD8+ T-cell population was found only in lymphoid tissues and resided predominantly in the T-cell zones along with naive CD8+ T cells. These PD-1+CD8+ T cells resembled stem cells during chronic LCMV infection, undergoing self-renewal and also differentiating into the terminally exhausted CD8+ T cells that were present in both lymphoid and non-lymphoid tissues. The proliferative burst after PD-1 blockade came almost exclusively from this CD8+ T-cell subset. Notably, the transcription factor TCF1 had a cell-intrinsic and essential role in the generation of this CD8+ T-cell subset. These findings provide a better understanding of T-cell exhaustion and have implications in the optimization of PD-1-directed immunotherapy in chronic infections and cancer.

Immune checkpoint blockade represents a major breakthrough in cancer therapy; however, responses are not universal. Genomic and immune features in pretreatment tumor biopsies have been reported to correlate with response in patients with melanoma and other cancers, but robust biomarkers have not been identified. We studied a cohort of patients with metastatic melanoma initially treated with cytotoxic T-lymphocyte–associated antigen-4 (CTLA4) blockade (n = 53) followed by programmed death-1 (PD-1) blockade at progression (n = 46), and analyzed immune signatures in longitudinal tissue samples collected at multiple time points during therapy. In this study, we demonstrate that adaptive immune signatures in tumor biopsy samples obtained early during the course of treatment are highly predictive of response to immune checkpoint blockade and also demonstrate differential effects on the tumor microenvironment induced by CTLA4 and PD-1 blockade. Importantly, potential mechanisms of therapeutic resistance to immune checkpoint blockade were also identified. Significance: These studies demonstrate that adaptive immune signatures in early on-treatment tumor biopsies are predictive of response to checkpoint blockade and yield insight into mechanisms of therapeutic resistance. These concepts have far-reaching implications in this age of precision medicine and should be explored in immune checkpoint blockade treatment across cancer types. Cancer Discov; 6(8); 827–37. ©2016 AACR. See related commentary by Teng et al., p. 818. This article is highlighted in the In This Issue feature, p. 803

Many pathogens, including Plasmodium spp., exploit the interaction of programmed death-1 (PD-1) with PD-1-ligand-1 (PD-L1) to "deactivate" T cell functions, but the role of PD-L2 remains unclear. We studied malarial infections to understand the contribution of PD-L2 to immunity. Here we have shown that higher PD-L2 expression on blood dendritic cells, from Plasmodium falciparum-infected individuals, correlated with lower parasitemia. Mechanistic studies in mice showed that PD-L2 was indispensable for establishing effective CD4(+) T cell immunity against malaria, because it not only inhibited PD-L1 to PD-1 activity but also increased CD3 and inducible co-stimulator (ICOS) expression on T cells. Importantly, administration of soluble multimeric PD-L2 to mice with lethal malaria was sufficient to dramatically improve immunity and survival. These studies show immuno-regulation by PD-L2, which has the potential to be translated into an effective treatment for malaria and other diseases where T cell immunity is ineffective or short-lived due to PD-1-mediated signaling.

Naive T cell stimulation activates anabolic metabolism to fuel the transition from quiescence to growth and proliferation. Here we show that naive CD4(+) T cell activation induces a unique program of mitochondrial biogenesis and remodeling. Using mass spectrometry, we quantified protein dynamics during T cell activation. We identified substantial remodeling of the mitochondrial proteome over the first 24 hr of T cell activation to generate mitochondria with a distinct metabolic signature, with one-carbon metabolism as the most induced pathway. Salvage pathways and mitochondrial one-carbon metabolism, fed by serine, contribute to purine and thymidine synthesis to enable T cell proliferation and survival. Genetic inhibition of the mitochondrial serine catabolic enzyme SHMT2 impaired T cell survival in culture and antigen-specific T cell abundance in vivo. Thus, during T cell activation, mitochondrial proteome remodeling generates specialized mitochondria with enhanced one-carbon metabolism that is critical for T cell activation and survival.

CD48, a member of the signaling lymphocyte activation molecule family, participates in adhesion and activation of immune cells. Although constitutively expressed on most hematopoietic cells, CD48 is upregulated on subsets of activated cells. CD48 can have activating roles on T cells, antigen presenting cells and granulocytes, by binding to CD2 or bacterial FimH, and through cell intrinsic effects. Interactions between CD48 and its high affinity ligand CD244 are more complex, with both stimulatory and inhibitory outcomes. CD244:CD48 interactions regulate target cell lysis by NK cells and CTLs, which are important for viral clearance and regulation of effector/memory T cell generation and survival. Here we review roles of CD48 in infection, tolerance, autoimmunity, and allergy, as well as the tools used to investigate this receptor. We discuss stimulatory and regulatory roles for CD48, its potential as a therapeutic target in human disease, and current challenges to investigation of this immunoregulatory receptor.

Summary The PD-1 pathway regulates dysfunctional T cells in chronic infection and cancer, but the role of this pathway during acute infection remains less clear. Here, we demonstrate that PD-1 signals are needed for optimal memory. Mice deficient in the PD-1 pathway exhibit impaired CD8+ T cell memory following acute influenza infection, including reduced virus-specific CD8+ T cell numbers and compromised recall responses. PD-1 blockade during priming leads to similar differences early post-infection but without the defect in memory formation, suggesting that timing and/or duration of PD-1 blockade could be tailored to modulate host responses. Our studies reveal a role for PD-1 as an integrator of CD8+ T cell signals that promotes CD8+ T cell memory formation and suggest PD-1 continues to fine-tune CD8+ T cells after they migrate into non-lymphoid tissues. These findings have important implications for PD-1-based immunotherapy, in which PD-1 inhibition may influence memory responses in patients.

Despite the significant clinical responses observed with checkpoint blockade, there is an urgent need to identify new therapeutic targets in immune cells for combination therapies. However, the use of functional genomic approaches in immune cells to discover immunotherapy targets is limited by inefficient vector delivery or perturbation of cell states. To circumvent these limitations, we developed CHIME: CHimeric IMmune Editing, a bone marrow chimeric CRISPR-Cas9 delivery system, to rapidly evaluate gene function in innate and adaptive immune cells in vivo without prior ex vivo manipulation. This approach enables efficient deletion of genes of interest in major immune lineages without altering immune development or function. To discover novel immunotherapy targets we performed an in vivo pooled genetic screen for negative regulators of CD8+ T-cell responses to LCMV Clone 13 viral infection. We found that deletion of the phosphatase Ptpn2 enhances CD8+ T-cell responses to chronic pathogens and cancer. In models of both chronic viral infection and transplantable tumors, Ptpn2 null CD8+ T cells expand more and show increased expression of effector genes compared to wild-type cells. Consistent with this, in the LCMV Clone 13 model, Ptpn2 deletion in CD8+ T cells affects the differentiation and expansion of exhausted subpopulations by increasing IFN-I signaling. Furthermore, deletion of Ptpn2 in the immune system induces a CD8+ T cell-dependent clearance of tumors and synergizes with PD-1 immune checkpoint blockade. Our results suggest that therapeutic inhibition of Ptpn2 in immune cells may enhance CD8+ T-cell effector function and mediate antitumor immunity to improve tumor control. More generally, these findings suggest that this genetic platform can enable rapid target discovery through pooled loss-of-function screening in immune cell lineages in vivo and presents a novel target for potential immune based therapies. Citation Format: Martin LaFleur, Thao Nguyen, Matthew Coxe, Brian Miller, Kathleen Yates, Jacob Gillis, Debattama Sen, John Doench, Nicholas Haining, Arlene Sharpe. CHIME screening identifies PTPN2 as a novel regulator of antitumor immunity [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B47.

Objective Active rheumatoid arthritis (RA) is accompanied by increased appendicular and axial bone loss, closely associated to the degree of inflammation. The programmed death-1 (PD-1) pathway is important for maintaining peripheral tolerance, and its ligand PD-L2 has recently been associated with bone morphogenetic protein activity. Here, we report that PD-L2 plays a central role in RA osteoimmunology. Methods Femoral bone mineral density (BMD) and trabecular bone microstructure were evaluated by micro-CT in wild type (WT) and PD-L2−/− mice. Osteoclasts were generated from RA synovial fluid mononuclear cells and peripheral blood monocytes. The effects of recombinant PD-L2, was evaluated by tartrate-resistant acid phosphatase (TRAP) activity and the development of bone erosions in the presence of anti-citrullinated protein antibodies (ACPA). Plasma soluble (s)PD-L2 levels were measured in patients with early (e)RA (n ​= ​103) treated with methotrexate alone or in combination with the TNF inhibitor Adalimumab. Results PD-L2−/− mice had a decreased BMD and deteriorated trabecular bone microstructure that was not related to the RANKL/OPG pathway. PD-L2 decreased TRAP activity in osteoclasts and decreased ACPA-induced erosions. In the RA synovial membrane PD-L2 was highly expressed especially in the lining layer and plasma sPD-L2 levels were increased in eRA patients and decreased with treatment. One-year sPD-L2 correlated inversely with erosive progression two years after treatment initiation with methotrexate and placebo. Conclusion PD-L2 regulates bone homeostasis in RA. Our findings provide new insight into the relationship between the immune system and bone homeostasis, and suggest a potential therapeutic target for limiting inflammatory bone loss in RA., Highlights • PD-L2 is closely related to bone homeostasis in a mouse model. • PD-L2 inhibits osteoclastogenesis and osteoclast activation in vitro. • PD-L2 is highly expressed by cells in the synovial membrane of rheumatoid arthritis. • PD-L2 is associated with less radiographic progression in patients with early rheumatoid arthritis.

Paterson et al. demonstrate that, in contrast to CTLA-4 germline knockout mice, conditional deletion on T reg cells during adulthood confers protection from EAE and does not increase resistance to tumors., Cytotoxic T lymphocyte antigen-4 (CTLA-4) is an essential negative regulator of T cell responses. Germline Ctla4 deficiency is lethal, making investigation of the function of CTLA-4 on mature T cells challenging. To elucidate the function of CTLA-4 on mature T cells, we have conditionally ablated Ctla4 in adult mice. We show that, in contrast to germline knockout mice, deletion of Ctla4 during adulthood does not precipitate systemic autoimmunity, but surprisingly confers protection from experimental autoimmune encephalomyelitis (EAE) and does not lead to increased resistance to MC38 tumors. Deletion of Ctla4 during adulthood was accompanied by activation and expansion of both conventional CD4+Foxp3− (T conv) and regulatory Foxp3+ (T reg cells) T cell subsets; however, deletion of CTLA-4 on T reg cells was necessary and sufficient for protection from EAE. CTLA-4 deleted T reg cells remained functionally suppressive. Deletion of Ctla4 on T reg cells alone or on all adult T cells led to major changes in the Ctla4 sufficient T conv cell compartment, including up-regulation of immunoinhibitory molecules IL-10, LAG-3 and PD-1, thereby providing a compensatory immunosuppressive mechanism. Collectively, our findings point to a profound role for CTLA-4 on T reg cells in limiting their peripheral expansion and activation, thereby regulating the phenotype and function of T conv cells.

Therapeutic antibodies targeting programmed cell death-1 (PD-1) activate tumor-specific immunity and have shown remarkable efficacy in the treatment of melanoma. Yet, little is known about tumor cell-intrinsic PD-1 pathway effects. Here we show that murine and human melanomas contain PD-1-expressing cancer subpopulations and demonstrate that melanoma cell-intrinsic PD-1 promotes tumorigenesis, even in mice lacking adaptive immunity. PD-1 inhibition on melanoma cells by RNA interference, blocking antibodies, or mutagenesis of melanoma-PD-1 signaling motifs suppresses tumor growth in immunocompetent, immunocompromised and PD-1-deficient tumor graft recipient mice. Conversely, melanoma-specific PD-1 overexpression enhances tumorigenicity, as does engagement of melanoma-PD-1 by its ligand, PD-L1, whereas melanoma-PD-L1 inhibition or knockout of host-PD-L1 attenuate growth of PD-1-positive melanomas. Mechanistically, the melanoma-PD-1 receptor modulates downstream effectors of mTOR signaling. Our results identify melanoma cell-intrinsic functions of the PD-1:PD-L1 axis in tumor growth and suggest that blocking melanoma-PD-1 might contribute to the striking clinical efficacy of anti-PD-1 therapy.

Summary Defective antibody production in aging is broadly attributed to immunosenescence. However, the precise immunological mechanisms remain unclear. Here, we demonstrate an increase in the ratio of inhibitory T follicular regulatory (TFR) cells to stimulatory T follicular helper (TFH) cells in aged mice. Aged TFH and TFR cells are phenotypically distinct from those in young mice, exhibiting increased programmed cell death protein-1 expression but decreased ICOS expression. Aged TFH cells exhibit defective antigen-specific responses, and programmed cell death protein-ligand 1 blockade can partially rescue TFH cell function. In contrast, young and aged TFR cells have similar suppressive capacity on a per-cell basis in vitro and in vivo. Together, these studies reveal mechanisms contributing to defective humoral immunity in aging: an increase in suppressive TFR cells combined with impaired function of aged TFH cells results in reduced T-cell-dependent antibody responses in aged mice.

SummaryAllergic asthma is caused by Th2-cell-type cytokines in response to allergen exposure. Type 2 innate lymphoid cells (ILC2s) are a newly identified subset of immune cells that, along with Th2 cells, contribute to the pathogenesis of asthma by producing copious amounts of IL-5 and IL-13, which cause eosinophilia and airway hyperreactivity (AHR), a cardinal feature of asthma. ILC2s express ICOS, a T cell costimulatory molecule with a currently unknown function. Here we showed that a lack of ICOS on murine ILC2s and blocking the ICOS:ICOS-ligand interaction in human ILC2s reduced AHR and lung inflammation. ILC2s expressed both ICOS and ICOS-ligand, and the ICOS:ICOS-ligand interaction promoted cytokine production and survival in ILC2s through STAT5 signaling. Thus, ICOS:ICOS-ligand signaling pathway is critically involved in ILC2 function and homeostasis.

A metabolic organ, the liver also has a central role in tolerance induction. Stromal cells lining the hepatic sinusoids, such as liver sinusoidal endothelial cells (LSECs) and hepatic stellate cells (HSCs), are the first liver cells to encounter gut-derived and systemic antigens, thereby shaping local and systemic tolerance. Recent studies have demonstrated that stromal cells can modulate immune responses by antigen-dependent and independent mechanisms. Stromal cells interfere with the function of other antigen-presenting cells (APCs) and induce non-responsive T cells as well as regulatory T cells and myeloid-derived suppressor cells (MDSCs). The immunosuppressive microenvironment thus created provides a means to protect the liver from tissue damage. Such tolerized surroundings, however, can be exploited by certain pathogens, promoting persistent liver infections.

Cutaneous leishmaniasis, caused mainly by Leishmania major , an obligate intracellular parasite, is a disfiguring disease characterized by large skin lesions and is transmitted by a sand fly vector. We previously showed that the chemokine receptor CXCR3 plays a critical role in mediating resistance to cutaneous leishmaniasis caused by Leishmania major . Furthermore, T cells from L. major -susceptible BALB/c but not L. major -resistant C57BL/6 mice fail to efficiently upregulate CXCR3 upon activation. We therefore examined whether transgenic expression of CXCR3 on T cells would enhance resistance to L. major infection in susceptible BALB/c mice. We generated BALB/c and C57BL/6 transgenic mice, which constitutively overexpressed CXCR3 under a CD2 promoter, and then examined the outcomes with L. major infection. Contrary to our hypothesis, transgenic expression of CXCR3 (CXCR3 Tg ) on T cells of BALB/c mice resulted in increased lesion sizes and parasite burdens compared to wild-type (WT) littermates after L. major infection. Restimulated lymph node cells from L. major -infected BALB/c-CXCR3 Tg mice produced more interleukin-4 (IL-4) and IL-10 and less gamma interferon (IFN-γ). Cells in draining lymph nodes from BALB/c-CXCR3 Tg mice showed enhanced Th2 and reduced Th1 cell accumulation associated with increased neutrophils and inflammatory monocytes. However, monocytes displayed an immature phenotype which correlated with increased parasite burdens. Interestingly, transgenic expression of CXCR3 on T cells did not impact the outcome of L. major infection in C57BL/6 mice, which mounted a predominantly Th1 response and spontaneously resolved their infection similar to WT littermates. Our findings demonstrate that transgenic expression of CXCR3 on T cells increases susceptibility of BALB/c mice to L. major .

Ox40 ligand (Ox40L)-Ox40 pathway has been shown to enhance Th2 responses and play a role in pathogenesis of cutaneous leishmaniasis (CL) caused by Leishmania major. Using Ox40l(-/-) BALB/c mice we analyzed the role of this pathway in determining the outcome to CL caused by L. mexicana and compared to L. major. Contrary to our expectations, Ox40l(-/-) mice were highly susceptible to both L. major (LV39) and L. mexicana (M379) and developed large non-healing lesions containing parasites comparable to Ox40l(+/+) BALB/c mice. Interestingly, upon in vitro stimulation with Leishmania antigen (LmAg), the lymph node cells from L. major infected Ox40l(-/-) mice produced significantly less IL-4 and IL-10 compared to Ox40l(+/+) mice. L. mexicana infected Ox40l(-/-) and Ox40l(+/+) mice did not show any difference in the production of IL-4 and IL-10. No difference was noted in the amount of Th1 cytokines IFN-ү and IL-12 produced by Ox40l(-/-) and Ox40l(+/+) mice infected with either parasite. These results indicate that the Ox40L-Ox40 pathway promotes Th2 bias only in L. major infection but not L. mexicana infection and this pathway is not critical for susceptibility to CL.

SummaryThe receptor CTLA-4 has been implicated in controlling B cell responses, but the mechanisms by which CTLA-4 regulates antibody production are not known. Here we showed deletion of CTLA-4 in adult mice increased Tfh and Tfr cell numbers and augmented B cell responses. In the effector phase, loss of CTLA-4 on Tfh cells resulted in heightened B cell responses, whereas loss of CTLA-4 on Tfr cells resulted in defective suppression of antigen-specific antibody responses. We also found that non-Tfr Treg cells could suppress B cell responses through CTLA-4 and that Treg and/or Tfr cells might downregulate B7-2 on B cells outside germinal centers as a means of suppression. Within the germinal center, however, Tfr cells potently suppress B cells through CTLA-4, but with a mechanism independent of altering B7-1 or B7-2. Thus, we identify multifaceted regulatory roles for CTLA-4 in Tfh, Tfr, and Treg cells, which together control humoral immunity.

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.

Immunotherapy with PD-1 checkpoint blockade is effective in only a minority of patients with cancer, suggesting that additional treatment strategies are needed. Here we use a pooled in vivo genetic screening approach using CRISPR-Cas9 genome editing in transplantable tumours in mice treated with immunotherapy to discover previously undescribed immunotherapy targets. We tested 2,368 genes expressed by melanoma cells to identify those that synergize with or cause resistance to checkpoint blockade. We recovered the known immune evasion molecules PD-L1 and CD47, and confirmed that defects in interferon-γ signalling caused resistance to immunotherapy. Tumours were sensitized to immunotherapy by deletion of genes involved in several diverse pathways, including NF-κB signalling, antigen presentation and the unfolded protein response. In addition, deletion of the protein tyrosine phosphatase PTPN2 in tumour cells increased the efficacy of immunotherapy by enhancing interferon-γ-mediated effects on antigen presentation and growth suppression. In vivo genetic screens in tumour models can identify new immunotherapy targets in unanticipated pathways.

Follicular regulatory T (TFR) cells are a newly defined regulatory T cell (Treg) subset that suppresses follicular helper T cell–mediated B cell responses in the germinal center reaction. The precise costimulatory signal requirements for proper TFR cell differentiation and function are still not known. Using conditional knockout strategies of CD28, we previously demonstrated that loss of CD28 signaling in Tregs caused autoimmunity in mice (termed CD28-ΔTreg mice), characterized by lymphadenopathy, accumulation of activated T cells, and cell-mediated inflammation of the skin and lung. In this study, we show that CD28 signaling is required for TFR cell differentiation. Treg-specific deletion of CD28 caused a reduction in TFR cell numbers and function, which resulted in increased germinal center B cells and Ab production. Moreover, residual CD28-deficient TFR cells showed a diminished suppressive capacity as assessed by their ability to inhibit Ab responses in vitro. Surprisingly, genetic deletion of B cells in CD28-ΔTreg mice prevented the development of lymphadenopathy and CD4+ T cell activation, and autoimmunity that mainly targeted skin and lung tissues. Thus, autoimmunity occurring in mice with CD28-deficient Tregs appears to be driven primarily by loss of TFR cell differentiation and function with resulting B cell–driven inflammation.

Follicular Tregs (Tfr cells) inhibit antibody production, whereas follicular Th cells (Tfh cells) stimulate it. Tfr cells are found in blood; however, relatively little is known about the developmental signals for these cells or their functions. Here we demonstrated that circulating Tfr and Tfh cells share properties of memory cells and are distinct from effector Tfr and Tfh cells found within lymph nodes (LNs). Circulating memory-like Tfh cells were potently reactivated by DCs, homed to germinal centers, and produced more cytokines than did effector LN Tfh cells. Circulating memory-like Tfr cells persisted for long periods of time in vivo and homed to germinal centers after reactivation. Effector LN Tfr cells suppressed Tfh cell activation and production of cytokines, including IL-21, and inhibited class switch recombination and B cell activation. The suppressive function of this population was not dependent on specific antigen. Similar to LN effector Tfr cells, circulating Tfr cells also suppressed B and Tfh cells, but with a much lower capacity. Our data indicate that circulating memory-like Tfr cells are less suppressive than LN Tfr cells and circulating memory-like Tfh cells are more potent than LN effector Tfh cells; therefore, these circulating populations can provide rapid and robust systemic B cell help during secondary antigen exposure.