X-CHIME enables combinatorial, inducible, lineage-specific and sequential knockout of genes in the immune system.

Annotation of immunologic gene function in vivo typically requires the generation of knockout mice, which is time consuming and low throughput. We previously developed CHimeric IMmune Editing (CHIME), a CRISPR-Cas9 bone marrow delivery system for constitutive, ubiquitous deletion of single genes. Here we describe X-CHIME, four new CHIME-based systems for modular and rapid interrogation of gene function combinatorially (C-CHIME), inducibly (I-CHIME), lineage-specifically (L-CHIME) or sequentially (S-CHIME). We use C-CHIME and S-CHIME to assess the consequences of combined deletion of Ptpn1 and Ptpn2, an embryonic lethal gene pair, in adult mice. We find that constitutive deletion of both PTPN1 and PTPN2 leads to bone marrow hypoplasia and lethality, while inducible deletion after immune development leads to enteritis and lethality. These findings demonstrate that X-CHIME can be used for rapid mechanistic evaluation of genes in distinct in vivo contexts and that PTPN1 and PTPN2 have some functional redundancy important for viability in adult mice.

Epigenetic tuning of PD-1 expression improves exhausted T cell function and viral control.

PD-1 is a key negative regulator of CD8+ T cell activation and is highly expressed by exhausted T cells in cancer and chronic viral infection. Although PD-1 blockade can improve viral and tumor control, physiological PD-1 expression prevents immunopathology and improves memory formation. The mechanisms driving high PD-1 expression in exhaustion are not well understood and could be critical to disentangling its beneficial and detrimental effects. Here, we functionally interrogated the epigenetic regulation of PD-1 using a mouse model with deletion of an exhaustion-specific PD-1 enhancer. Enhancer deletion exclusively alters PD-1 expression in CD8+ T cells in chronic infection, creating a 'sweet spot' of intermediate expression where T cell function is optimized compared to wild-type and Pdcd1-knockout cells. This permits improved control of chronic infection without additional immunopathology. Together, these results demonstrate that tuning PD-1 via epigenetic editing can reduce CD8+ T cell dysfunction while avoiding excess immunopathology.

Control of gasdermin D oligomerization and pyroptosis by the Ragulator-Rag-mTORC1 pathway.

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).

Obesity Shapes Metabolism in the Tumor Microenvironment to Suppress Anti-Tumor 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.

LSD1 Ablation Stimulates Anti-tumor Immunity and Enables Checkpoint Blockade.

Chromatin regulators play a broad role in regulating gene expression and, when gone awry, can lead to cancer. Here, we demonstrate that ablation of the histone demethylase LSD1 in cancer cells increases repetitive element expression, including endogenous retroviral elements (ERVs), and decreases expression of RNA-induced silencing complex (RISC) components. Significantly, this leads to double-stranded RNA (dsRNA) stress and activation of type 1 interferon, which stimulates anti-tumor T cell immunity and restrains tumor growth. Furthermore, LSD1 depletion enhances tumor immunogenicity and T cell infiltration in poorly immunogenic tumors and elicits significant responses of checkpoint blockade-refractory mouse melanoma to anti-PD-1 therapy. Consistently, TCGA data analysis shows an inverse correlation between LSD1 expression and CD8+ T cell infiltration in various human cancers. Our study identifies LSD1 as a potent inhibitor of anti-tumor immunity and responsiveness to immunotherapy and suggests LSD1 inhibition combined with PD-(L)1 blockade as a novel cancer treatment strategy.

PTPN2 regulates the generation of exhausted CD8+ T cell subpopulations and restrains tumor immunity.

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.

Fibroblastic reticular cells enhance T cell metabolism and survival via epigenetic remodeling.

Lymph node fibroblastic reticular cells (FRCs) respond to signals from activated T cells by releasing nitric oxide, which inhibits T cell proliferation and restricts the size of the expanding T cell pool. Whether interactions with FRCs also support the function or differentiation of activated CD8+ T cells is not known. Here we report that encounters with FRCs enhanced cytokine production and remodeled chromatin accessibility in newly activated CD8+ T cells via interleukin-6. These epigenetic changes facilitated metabolic reprogramming and amplified the activity of pro-survival pathways through differential transcription factor activity. Accordingly, FRC conditioning significantly enhanced the persistence of virus-specific CD8+ T cells in vivo and augmented their differentiation into tissue-resident memory T cells. Our study demonstrates that FRCs play a role beyond restricting T cell expansion-they can also shape the fate and function of CD8+ T cells.

Subsets of exhausted CD8+ T cells differentially mediate tumor control and respond to checkpoint blockade.

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.

Author Correction: Subsets of exhausted CD8+ T cells differentially mediate tumor control and respond to checkpoint blockade.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Neuropeptide signalling orchestrates T cell differentiation.

The balance between T helper type 1 (TH1) cells and other TH cells is critical for antiviral and anti-tumour responses1-3, but how this balance is achieved remains poorly understood. Here we dissected the dynamic regulation of TH1 cell differentiation during in vitro polarization, and during in vivo differentiation after acute viral infection. We identified regulators modulating T helper cell differentiation using a unique TH1-TH2 cell dichotomous culture system and systematically validated their regulatory functions through multiple in vitro and in vivo CRISPR screens. We found that RAMP3, a component of the receptor for the neuropeptide CGRP (calcitonin gene-related peptide), has a cell-intrinsic role in TH1 cell fate determination. Extracellular CGRP signalling through the receptor RAMP3-CALCRL restricted the differentiation of TH2 cells, but promoted TH1 cell differentiation through the activation of downstream cAMP response element-binding protein (CREB) and activating transcription factor 3 (ATF3). ATF3 promoted TH1 cell differentiation by inducing the expression of Stat1, a key regulator of TH1 cell differentiation. After viral infection, an interaction between CGRP produced by neurons and RAMP3 expressed on T cells enhanced the anti-viral IFNγ-producing TH1 and CD8+ T cell response, and timely control of acute viral infection. Our research identifies a neuroimmune circuit in which neurons participate in T cell fate determination by producing the neuropeptide CGRP during acute viral infection, which acts on RAMP3-expressing T cells to induce an effective anti-viral TH1 cell response.

Targeting PD-L2-RGMb overcomes microbiome-related immunotherapy resistance.

The gut microbiota is a crucial regulator of anti-tumour immunity during immune checkpoint inhibitor therapy. Several bacteria that promote an anti-tumour response to immune checkpoint inhibitors have been identified in mice1-6. Moreover, transplantation of faecal specimens from responders can improve the efficacy of anti-PD-1 therapy in patients with melanoma7,8. However, the increased efficacy from faecal transplants is variable and how gut bacteria promote anti-tumour immunity remains unclear. Here we show that the gut microbiome downregulates PD-L2 expression and its binding partner repulsive guidance molecule b (RGMb) to promote anti-tumour immunity and identify bacterial species that mediate this effect. PD-L1 and PD-L2 share PD-1 as a binding partner, but PD-L2 can also bind RGMb. We demonstrate that blockade of PD-L2-RGMb interactions can overcome microbiome-dependent resistance to PD-1 pathway inhibitors. Antibody-mediated blockade of the PD-L2-RGMb pathway or conditional deletion of RGMb in T cells combined with an anti-PD-1 or anti-PD-L1 antibody promotes anti-tumour responses in multiple mouse tumour models that do not respond to anti-PD-1 or anti-PD-L1 alone (germ-free mice, antibiotic-treated mice and even mice colonized with stool samples from a patient who did not respond to treatment). These studies identify downregulation of the PD-L2-RGMb pathway as a specific mechanism by which the gut microbiota can promote responses to PD-1 checkpoint blockade. The results also define a potentially effective immunological strategy for treating patients who do not respond to PD-1 cancer immunotherapy.

In Vivo CRISPR Screening Reveals CHD7 as a Positive Regulator of Short-lived Effector Cells.

CD8+ T cells differentiate into two subpopulations in response to acute viral infection: memory precursor effector cells (MPECs) and short-lived effector cells (SLECs). MPECs and SLECs are epigenetically distinct; however, the epigenetic regulators required for formation of these subpopulations are mostly unknown. In this study, we performed an in vivo CRISPR screen in murine naive CD8+ T cells to identify the epigenetic regulators required for MPEC and SLEC formation, using the acute lymphocytic choriomeningitis virus Armstrong infection model. We identified the ATP-dependent chromatin remodeler CHD7 (chromodomain-helicase DNA-binding protein 7) as a positive regulator of SLEC formation, as knockout (KO) of Chd7 reduced SLECs numerically. In contrast, KO of Chd7 increased the formation of central memory T cells following pathogen clearance yet attenuated memory cell expansion following a rechallenge. These findings establish CHD7 as a novel positive regulator of SLEC and a negative regulator of central memory T cell formation.

In vivo CRISPR screening identifies Ptpn2 as a cancer immunotherapy target.

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.

Immuno-PET identifies the myeloid compartment as a key contributor to the outcome of the antitumor response under PD-1 blockade.

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 89Zr-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.

Inhibitors of the PD-1 Pathway in Tumor Therapy.

The programmed death 1 (PD-1) pathway delivers inhibitory signals that function as a brake for immune responses. This pathway limits the initiation and duration of immune responses, thereby protecting tissues from immune-mediated damage and autoimmune diseases. However, the PD-1 pathway also inhibits immune responses to tumors. The critical role of PD-1 in preventing antitumor immunity is demonstrated by the transformative effects of PD-1 pathway blockade in a broad range of cancers with the hallmark of durability of response. Despite this success, most patients do not respond to PD-1 monotherapy, and some patients experience adverse events. In this review, we discuss the functions of the PD-1 pathway and its translation to cancer immunotherapy. We also consider current challenges and opportunities for PD-1 cancer immunotherapy, including mechanisms of response and resistance, identification of biomarkers of response to PD-1 therapy, characterization and treatment of PD-1 therapy-related adverse events, and development of safe and effective combination therapies.

Framework for in vivo T cell screens.

In vivo T cell screens are a powerful tool for elucidating complex mechanisms of immunity, yet there is a lack of consensus on the screen design parameters required for robust in vivo screens: gene library size, cell transfer quantity, and number of mice. Here, we describe the Framework for In vivo T cell Screens (FITS) to provide experimental and analytical guidelines to determine optimal parameters for diverse in vivo contexts. As a proof-of-concept, we used FITS to optimize the parameters for a CD8+ T cell screen in the B16-OVA tumor model. We also included unique molecular identifiers (UMIs) in our screens to (1) improve statistical power and (2) track T cell clonal dynamics for distinct gene knockouts (KOs) across multiple tissues. These findings provide an experimental and analytical framework for performing in vivo screens in immune cells and illustrate a case study for in vivo T cell screens with UMIs.

Formate Supplementation Enhances Antitumor CD8+ T-cell Fitness and Efficacy of PD-1 Blockade.

The tumor microenvironment (TME) restricts antitumor CD8+ T-cell function and immunotherapy responses. Cancer cells compromise the metabolic fitness of CD8+ T cells within the TME, but the mechanisms are largely unknown. Here we demonstrate that one-carbon (1C) metabolism is enhanced in T cells in an antigen-specific manner. Therapeutic supplementation of 1C metabolism using formate enhances CD8+ T-cell fitness and antitumor efficacy of PD-1 blockade in B16-OVA tumors. Formate supplementation drives transcriptional alterations in CD8+ T-cell metabolism and increases gene signatures for cellular proliferation and activation. Combined formate and anti-PD-1 therapy increases tumor-infiltrating CD8+ T cells, which are essential for enhanced tumor control. Our data demonstrate that formate provides metabolic support to CD8+ T cells reinvigorated by anti-PD-1 to overcome a metabolic vulnerability in 1C metabolism in the TME to further improve T-cell function. SIGNIFICANCE: This study identifies that deficiencies in 1C metabolism limit the efficacy of PD-1 blockade in B16-OVA tumors. Supplementing 1C metabolism with formate during anti-PD-1 therapy enhances CD8+ T-cell fitness in the TME and CD8+ T-cell-mediated tumor clearance. These findings demonstrate that formate supplementation can enhance exhausted CD8+ T-cell function. See related commentary by Lin et al., p. 2507. This article is featured in Selected Articles from This Issue, p. 2489.

CRISPR Screens to Identify Regulators of Tumor Immunity.

Cancer immunotherapies, such as immune checkpoint blockade (ICB), have been used in a wide range of tumor types with immense clinical benefit. However, ICB does not work in all patients, and attempts to combine ICB with other immune-based therapies have not lived up to their initial promise. Thus, there is a significant unmet need to discover new targets and combination therapies to extend the benefits of immunotherapy to more patients. Systems biology approaches are well suited for addressing this problem because these approaches enable evaluation of many gene targets simultaneously and ranking their relative importance for a phenotype of interest. As such, loss-of-function CRISPR screens are an emerging set of tools being used to prioritize gene targets for modulating pathways of interest in tumor and immune cells. This review describes the first screens performed to discover cancer immunotherapy targets and the technological advances that will enable next-generation screens.

Batf-mediated epigenetic control of effector CD8+ T cell differentiation.

The response of naive CD8+ T cells to their cognate antigen involves rapid and broad changes to gene expression that are coupled with extensive chromatin remodeling, but the mechanisms governing these changes are not fully understood. Here, we investigated how these changes depend on the basic leucine zipper ATF-like transcription factor Batf, which is essential for the early phases of the process. Through genome scale profiling, we characterized the role of Batf in chromatin organization at several levels, including the accessibility of key regulatory regions, the expression of their nearby genes, and the interactions that these regions form with each other and with key transcription factors. We identified a core network of transcription factors that cooperated with Batf, including Irf4, Runx3, and T-bet, as indicated by their colocalization with Batf and their binding in regions whose accessibility, interactions, and expression of nearby genes depend on Batf. We demonstrated the synergistic activity of this network by overexpressing the different combinations of these genes in fibroblasts. Batf and Irf4, but not Batf alone, were sufficient to increase accessibility and transcription of key loci, normally associated with T cell function. Addition of Runx3 and T-bet further contributed to fine-tuning of these changes and was essential for establishing chromatin loops characteristic of T cells. These data provide a resource for studying the epigenomic and transcriptomic landscape of effector differentiation of cytotoxic T cells and for investigating the interdependency between transcription factors and its effects on the epigenome and transcriptome of primary cells.