Human regulatory T (Treg) cells are essential for immune homeostasis. The transcription factor FOXP3 maintains Treg cell identity, yet the complete set of key transcription factors that control Treg cell gene expression remains unknown. Here, we used pooled and arrayed Cas9 ribonucleoprotein screens to identify transcription factors that regulate critical proteins in primary human Treg cells under basal and proinflammatory conditions. We then generated 54,424 single-cell transcriptomes from Treg cells subjected to genetic perturbations and cytokine stimulation, which revealed distinct gene networks individually regulated by FOXP3 and PRDM1, in addition to a network coregulated by FOXO1 and IRF4. We also discovered that HIVEP2, to our knowledge not previously implicated in Treg cell function, coregulates another gene network with SATB1 and is important for Treg cell-mediated immunosuppression. By integrating CRISPR screens and single-cell RNA-sequencing profiling, we have uncovered transcriptional regulators and downstream gene networks in human Treg cells that could be targeted for immunotherapies.

Regulatory T (Treg) cells are required to control immune responses and maintain homeostasis, but are a significant barrier to antitumour immunity1. Conversely, Treg instability, characterized by loss of the master transcription factor Foxp3 and acquisition of proinflammatory properties2, can promote autoimmunity and/or facilitate more effective tumour immunity3,4. A comprehensive understanding of the pathways that regulate Foxp3 could lead to more effective Treg therapies for autoimmune disease and cancer. The availability of new functional genetic tools has enabled the possibility of systematic dissection of the gene regulatory programs that modulate Foxp3 expression. Here we developed a CRISPR-based pooled screening platform for phenotypes in primary mouse Treg cells and applied this technology to perform a targeted loss-of-function screen of around 500 nuclear factors to identify gene regulatory programs that promote or disrupt Foxp3 expression. We identified several modulators of Foxp3 expression, including ubiquitin-specific peptidase 22 (Usp22) and ring finger protein 20 (Rnf20). Usp22, a member of the deubiquitination module of the SAGA chromatin-modifying complex, was revealed to be a positive regulator that stabilized Foxp3 expression; whereas the screen suggested that Rnf20, an E3 ubiquitin ligase, can serve as a negative regulator of Foxp3. Treg-specific ablation of Usp22 in mice reduced Foxp3 protein levels and caused defects in their suppressive function that led to spontaneous autoimmunity but protected against tumour growth in multiple cancer models. Foxp3 destabilization in Usp22-deficient Treg cells could be rescued by ablation of Rnf20, revealing a reciprocal ubiquitin switch in Treg cells. These results reveal previously unknown modulators of Foxp3 and demonstrate a screening method that can be broadly applied to discover new targets for Treg immunotherapies for cancer and autoimmune disease.

Monoclonal antibodies (abs) targeting the programmed cell death 1 (PD-1) immune checkpoint pathway have revolutionized tumor therapy. Because T-cell-directed PD-1 blockade boosts tumor immunity, anti-PD-1 abs have been developed for examining T-cell-PD-1 functions. More recently, PD-1 expression has also been reported directly on cancer cells of various etiology, including in melanoma. Nevertheless, there is a paucity of studies validating anti-PD-1 ab clone utility in specific assay types for characterizing tumor cell-intrinsic PD-1. Here, we demonstrate reactivity of several anti-murine PD-1 ab clones and recombinant PD-L1 with live B16-F10 melanoma cells and YUMM lines using multiple independent methodologies, positive and negative PD-1-specific controls, including PD-1-overexpressing and PD-1 knockout cells. Flow cytometric analyses with two separate anti-PD-1 ab clones, 29F.1A12 and RMP1-30, revealed PD-1 surface protein expression on live murine melanoma cells, which was corroborated by marked enrichment in PD-1 gene (Pdcd1) expression. Immunoblotting, immunoprecipitation, and mass spectrometric sequencing confirmed PD-1 protein expression by B16-F10 cells. Recombinant PD-L1 also recognized melanoma cell-expressed PD-1, the blockade of which by 29F.1A12 fully abrogated PD-1:PD-L1 binding. Together, our data provides multiple lines of evidence establishing PD-1 expression by live murine melanoma cells and validates ab clones and assay systems for tumor cell-directed PD-1 pathway investigations.

A persistent concern with CRISPR-Cas9 gene editing has been the potential to generate mutations at off-target genomic sites. While CRISPR-engineering mice to delete a ~360 bp intronic enhancer, here we discovered a founder line that had marked immune dysregulation caused by a 24 kb tandem duplication of the sequence adjacent to the on-target deletion. Our results suggest unintended repair of on-target genomic cuts can cause pathogenic "bystander" mutations that escape detection by routine targeted genotyping assays.

Hermansky-Pudlak syndrome (HPS) is a group of rare autosomal recessive disorders characterized by oculocutaneous albinism, a bleeding tendency, and sporadic pulmonary fibrosis, granulomatous colitis or infections. Nine HPS-causing genes have been identified in humans. HPS-1 is the most severe subtype with a prevalence of ~1/1800 in northwest Puerto Rico due to a founder mutation in the HPS1 gene. Mutations in HPS genes affect the biogenesis of lysosome-related organelles such as melanosomes in melanocytes and platelet dense granules. Two of these genes (HPS1 and HPS4) encode the HPS1 and HPS4 proteins, which assemble to form a complex known as Biogenesis of Lysosome-related Organelle Complex 3 (BLOC-3). We report the identification of the interacting regions in HPS1 and HPS4 required for the formation of this complex. Two regions in HPS1, spanning amino acids 1-249 and 506-700 are required for binding to HPS4; the middle portion of HPS1 (residues 250-505) is not required for this interaction. Further interaction studies showed that the N-termini of HPS1 and HPS4 interact with each other and that a discrete region of HPS4 (residues 340-528) interacts with both the N- and C-termini of the HPS1 protein. Several missense mutations found in HPS-1 patients did not affect interaction with HPS4, but some mutations involving regions interacting with HPS4 caused instability of HPS1. These observations extend our understanding of BLOC-3 assembly and represent an important first step in the identification of domains responsible for the biogenesis of lysosome-related organelles.

Although the presence of tumor-infiltrating lymphocytes (TILs) indicates an endogenous antitumor response, immune regulatory pathways can subvert the effector phase and enable tumor escape. Negative regulatory pathways include extrinsic suppression mechanisms, but also a T cell-intrinsic dysfunctional state. A more detailed study has been hampered by a lack of cell surface markers defining tumor-specific dysfunctional TILs, and PD-1 alone is not sufficient. Recently, we identified the transcription factor Egr2 as a critical component in controlling the anergic state in vitro. In this study, we show that the Egr2-driven cell surface proteins LAG-3 and 4-1BB can identify dysfunctional tumor antigen-specific CD8+ TIL. Co-expression of 4-1BB and LAG-3 was seen on a majority of CD8+ TILs, but not in lymphoid organs. Functional analysis revealed defective IL-2 and TNF production yet retained expression of IFN-γ and regulatory T cell-recruiting chemokines. Transcriptional and phenotypic characterization revealed coexpression of multiple additional co-stimulatory and co-inhibitory receptors. Administration of anti-LAG-3 plus anti-4-1BB mAbs was therapeutic against tumors in vivo, which correlated with phenotypic normalization. Our results indicate that coexpression of LAG-3 and 4-1BB characterize dysfunctional T cells within tumors, and that targeting these receptors has therapeutic utility.

The challenge of linking intergenic mutations to target genes has limited molecular understanding of human diseases. Here we show that H3K27ac HiChIP generates high-resolution contact maps of active enhancers and target genes in rare primary human T cell subtypes and coronary artery smooth muscle cells. Differentiation of naive T cells into T helper 17 cells or regulatory T cells creates subtype-specific enhancer-promoter interactions, specifically at regions of shared DNA accessibility. These data provide a principled means of assigning molecular functions to autoimmune and cardiovascular disease risk variants, linking hundreds of noncoding variants to putative gene targets. Target genes identified with HiChIP are further supported by CRISPR interference and activation at linked enhancers, by the presence of expression quantitative trait loci, and by allele-specific enhancer loops in patient-derived primary cells. The majority of disease-associated enhancers contact genes beyond the nearest gene in the linear genome, leading to a fourfold increase in the number of potential target genes for autoimmune and cardiovascular diseases.

PD-1/PD-L1 blockade can promote robust tumor regression yet secondary resistance often occurs as immune selective pressure drives outgrowth of resistant tumor clones. Here using a genome-wide CRISPR screen in B16.SIY melanoma cells, we confirm Ifngr2 and Jak1 as important genes conferring sensitivity to T cell-mediated killing in vitro. However, when implanted into mice, these Ifngr2- and Jak1-deficient tumors paradoxically are better controlled immunologically. This phenotype maps to defective PD-L1 upregulation on mutant tumor cells, which improves anti-tumor efficacy of CD8+ T cells. To reconcile these observations with clinical reports of anti-PD-1 resistance linked to emergence of IFN-γ signaling mutants, we show that when mixed with wild-type tumor cells, IFN-γ-insensitive tumor cells indeed grow out, which depends upon PD-L1 expression by wild-type cells. Our results illustrate the complexity of functions for IFN-γ in anti-tumor immunity and demonstrate that intratumor heterogeneity and clonal cooperation can contribute to immunotherapy resistance.

The developmental programs that generate a broad repertoire of regulatory T cells (Treg cells) able to respond to both self antigens and non-self antigens remain unclear. Here we found that mature Treg cells were generated through two distinct developmental programs involving CD25+ Treg cell progenitors (CD25+ TregP cells) and Foxp3lo Treg cell progenitors (Foxp3lo TregP cells). CD25+ TregP cells showed higher rates of apoptosis and interacted with thymic self antigens with higher affinity than did Foxp3lo TregP cells, and had a T cell antigen receptor repertoire and transcriptome distinct from that of Foxp3lo TregP cells. The development of both CD25+ TregP cells and Foxp3lo TregP cells was controlled by distinct signaling pathways and enhancers. Transcriptomics and histocytometric data suggested that CD25+ TregP cells and Foxp3lo TregP cells arose by coopting negative-selection programs and positive-selection programs, respectively. Treg cells derived from CD25+ TregP cells, but not those derived from Foxp3lo TregP cells, prevented experimental autoimmune encephalitis. Our findings indicate that Treg cells arise through two distinct developmental programs that are both required for a comprehensive Treg cell repertoire capable of establishing immunotolerance.

Mechanisms implicated in robust transplantation tolerance at the cellular level can be broadly categorized into those that inhibit alloreactive T cells intrinsically (clonal deletion and dysfunction) or extrinsically through regulation. Here, we investigated whether additional population-level mechanisms control T cells by examining whether therapeutically induced peripheral transplantation tolerance could influence T cell populations' avidity for alloantigens. Whereas T cells with high avidity preferentially accumulated during acute rejection of allografts, the alloreactive T cells in tolerant recipients retained a low-avidity profile, comparable to naive mice despite evidence of activation. These contrasting avidity profiles upon productive versus tolerogenic stimulation were durable and persisted upon alloantigen re-encounter in the absence of any immunosuppression. Thus, peripheral transplantation tolerance involves control of alloreactive T cells at the population level, in addition to the individual cell level. Controlling expansion or eliminating high-affinity, donor-specific T cells long term may be desirable to achieve robust transplantation tolerance in the clinic.

RasGRP1 is a Ras guanine nucleotide exchange factor, and an essential regulator of lymphocyte receptor signaling. In mice, Rasgrp1 deletion results in defective T lymphocyte development. RASGRP1-deficient patients suffer from immune deficiency, and the RASGRP1 gene has been linked to autoimmunity. However, how RasGRP1 levels are regulated, and if RasGRP1 dosage alterations contribute to autoimmunity remains unknown. We demonstrate that diminished Rasgrp1 expression caused defective T lymphocyte selection in C57BL/6 mice, and that the severity of inflammatory disease inversely correlates with Rasgrp1 expression levels. In patients with autoimmunity, active inflammation correlated with decreased RASGRP1 levels in CD4+ T cells. By analyzing H3K27 acetylation profiles in human T cells, we identified a RASGRP1 enhancer that harbors autoimmunity-associated SNPs. CRISPR-Cas9 disruption of this enhancer caused lower RasGRP1 expression, and decreased binding of RUNX1 and CBFB transcription factors. Analyzing patients with autoimmunity, we detected reduced RUNX1 expression in CD4+ T cells. Lastly, we mechanistically link RUNX1 to transcriptional regulation of RASGRP1 to reveal a key circuit regulating RasGRP1 expression, which is vital to prevent inflammatory disease.

Proper activation of cytotoxic T cells via the T cell receptor and the costimulatory receptor CD28 is essential for adaptive immunity against viruses, many intracellular bacteria and cancers. Through biochemical analysis of RNA:protein interactions, we uncovered a non-coding RNA circuit regulating activation and differentiation of cytotoxic T cells composed of the long non-coding RNA Malat1 (Metastasis Associated Lung Adenocarcinoma Transcript 1) and the microRNA family miR-15/16. miR-15/16 is a widely and highly expressed tumor suppressor miRNA family important for cell proliferation and survival. miR-15/16 also play important roles in T cell responses to viral infection, including the regulation of antigen-specific T cell expansion and T cell memory. Comparative Argonaute-2 high throughput sequencing of crosslinking immunoprecipitation (Ago2 HITS-CLIP, or AHC) combined with gene expression profiling in normal and miR-15/16-deficient T cells revealed a large network of several hundred direct miR-15/16 target mRNAs, many with functional relevance for T cell activation, survival and memory formation. Among these targets, the long non-coding RNA Malat1 contained the largest absolute magnitude miR-15/16-dependent AHC peak in T cells. This binding site was also among the strongest lncRNA:miRNA interactions detected in the T cell transcriptome. We used CRISPR targeting with homology directed repair to generate mice with a 5-nucleotide mutation in the miR-15/16 binding site in Malat1. This mutation interrupted Malat1:miR-15/16 interaction, and enhanced the repression of other miR-15/16 target genes, including CD28. Interrupting Malat1 interaction with miR-15/16 decreased cytotoxic T cell activation, including the expression of IL-2 and a broader CD28-responsive gene program. Accordingly, Malat1 mutation diminished memory cell persistence following LCMV Armstrong and Listeria monocytogenes infection. This study marks a significant advance in the study of long noncoding RNAs in the immune system by ascribing cell-intrinsic, sequence-specific in vivo function to Malat1. These findings have implications for T cell-mediated autoimmune diseases, antiviral and anti-tumor immunity, as well as lung adenocarcinoma and other malignancies where Malat1 is overexpressed.