BACKGROUND: Insight into the genetic basis for many common autoimmune disorders has been uncovered by genome-wide association studies (GWAS), but this alone does not reveal causal variants, effector genes, or the cell types impacted by disease-associated variation. RESULTS: Here, we generate 3D genomic datasets consisting of promoter-focused Capture-C, Hi-C, ATAC-seq, and RNA-seq and integrate this data with GWAS of 16 autoimmune traits to physically map disease-associated variants to the effector genes they likely regulate in 57 human cell types. The majority of variants implicated by these cis-regulatory architectures are trait-specific, but nearly half of the target genes connected to these variants are shared across multiple autoimmune disorders in multiple cell types, leading to enrichment of similar biological networks. While this suggests a high level of genetic diversity and complexity that converges at the level of target gene and cell type, some trait-specific pathways representing potential areas for disease-specific intervention were identified. We pharmacologically validate squalene synthase, a cholesterol biosynthetic enzyme encoded by the FDFT1 gene implicated by our approach and supported by prior eQTL data in multiple sclerosis and systemic lupus erythematosus, as a novel immunomodulatory drug target controlling T cell inflammatory cytokine production and aiding B cell antibody production in a human lymphoid organoid model. CONCLUSIONS: These data represent a comprehensive resource for basic discovery of gene cis-regulatory mechanisms, and the analyses reported reveal mechanisms by which autoimmune-associated variants act to regulate gene expression, function, and pathology across multiple, distinct tissues and cell types.

Ikaros is a transcriptional factor required for conventional T cell development, differentiation, and anergy. While the related factors Helios and Eos have defined roles in regulatory T cells (Treg), a role for Ikaros has not been established. To determine the function of Ikaros in the Treg lineage, we generated mice with Treg-specific deletion of the Ikaros gene (Ikzf1). We find that Ikaros cooperates with Foxp3 to establish a major portion of the Treg epigenome and transcriptome. Ikaros-deficient Treg exhibit Th1-like gene expression with abnormal production of IL-2, IFNg, TNFa, and factors involved in Wnt and Notch signaling. While Ikzf1-Treg-cko mice do not develop spontaneous autoimmunity, Ikaros-deficient Treg are unable to control conventional T cell-mediated immune pathology in response to TCR and inflammatory stimuli in models of IBD and organ transplantation. These studies establish Ikaros as a core factor required in Treg for tolerance and the control of inflammatory immune responses.

Genome-wide association studies (GWAS) have identified hundreds of genetic signals associated with autoimmune disease. The majority of these signals are located in non-coding regions and likely impact cis-regulatory elements (cRE). Because cRE function is dynamic across cell types and states, profiling the epigenetic status of cRE across physiological processes is necessary to characterize the molecular mechanisms by which autoimmune variants contribute to disease risk. We localized risk variants from 15 autoimmune GWAS to cRE active during TCR-CD28 co-stimulation of naïve human CD4+ T cells. To characterize how dynamic changes in gene expression correlate with cRE activity, we measured transcript levels, chromatin accessibility, and promoter–cRE contacts across three phases of naive CD4+ T cell activation using RNA-seq, ATAC-seq, and HiC. We identified ~1200 protein-coding genes physically connected to accessible disease-associated variants at 423 GWAS signals, at least one-third of which are dynamically regulated by activation. From these maps, we functionally validated a novel stretch of evolutionarily conserved intergenic enhancers whose activity is required for activation-induced IL2 gene expression in human and mouse, and is influenced by autoimmune-associated genetic variation. The set of genes implicated by this approach are enriched for genes controlling CD4+ T cell function and genes involved in human inborn errors of immunity, and we pharmacologically validated eight implicated genes as novel regulators of T cell activation. These studies directly show how autoimmune variants and the genes they regulate influence processes involved in CD4+ T cell proliferation and activation.

Abstract Genome-wide association studies (GWAS) have identified hundreds of genetic loci associated with autoimmune disease, but the causal variants and their contribution to immune dysregulation remain largely unknown. We measured the dynamics of chromosome conformation, chromatin accessibility, and gene transcription across three phases of naive human CD4+ T cell activation and co-localized active cis-regulatory elements with the 95% credible set of variants from 15 autoimmune GWAS. Reorganization of chromosome structure placed these elements in direct contact with ~1,200 protein-coding genes, at least one-third of which were dynamically regulated by stimulation. The set of implicated genes is enriched for high-throughput CRISPR screen targets that control multiple aspects of CD4+ T cell activation, and we pharmacologically validated eight novel gene products as potent regulators of T cell proliferation. These maps also allowed the identification and functional validation of a novel stretch of intergenic enhancers whose activity is required for IL2 gene expression and is influenced by autoimmune-associated genetic variation. This study represents a powerful strategy and resource for assigning physiologic relevance to autoimmune-risk variants and identifying novel genes that control T cell activation and function.

BACKGROUND:SARS-CoV-2 infection results in a broad spectrum of COVID-19 disease, from mild or no symptoms to hospitalization and death. COVID-19 disease severity has been associated with some pre-existing conditions and the magnitude of the adaptive immune response to SARS-CoV-2, and a recent genome-wide association study (GWAS) of the risk of critical illness revealed a significant genetic component. To gain insight into how human genetic variation attenuates or exacerbates disease following SARS-CoV-2 infection, we implicated putatively functional COVID risk variants in the cis-regulatory landscapes of human immune cell types with established roles in disease severity and used high-resolution chromatin conformation capture to map these disease-associated elements to their effector genes. RESULTS:This functional genomic approach implicates 16 genes involved in viral replication, the interferon response, and inflammation. Several of these genes (PAXBP1, IFNAR2, OAS1, OAS3, TNFAIP8L1, GART) were differentially expressed in immune cells from patients with severe versus moderate COVID-19 disease, and we demonstrate a previously unappreciated role for GART in T cell-dependent antibody-producing B cell differentiation in a human tonsillar organoid model. CONCLUSIONS:This study offers immunogenetic insight into the basis of COVID-19 disease severity and implicates new targets for therapeutics that limit SARS-CoV-2 infection and its resultant life-threatening inflammation.

Systemic lupus erythematosus (SLE) is mediated by autoreactive antibodies that damage multiple tissues. Genome-wide association studies (GWAS) link >60 loci with SLE risk, but the causal variants and effector genes are largely unknown. We generated high-resolution spatial maps of SLE variant accessibility and gene connectivity in human follicular helper T cells (TFH), a cell type required for anti-nuclear antibodies characteristic of SLE. Of the ~400 potential regulatory variants identified, 90% exhibit spatial proximity to genes distant in the 1D genome sequence, including variants that loop to regulate the canonical TFH genes BCL6 and CXCR5 as confirmed by genome editing. SLE ‘variant-to-gene’ maps also implicate genes with no known role in TFH/SLE disease biology, including the kinases HIPK1 and MINK1. Targeting these kinases in TFH inhibits production of IL-21, a cytokine crucial for class-switched B cell antibodies. These studies offer mechanistic insight into the SLE-associated regulatory architecture of the human genome.

Ikaros (encoded by Ikzf1) is a lymphocyte-specific transcription factor that controls a wide spectrum of T cell functions, including T helper differentiation, cytokine production, and tolerance in response to antigenic stimuli. Using heterozygous and dominant-negative mouse models, we have shown previously that Ikaros controls IL-2 production by conventional T cells, T helper differentiation, CD8+ effector differentiation, and anergy induction. While the role of Ikaros in many T cell subsets has been established, its role in Treg-mediated peripheral tolerance has not been examined. To examine the Treg-specific role of Ikaros, we generated Ikzf1-fl/fl-Foxp3-YFP-Cre+ mouse with conditional deletion of Ikaros in Tregs. Ikzf1-fl/fl-Foxp3-YFP-Cre+ mouse exhibited enlarged secondary lymphoid tissues, with increased frequencies of effector memory Tconv and Treg. We show that Ikaros expression by Tregs is required for immune tolerance in an allogenic cardiac transplant model. While CD28- and CD40L-blockade induced long-term cardiac allograft tolerance in control recipients, Ikzf1-fl/fl-Foxp3-YFP-Cre+ recipients rejected the allografts in a short duration (p<0.01). We show that Ikaros forms a complex with Foxp3, which is required for Foxp3 to bind to and repress inflammatory cytokine genes. Ikaros is also required for normal iTreg development in vitro, as TGF-B/IL-2-induced iTreg development is impaired in conventional CD4+ T cells with reduced Ikaros activity. These studies suggest that Ikaros interacts with Foxp3 and directs Foxp3 to exert its suppressive function in Tregs.

Systemic lupus erythematosus (SLE) is a complex inflammatory disease mediated by autoreactive antibodies that damages multiple tissues in children and adults. Genome-wide association studies (GWAS) have statistically implicated hundreds of loci in the susceptibility to human disease, including SLE, but the majority have failed to identify the causal variants or the effector genes. As a physicochemical approach to detecting functional variants and connecting them to target genes, we generated comprehensive, high-resolution maps of SLE variant accessibility and gene connectivity in the context of the three-dimensional chromosomal architecture of human tonsillar follicular helper T cells (TFH), a cell type required for the production of anti-nuclear antibodies characteristic of SLE. These spatial epigenomic maps identified a shortlist of over 400 potentially functional variants across 48 GWAS-implicated SLE loci. Twenty percent of these variants were located in open promoters of highly-expressed TFH genes, while 80% reside in non-promoter genomic regions that are connected in 3D to genes that likewise tend to be highly expressed in TFH. Importantly, we find that 90% of SLE-associated variants exhibit spatial proximity to genes that are not nearby in the 1D sequence of the genome, and over 60% of variants ‘skip’ the nearest gene to physically interact only with the promoters of distant genes. Gene ontology confirmed that genes in spatial proximity to SLE variants reside in highly SLE-relevant networks, including accessible variants that loop 200-1000 kb to interact with the promoters of the canonical TFH genes BCL6 and CXCR5 . CRISPR-Cas9 genome editing confirmed that these variants reside in novel, distal regulatory elements required for normal BCL6 and CXCR5 expression by T cells. Furthermore, SLE-associated SNP-promoter interactomes implicated a set of novel genes with no known role in TFH or SLE disease biology, including the homeobox-interacting protein kinase HIPK1 and the Ste kinase homolog MINK1. Targeting these kinases in primary human TFH cells inhibited production of IL-21, a requisite cytokine for production of class-switched antibodies by B cells. This 3D-variant-to-gene mapping approach gives mechanistic insight into the SLE-associated regulatory architecture of the human genome.

Interleukin-2 is a potent T cell growth factor with crucial roles in both immunity and tolerance. The IL2 gene is regulated by multiple signaling pathways, transcription factors, and epigenetic processes operating at the 500 bp upstream regulatory region (URR). Genome-wide association studies (GWAS) in humans have shown that genetic variation in the IL2 region is associated with susceptibility to autoimmune diseases. This region harbors several conserved noncoding sequences. We are performing a comprehensive epigenomic and functional interrogation of the human IL2-IL21 multi-locus region, using a combination of open chromatin mapping (ATAC-seq), recombinant promoter-reporter assays, 3-dimensional chromosome conformation capture (Capture-C), and CRISPR/CAS9 genome editing. ATAC-seq analysis identified TCR/CD28 activation-dependent chromatin remodeling at −46, −51, −80, −83, −-85, −121, and −128 kb upstream of IL2 in human T cells. Chromatin conformation capture indicates that several of these distal regions physically interact with IL2, and are capable of enhancing transcription from the IL2-URR in recombinant reporter assays. The −128 kb element showed autoimmune disease-associated allelic variation in enhancer activity, and CRISPR/CAS9-mediated deletion of this element in human T cells resulted in a 2–3 fold reduction in activation-induced IL-2 production. These results suggest that the IL2 locus has evolved an extensive regulatory architecture that allows for variable production of IL-2, and provide insight into why most autoimmune IL2 polymorphisms are located far away from the gene.

IL-2 is a key immunoregulatory cytokine with pleotropic effects on many immune cell types. Its expression is highly regulated in T cells through multiple signaling pathways, chromatin remodeling, DNA methylation, and cooperative transcription factor binding. Recently, we identified an evolutionarily conserved intergenic enhancer 83 kb upstream of the Il2 gene that augments transcription from the Il2 promoter and upstream regulatory region (URR) by >50-fold in recombinant reporter assays. Upon TCR/CD28 co-stimulation of primary CD4+ T cells, a long-range chromosomal loop is established between the −83 kb CNS and the endogenous Il2 promoter, and both regions become epigenetically marked by histone acetylation and methylation. To determine the relative contribution of the URR vs. this new distal element to inducible transcription of the Il2 gene, we deleted the URR or the −83 kb CNS in mice using CRISPR/CAS genome editing. Deletion of the URR (~500 bp beyond the TATA element) completely abolished the Il2 transcription by CD4+ T cells, while a ~500 bp deletion of the −83 kb CNS resulted in a 2- to 4-fold decrease in IL-2 production. Young mice deficient for the −83 kb enhancer exhibited no major defects in lymphoid development, however, mutant CD4+ T cells showed reduced proliferative capacity in vitro. Furthermore, TGFB-induced iTreg differentiation from mutant conventional CD4+ precursors was strongly impaired, but could be fully restored by addition of exogenous IL-2. These results show that induction of Il2 depends upon cooperation between the promoter-URR and at least one intergenic enhancer, and suggest mechanisms by which graded expression of Il2 might be achieved through ‘modular’ recruitment of distinct enhancer elements.

Foxp3 + T regulatory (T reg ) cells suppress immune cell activation and establish normal immune homeostasis. How T reg cells maintain their identity is not completely understood. Here we show that Ndfip1, a coactivator of Nedd4-family E3 ubiquitin ligases, is required for T reg cell stability and function. Ndfip1 deletion in T reg cells results in autoinflammatory disease. Ndfip1-deficient T reg cells are highly proliferative and are more likely to lose Foxp3 expression to become IL-4-producing T H 2 effector cells. Proteomic analyses indicate altered metabolic signature of Ndfip1-deficient T reg cells and metabolic profiling reveals elevated glycolysis and increased mTORC1 signalling. Ndfip1 restricts T reg cell metabolism and IL-4 production via distinct mechanisms, as IL-4 deficiency does not prevent hyperproliferation or elevated mTORC1 signalling in Ndfip1-deficient T reg cells. Thus, Ndfip1 preserves T reg lineage stability and immune homeostasis by preventing the expansion of highly proliferative and metabolically active T reg cells and by preventing pathological secretion of IL-4 from T reg cells.

Abstract Recent success of adoptive transfer of T cells expressing chimeric antigen receptors (CARs) in patients with blood-borne malignancies has resulted in growing enthusiasm to apply the same approach to treat solid tumors. CAR-expressing T cells, however, will likely face many challenges that will limit their efficacy in solid tumors. One possible obstacle will be the intrinsic negative feedback system that prevent continuing T cell responses. The transcription factor Ikaros, which has been shown to suppress endogenous T effector functions through epigenetic remodeling of cytokine gene loci, may be one such negative regulator. Here we show that anti-mesothelin CAR (mesoCAR) T cells with reduced levels of Ikaros consistently mediate better in vitro cell killing and in vivo tumor regression than their wild-type counterparts. This increased anti-tumor activity was accompanied by increased persistence of mesoCAR T cells, as well as increased IFN-γ production. Enhanced cytolytic activity was also evidenced in mesoCAR T cells with reduced Ikaros, which could be partially explained by increased CD107a upregulation and Granzyme B production. Reduction in the Ikaros level in mesoCAR T cells did not affect T cell receptor and CAR signaling, but was able to render them more resistant to soluble inhibitory factors like TGF-β and adenosine. In summary, inhibition of the transcriptional repressor Ikaros in CAR T cells could be an attractive strategy to overcome immunosuppressive tumor microenvironment and enhance their tumoricidal capacity. Further clinical development is warranted. Citation Format: Shaun O'Brien, Rajan M. Thomas, Steven M. Albelda, Andrew D. Wells, Liang-Chuan S. Wang. Inhibition of the transcription factor Ikaros augments the tumoricidal capacity of CD8+ T cells expressing chimeric antigen receptor. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B12.

Abstract Recent success of adoptive transfer of T cells expressing chimeric antigen receptors (CARs) in patients with blood-borne malignancies has resulted in growing enthusiasm to apply the same approach to treat solid tumors. CAR-expressing T cells, however, will likely face many challenges that will limit their efficacy in solid tumors. One possible obstacles will be the intrinsic negative feedback systems that prevent continuing T cell responses. The transcription factor Ikaros which has been shown to suppress endogenous T effector functions through epigenetic remodeling of cytokine gene loci may be one such negative regulator. Here we show that anti-mesothelin CAR (mesoCAR) T cells with reduced levels of Ikaros consistently mediate better in vitro cell killing and in vivo tumor regression than their wild-type counterparts. This increased anti-tumor activity was accompanied by increased persistence of mesoCAR T cells, as well as increased IFN-g. Enhanced cytolytic activity was also evidenced in mesoCAR T cells with reduced Ikaros, which could be partially explained by increased CD107a upregulation and Granzyme B production. Reduction in the Ikaros level in mesoCAR T cells did not affect T cell receptor and CAR signaling, but was able to render them more resistant to soluble inhibitory factors like TGF-B and adenosine. In summary, inhibition of the transcriptional repressor Ikaros in CAR T cells could be an attractive strategy to overcome immunosuppressive tumor microenvironments and enhance their tumoricidal capacity. Further clinical development is warranted. Citation Format: Shaun O'Brien, Liang-Chuan Wang, Kheng Newick, Rajan M. Thomas, Andrew Wells, Steven M. Albelda. Inhibition of the transcription factor Ikaros augments the tumoricidal capacity of CD8+ T cells expressing chimeric antigen receptors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3142. doi:10.1158/1538-7445.AM2015-3142

Naive CD4(+) T cells require signals from the TCR and CD28 to produce IL-2, expand, and differentiate. However, these same signals are not sufficient to induce autocrine IL-2 production by naive CD8(+) T cells, which require cytokines provided by other cell types to drive their differentiation. The basis for failed autocrine IL-2 production by activated CD8(+) cells is unclear. We find that Ikaros, a transcriptional repressor that silences IL-2 in anergic CD4(+) T cells, also restricts autocrine IL-2 production by CD8(+) T cells. We find that CD8(+) T cell activation in vitro in the absence of exogenous cytokines and CD4 help leads to marked induction of Ikaros, a known repressor of the Il2 gene. Naive murine CD8 T cells haplo-insufficient for Ikzf1 failed to upregulate Ikaros, produced autocrine IL-2, and differentiated in an IL-2-dependent manner into IFN-γ-producing CTLs in response to TCR/CD28 stimulation alone. Furthermore, Ikzf1 haplo-insufficient CD8(+) T cells were more effective at controlling Listeria infection and B16 melanoma growth in vivo, and they could provide help to neighboring, non-IL-2-producing cells to differentiate into IFN-γ-producing effectors. Therefore, by repressing autocrine IL-2 production, Ikaros ensures that naive CD8(+) T cells remain dependent on licensing by APCs and CD4(+) T cells, and it may therefore act as a cell-intrinsic safeguard against inappropriate CTL differentiation and immunopathology.