Cross-disorder genetic analysis of immune diseases reveals distinct gene associations that converge on common pathways.

Genome-wide association studies (GWAS) have mapped thousands of susceptibility loci associated with immune-mediated diseases. To assess the extent of the genetic sharing across nine immune-mediated diseases we apply genomic structural equation modelling to GWAS data from European populations. We identify three disease groups: gastrointestinal tract diseases, rheumatic and systemic diseases, and allergic diseases. Although loci associated with the disease groups are highly specific, they converge on perturbing the same pathways. Finally, we test for colocalization between loci and single-cell eQTLs derived from peripheral blood mononuclear cells. We identify the causal route by which 46 loci predispose to three disease groups and find evidence for eight genes being candidates for drug repurposing. Taken together, here we show that different constellations of diseases have distinct patterns of genetic associations, but that associated loci converge on perturbing different nodes in T cell activation and signalling pathways.

Genetic variants associated with psychiatric disorders are enriched at epigenetically active sites in lymphoid cells.

Multiple psychiatric disorders have been associated with abnormalities in both the innate and adaptive immune systems. The role of these abnormalities in pathogenesis, and whether they are driven by psychiatric risk variants, remains unclear. We test for enrichment of GWAS variants associated with multiple psychiatric disorders (cross-disorder or trans-diagnostic risk), or 5 specific disorders (cis-diagnostic risk), in regulatory elements in immune cells. We use three independent epigenetic datasets representing multiple organ systems and immune cell subsets. Trans-diagnostic and cis-diagnostic risk variants (for schizophrenia and depression) are enriched at epigenetically active sites in brain tissues and in lymphoid cells, especially stimulated CD4+ T cells. There is no evidence for enrichment of either trans-risk or cis-risk variants for schizophrenia or depression in myeloid cells. This suggests a possible model where environmental stimuli activate T cells to unmask the effects of psychiatric risk variants, contributing to the pathogenesis of mental health disorders.

GWAS and autoimmunity: What have we learned and what next.

Autoimmune diseases are common conditions characterized by loss of tolerance, female predominance and a remarkable heterogeneity among different populations. Most often they are polygenic and several genetic loci have been linked with the risk of developing autoimmune diseases. However, causal inference is difficult. When the genomic revolution began there were high hopes of translating fast genetic analyses to the bedside but this has proven to be challenging. Nonetheless, over the last decade, fine-mapping strategies have greatly improved; one of the most significant research lines focuses on the in vivo and ex vivo definition of the effect of genetic variants within the target tissues and within specific subpopulations of immune cells that are involved in the disease pathogenesis. This strategy also includes the longitudinal tracking of a large number of immunophenotypes in many individuals to build a large reference atlas for variant characterization. In this review, we discuss the results obtained by GWAS in autoimmune diseases and review recent advances in fine mapping strategies. More importantly, we discuss gaps and future directions.

Differences in CD80 and CD86 transendocytosis reveal CD86 as a key target for CTLA-4 immune regulation.

CD28 and CTLA-4 (CD152) play essential roles in regulating T cell immunity, balancing the activation and inhibition of T cell responses, respectively. Although both receptors share the same ligands, CD80 and CD86, the specific requirement for two distinct ligands remains obscure. In the present study, we demonstrate that, although CTLA-4 targets both CD80 and CD86 for destruction via transendocytosis, this process results in separate fates for CTLA-4 itself. In the presence of CD80, CTLA-4 remained ligand bound, and was ubiquitylated and trafficked via late endosomes and lysosomes. In contrast, in the presence of CD86, CTLA-4 detached in a pH-dependent manner and recycled back to the cell surface to permit further transendocytosis. Furthermore, we identified clinically relevant mutations that cause autoimmune disease, which selectively disrupted CD86 transendocytosis, by affecting either CTLA-4 recycling or CD86 binding. These observations provide a rationale for two distinct ligands and show that defects in CTLA-4-mediated transendocytosis of CD86 are associated with autoimmunity.

Immune disease risk variants regulate gene expression dynamics during CD4+ T cell activation.

During activation, T cells undergo extensive gene expression changes that shape the properties of cells to exert their effector function. Understanding the regulation of this process could help explain how genetic variants predispose to immune diseases. Here, we mapped genetic effects on gene expression (expression quantitative trait loci (eQTLs)) using single-cell transcriptomics. We profiled 655,349 CD4+ T cells, capturing transcriptional states of unstimulated cells and three time points of cell activation in 119 healthy individuals. This identified 38 cell clusters, including transient clusters that were only present at individual time points of activation. We found 6,407 genes whose expression was correlated with genetic variation, of which 2,265 (35%) were dynamically regulated during activation. Furthermore, 127 genes were regulated by variants associated with immune-mediated diseases, with significant enrichment for dynamic effects. Our results emphasize the importance of studying context-specific gene expression regulation and provide insights into the mechanisms underlying genetic susceptibility to immune-mediated diseases.

Immune disease variants modulate gene expression in regulatory CD4+ T cells.

Identifying cellular functions dysregulated by disease-associated variants could implicate novel pathways for drug targeting or modulation in cell therapies. However, follow-up studies can be challenging if disease-relevant cell types are difficult to sample. Variants associated with immune diseases point toward the role of CD4+ regulatory T cells (Treg cells). We mapped genetic regulation (quantitative trait loci [QTL]) of gene expression and chromatin activity in Treg cells, and we identified 133 colocalizing loci with immune disease variants. Colocalizations of immune disease genome-wide association study (GWAS) variants with expression QTLs (eQTLs) controlling the expression of CD28 and STAT5A, involved in Treg cell activation and interleukin-2 (IL-2) signaling, support the contribution of Treg cells to the pathobiology of immune diseases. Finally, we identified seven known drug targets suitable for drug repurposing and suggested 63 targets with drug tractability evidence among the GWAS signals that colocalized with Treg cell QTLs. Our study is the first in-depth characterization of immune disease variant effects on Treg cell gene expression modulation and dysregulation of Treg cell function.

CD86 Is a Selective CD28 Ligand Supporting FoxP3+ Regulatory T Cell Homeostasis in the Presence of High Levels of CTLA-4.

CD80 and CD86 are expressed on antigen presenting cells and are required to engage their shared receptor, CD28, for the costimulation of CD4 T cells. It is unclear why two stimulatory ligands with overlapping roles have evolved. CD80 and CD86 also bind the regulatory molecule CTLA-4. We explored the role of CD80 and CD86 in the homeostasis and proliferation of CD4+FoxP3+ regulatory T cells (Treg), which constitutively express high levels of CTLA-4 yet are critically dependent upon CD28 signals. We observed that CD86 was the dominant ligand for Treg proliferation, survival, and maintenance of a regulatory phenotype, with higher expression of CTLA-4, ICOS, and OX40. We also explored whether CD80-CD28 interactions were specifically compromised by CTLA-4 and found that antibody blockade, clinical deficiency of CTLA-4 and CRISPR-Cas9 deletion of CTLA-4 all improved Treg survival following CD80 stimulation. Taken together, our data suggest that CD86 is the dominant costimulatory ligand for Treg homeostasis, despite its lower affinity for CD28, because CD80-CD28 interactions are selectively impaired by the high levels of CTLA-4. These data suggest a cell intrinsic role for CTLA-4 in regulating CD28 costimulation by direct competition for CD80, and indicate that that CD80 and CD86 have discrete roles in CD28 costimulation of CD4 T cells in the presence of high levels of CTLA-4.

Genomic profiling of T-cell activation suggests increased sensitivity of memory T cells to CD28 costimulation.

T-cell activation is a critical driver of immune responses. The CD28 costimulation is an essential regulator of CD4 T-cell responses, however, its relative importance in naive and memory T cells is not fully understood. Using different model systems, we observe that human memory T cells are more sensitive to CD28 costimulation than naive T cells. To deconvolute how the T-cell receptor (TCR) and CD28 orchestrate activation of human T cells, we stimulate cells using varying intensities of TCR and CD28 and profiled gene expression. We show that genes involved in cell cycle progression and division are CD28-driven in memory cells, but under TCR control in naive cells. We further demonstrate that T-helper differentiation and cytokine expression are controlled by CD28. Using chromatin accessibility profiling, we observe that AP1 transcriptional regulation is enriched when both TCR and CD28 are engaged, whereas open chromatin near CD28-sensitive genes is enriched for NF-kB motifs. Lastly, we show that CD28-sensitive genes are enriched in GWAS regions associated with immune diseases, implicating a role for CD28 in disease development. Our study provides important insights into the differential role of costimulation in naive and memory T-cell responses and disease susceptibility.

Single-cell transcriptomics identifies an effectorness gradient shaping the response of CD4+ T cells to cytokines.

Naïve CD4+ T cells coordinate the immune response by acquiring an effector phenotype in response to cytokines. However, the cytokine responses in memory T cells remain largely understudied. Here we use quantitative proteomics, bulk RNA-seq, and single-cell RNA-seq of over 40,000 human naïve and memory CD4+ T cells to show that responses to cytokines differ substantially between these cell types. Memory T cells are unable to differentiate into the Th2 phenotype, and acquire a Th17-like phenotype in response to iTreg polarization. Single-cell analyses show that T cells constitute a transcriptional continuum that progresses from naïve to central and effector memory T cells, forming an effectorness gradient accompanied by an increase in the expression of chemokines and cytokines. Finally, we show that T cell activation and cytokine responses are influenced by the effectorness gradient. Our results illustrate the heterogeneity of T cell responses, furthering our understanding of inflammation.

CD80 on Human T Cells Is Associated With FoxP3 Expression and Supports Treg Homeostasis.

CD80 and CD86 are expressed on antigen presenting cells (APCs) and their role in providing costimulation to T cells is well established. However, it has been shown that these molecules can also be expressed by T cells, but the significance of this observation remains unknown. We have investigated stimuli that control CD80 and CD86 expression on T cells and show that in APC-free conditions around 40% of activated, proliferating CD4+ T cells express either CD80, CD86 or both. Expression of CD80 and CD86 was strongly dependent upon provision of CD28 costimulation as ligands were not expressed following TCR stimulation alone. Furthermore, we observed that CD80+ T cells possessed the hallmarks of induced regulatory T cells (iTreg), expressing Foxp3 and high levels of CTLA-4 whilst proliferating less extensively. In contrast, CD86 was preferentially expressed on INF-γ producing cells, which proliferated more extensively and had characteristics of effector T cells. Finally, we demonstrated that CD80 expressed on T cells inhibits CTLA-4 function and facilitates the growth of iTreg. Together these data establish endogenous expression of CD80 and CD86 by activated T cells is not due to ligand capture by transendocytosis and highlight clear differences in their expression patterns and associated functions.

Chromatin activity at GWAS loci identifies T cell states driving complex immune diseases.

Immune-disease-associated variants are enriched in active chromatin regions of T cells and macrophages. However, whether these variants function in specific cell states is unknown. Here we stimulated T cells and macrophages in the presence of 13 cytokines and profiled active and open chromatin regions. T cell activation induced major chromatin remodeling, while the presence of cytokines fine-tuned the magnitude of changes. We developed a statistical method that accounts for subtle changes in the chromatin landscape to identify SNP enrichment across cell states. Our results point towards the role of immune-disease-associated variants in early rather than late activation of memory CD4+ T cells, with modest differences across cytokines. Furthermore, variants associated with inflammatory bowel disease are enriched in type 1 T helper (TH1) cells, whereas variants associated with Alzheimer's disease are enriched in different macrophage cell states. Our results represent an in-depth analysis of immune-disease-associated variants across a comprehensive panel of activation states of T cells and macrophages.

Genetic variation at the CD28 locus and its impact on expansion of pro-inflammatory CD28 negative T cells in healthy individuals.

The CD28 locus is associated with susceptibility to a variety of autoimmune and immune-mediated inflammatory diseases including primary sclerosing cholangitis (PSC). Previously, we linked the CD28 pathway in PSC disease pathology and found that vitamin D could maintain CD28 expression. Here, we assessed whether the PSC-associated CD28 risk variant A (rs7426056) affects CD28 expression and T cell function in healthy individuals (n = 14 AA, n = 14 AG, n = 14 GG). Homozygotes for the PSC disease risk allele (AA) showed significantly lower CD28 mRNA expression ex-vivo than either GG or AG (p < 0.001) in total peripheral blood mononuclear cells. However, the CD28 risk variant alone was not sufficient to explain CD28 protein loss on CD4+ T cells. All genotypes responded equally to vitamin D as indicated by induction of a regulatory phenotype and an increased anti-inflammatory/pro-inflammatory cytokine ratio. A genotypic effect on response to TNFα stimuli was detected, which was inhibited by vitamin D. Together our results show: (a) an altered gene expression in carriers of the susceptible CD28 variant, (b) no differences in protein levels on CD4+ T cells, and

Immunogenomic approaches to understand the function of immune disease variants.

Mapping hundreds of genetic variants through genome wide association studies provided an opportunity to gain insights into the pathobiology of immune-mediated diseases. However, as most of the disease variants fall outside the gene coding sequences the functional interpretation of the exact role of the associated variants remains to be determined. The integration of disease-associated variants with large scale genomic maps of cell-type-specific gene regulation at both chromatin and transcript levels deliver examples of functionally prioritized causal variants and genes. In particular, the enrichment of disease variants with histone marks can point towards the cell types most relevant to disease development. Furthermore, chromatin contact maps that link enhancers to promoter regions in a direct way allow the identification of genes that can be regulated by the disease variants. Candidate genes implicated with such approaches can be further examined through the correlation of gene expression with genotypes. Additionally, in the context of immune-mediated diseases it is important to combine genomics with immunology approaches. Genotype correlations with the immune system as a whole, as well as with cellular responses to different stimuli, provide a valuable platform for understanding the functional impact of disease-associated variants. The intersection of immunogenomic resources with disease-associated variants paints a detailed picture of disease causal mechanisms. Here, we provide an overview of recent studies that combine these approaches to identify disease vulnerable pathways.

Identifying functional defects in patients with immune dysregulation due to LRBA and CTLA-4 mutations.

Heterozygous CTLA-4 deficiency has been reported as a monogenic cause of common variable immune deficiency with features of immune dysregulation. Direct mutation in CTLA-4 leads to defective regulatory T-cell (Treg) function associated with impaired ability to control levels of the CTLA-4 ligands, CD80 and CD86. However, additional mutations affecting the CTLA-4 pathway, such as those recently reported for LRBA, indirectly affect CTLA-4 expression, resulting in clinically similar disorders. Robust phenotyping approaches sensitive to defects in the CTLA-4 pathway are therefore required to inform understanding of such immune dysregulation syndromes. Here, we describe assays capable of distinguishing a variety of defects in the CTLA-4 pathway. Assessing total CTLA-4 expression levels was found to be optimal when restricting analysis to the CD45RA-Foxp3+ fraction. CTLA-4 induction following stimulation, and the use of lysosomal-blocking compounds, distinguished CTLA-4 from LRBA mutations. Short-term T-cell stimulation improved the capacity for discriminating the Foxp3+ Treg compartment, clearly revealing Treg expansions in these disorders. Finally, we developed a functionally orientated assay to measure ligand uptake by CTLA-4, which is sensitive to ligand-binding or -trafficking mutations, that would otherwise be difficult to detect and that is appropriate for testing novel mutations in CTLA-4 pathway genes. These approaches are likely to be of value in interpreting the functional significance of mutations in the CTLA-4 pathway identified by gene-sequencing approaches.

A CD80-Biased CTLA4-Ig Fusion Protein with Superior In Vivo Efficacy by Simultaneous Engineering of Affinity, Selectivity, Stability, and FcRn Binding.

Affinity- and stability-engineered variants of CTLA4-Ig fusion molecules with enhanced pharmacokinetic profiles could yield improved therapies with the potential of higher efficacy and greater convenience to patients. In this study, to our knowledge, we have, for the first time, used in vitro evolution to simultaneously optimize CTLA4 affinity and stability. We selected for improved binding to both ligands, CD80 and CD86, and screened as dimeric Fc fusions directly in functional assays to identify variants with stronger suppression of in vitro T cell activation. The majority of CTLA4 molecules showing the largest potency gains in primary in vitro and ex vivo human cell assays, using PBMCs from type 1 diabetes patients, had significant improvements in CD80, but only modest gains in CD86 binding. We furthermore observed different potency rankings between our lead molecule MEDI5265, abatacept, and belatacept, depending on which type of APC was used, with MEDI5265 consistently being the most potent. We then created fusions of both stability- and potency-optimized CTLA4 moieties with human Fc variants conferring extended plasma t1/2 In a cynomolgus model of T cell-dependent Ab response, the CTLA4-Ig variant MEDI5265 could be formulated at >100 mg/ml for s.c. administration and showed superior efficacy and significantly prolonged serum t1/2 The combination of higher stability and potency with prolonged pharmacokinetics could be compatible with very infrequent, s.c. dosing while maintaining a similar level of immune suppression to more frequently and i.v. administered licensed therapies.

Gender-related differences in susceptibility to oxidative stress in healthy middle-aged Serbian adults.

Gender-related differences in the association between polymorphism of xenobiotic-metabolising enzymes or non-genetic biomarkers and susceptibility to oxidative stress was assessed in healthy middle-aged Serbian adults, by urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG/creatinine) and total antioxidant status in serum (TAOS). Females were more susceptible to oxidative stress. In both genders, positive predictor of the antioxidative protection was serum triglyceride, while BMI <25 kg/m(2) was associated with oxidative stress. Susceptibility to oxidative stress in males was associated with GSTT1*null allele and increased serum iron, but in females, it was decreased serum bilirubin. Early identification of the risk factors could be important in the prevention of oxidative stress-related diseases.

1,25(OH)2D3 Promotes the Efficacy of CD28 Costimulation Blockade by Abatacept.

Inhibition of the CD28:CD80/CD86 T cell costimulatory pathway has emerged as an effective strategy for the treatment of T cell-mediated inflammatory diseases. However, patient responses to CD28-ligand blockade by abatacept (CTLA-4-Ig) in conditions such as rheumatoid arthritis are variable and often suboptimal. In this study, we show that the extent to which abatacept suppresses T cell activation is influenced by the strength of TCR stimulation, with high-strength TCR stimulation being associated with relative abatacept insensitivity. Accordingly, cyclosporin A, an inhibitor of T cell stimulation via the TCR, synergized with abatacept to inhibit T cell activation. We also observed that 1,25-dihydroxyvitamin D3 enhanced the inhibition of T cell activation by abatacept, strongly inhibiting T cell activation driven by cross-linked anti-CD3, but with no effect upon anti-CD28 driven stimulation. Thus, like cyclosporin A, 1,25-dihydroxyvitamin D3 inhibits TCR-driven activation, thereby promoting abatacept sensitivity. Vitamin D3 supplementation may therefore be a useful adjunct for the treatment of conditions such as rheumatoid arthritis in combination with abatacept to promote the efficacy of treatment.

Vitamin D Antagonises the Suppressive Effect of Inflammatory Cytokines on CTLA-4 Expression and Regulatory Function.

The immune suppressive protein CTLA-4 is constitutively expressed by Tregs and induced in effector T cells upon activation. Its crucial role in adaptive immunity is apparent from the fatal autoimmune pathology seen in CTLA-4 knockout mice. However, little is known regarding factors that regulate CTLA-4 expression and their effect upon its function to remove CD80 and CD86 from antigen presenting cells by transendocytosis. Th17 cells are emerging as significant players in autoimmunity as well as other diseases. Therefore, in this study we have examined the effects of Th17 polarising conditions on CTLA-4 expression and function in human T cells and show that Th17 conditions can suppress the expression of CTLA-4 and its transendocytic function. In contrast to Th17 cells, vitamin D is inversely associated with autoimmune disease. We have previously shown a striking ability of 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) to enhance CTLA-4, however, its effects upon B7 transendocytosis and its activity in the context of inflammation remained unknown. Here we show that induction of CTLA-4 by 1,25(OH)2D3 can actually be enhanced in the presence of Th17 polarising cytokines. Furthermore, its transendocytic function was maintained such that T cells generated in the presence of Th17 conditions and 1,25(OH)2D3 were highly effective at capturing CTLA-4 ligands from antigen presenting cells and suppressing T cell division. Taken together, these data reveal an inhibitory effect of Th17 polarising conditions upon CTLA-4-mediated regulation and show that 1,25(OH)2D3 counteracts this effect. Given the importance of CTLA-4-mediated suppression in the control of autoimmune diseases, our novel data highlight the importance of vitamin D in inflammatory settings.

A transendocytosis perspective on the CD28/CTLA-4 pathway.

T cell activation is a key event in the adaptive immune response and vital to the generation of both cellular and humoral immunity. Activation is required not only for effective CD4 T cell responses but also to provide help for B cells and the generation of cytotoxic T cell responses. Unsurprisingly, impaired T cell activation results in infectious pathology, whereas dysregulated activation can result in autoimmunity. The decision to activate is therefore tightly regulated and the CD28/CTLA-4 pathway represents this apical decision point at the molecular level. In particular, CTLA-4 (CD152) is an essential checkpoint control for autoimmunity; however, the molecular mechanism(s) by which CTLA-4 achieves its regulatory function are not well understood, especially how it functionally intersects with the CD28 pathway. In this chapter, we review the established molecular and cellular concepts relating to CD28 and CTLA-4 biology, and attempt to integrate these by discussing the transendocytosis of ligands as a new model of CTLA-4 function.