The Identity of Human Tissue-Emigrant CD8+ T Cells.

Lymphocyte migration is essential for adaptive immune surveillance. However, our current understanding of this process is rudimentary, because most human studies have been restricted to immunological analyses of blood and various tissues. To address this knowledge gap, we used an integrated approach to characterize tissue-emigrant lineages in thoracic duct lymph (TDL). The most prevalent immune cells in human and non-human primate efferent lymph were T cells. Cytolytic CD8+ T cell subsets with effector-like epigenetic and transcriptional signatures were clonotypically skewed and selectively confined to the intravascular circulation, whereas non-cytolytic CD8+ T cell subsets with stem-like epigenetic and transcriptional signatures predominated in tissues and TDL. Moreover, these anatomically distinct gene expression profiles were recapitulated within individual clonotypes, suggesting parallel differentiation programs independent of the expressed antigen receptor. Our collective dataset provides an atlas of the migratory immune system and defines the nature of tissue-emigrant CD8+ T cells that recirculate via TDL.

Distinct Regulation of Th17 and Th1 Cell Differentiation by Glutaminase-Dependent Metabolism.

Activated T cells differentiate into functional subsets with distinct metabolic programs. Glutaminase (GLS) converts glutamine to glutamate to support the tricarboxylic acid cycle and redox and epigenetic reactions. Here, we identify a key role for GLS in T cell activation and specification. Though GLS deficiency diminished initial T cell activation and proliferation and impaired differentiation of Th17 cells, loss of GLS also increased Tbet to promote differentiation and effector function of CD4 Th1 and CD8 CTL cells. This was associated with altered chromatin accessibility and gene expression, including decreased PIK3IP1 in Th1 cells that sensitized to IL-2-mediated mTORC1 signaling. In vivo, GLS null T cells failed to drive Th17-inflammatory diseases, and Th1 cells had initially elevated function but exhausted over time. Transient GLS inhibition, however, led to increased Th1 and CTL T cell numbers. Glutamine metabolism thus has distinct roles to promote Th17 but constrain Th1 and CTL effector cell differentiation.

Antagonism of B cell enhancer networks by STAT5 drives leukemia and poor patient survival.

The transcription factor STAT5 has a critical role in B cell acute lymphoblastic leukemia (B-ALL). How STAT5 mediates this effect is unclear. Here we found that activation of STAT5 worked together with defects in signaling components of the precursor to the B cell antigen receptor (pre-BCR), including defects in BLNK, BTK, PKCß, NF-κB1 and IKAROS, to initiate B-ALL. STAT5 antagonized the transcription factors NF-κB and IKAROS by opposing regulation of shared target genes. Super-enhancers showed enrichment for STAT5 binding and were associated with an opposing network of transcription factors, including PAX5, EBF1, PU.1, IRF4 and IKAROS. Patients with a high ratio of active STAT5 to NF-κB or IKAROS had more-aggressive disease. Our studies indicate that an imbalance of two opposing transcriptional programs drives B-ALL and suggest that restoring the balance of these pathways might inhibit B-ALL.

Foxp3 and Toll-like receptor signaling balance Treg cell anabolic metabolism for suppression.

CD4+ effector T cells (Teff cells) and regulatory T cells (Treg cells) undergo metabolic reprogramming to support proliferation and immunological function. Although signaling via the lipid kinase PI(3)K (phosphatidylinositol-3-OH kinase), the serine-threonine kinase Akt and the metabolic checkpoint kinase complex mTORC1 induces both expression of the glucose transporter Glut1 and aerobic glycolysis for Teff cell proliferation and inflammatory function, the mechanisms that regulate Treg cell metabolism and function remain unclear. We found that Toll-like receptor (TLR) signals that promote Treg cell proliferation increased PI(3)K-Akt-mTORC1 signaling, glycolysis and expression of Glut1. However, TLR-induced mTORC1 signaling also impaired Treg cell suppressive capacity. Conversely, the transcription factor Foxp3 opposed PI(3)K-Akt-mTORC1 signaling to diminish glycolysis and anabolic metabolism while increasing oxidative and catabolic metabolism. Notably, Glut1 expression was sufficient to increase the number of Treg cells, but it reduced their suppressive capacity and Foxp3 expression. Thus, inflammatory signals and Foxp3 balance mTORC1 signaling and glucose metabolism to control the proliferation and suppressive function of Treg cells.

The common variable immunodeficiency IgM repertoire narrowly recognizes erythrocyte and platelet glycans.

BACKGROUND: Autoimmune cytopenias (AICs) regularly occur in profoundly IgG-deficient patients with common variable immunodeficiency (CVID). The isotypes, antigenic targets, and origin(s) of their disease-causing autoantibodies are unclear. OBJECTIVE: We sought to determine reactivity, clonality, and provenance of AIC-associated IgM autoantibodies in patients with CVID. METHODS: We used glycan arrays, patient erythrocytes, and platelets to determine targets of CVID IgM autoantibodies. Glycan-binding profiles were used to identify autoreactive clones across B-cell subsets, specifically circulating marginal zone (MZ) B cells, for sorting and IGH sequencing. The locations, transcriptomes, and responses of tonsillar MZ B cells to different TH- cell subsets were determined by confocal microscopy, RNA-sequencing, and cocultures, respectively. RESULTS: Autoreactive IgM coated erythrocytes and platelets from many CVID patients with AICs (CVID+AIC). On glycan arrays, CVID+AIC plasma IgM narrowly recognized erythrocytic i antigens and platelet i-related antigens and failed to bind hundreds of pathogen- and tumor-associated carbohydrates. Polyclonal i antigen-recognizing B-cell receptors were highly enriched among CVID+AIC circulating MZ B cells. Within tonsillar tissues, MZ B cells secreted copious IgM when activated by the combination of IL-10 and IL-21 or when cultured with IL-10/IL-21-secreting FOXP3-CD25hi T follicular helper (Tfh) cells. In lymph nodes from immunocompetent controls, MZ B cells, plentiful FOXP3+ regulatory T cells, and rare FOXP3-CD25+ cells that represented likely CD25hi Tfh cells all localized outside of germinal centers. In CVID+AIC lymph nodes, cellular positions were similar but CD25hi Tfh cells greatly outnumbered regulatory cells. CONCLUSIONS: Our findings indicate that glycan-reactive IgM autoantibodies produced outside of germinal centers may contribute to the autoimmune pathogenesis of CVID.

A UVB-responsive common variant at chromosome band 7p21.1 confers tanning response and melanoma risk via regulation of the aryl hydrocarbon receptor, AHR.

Genome-wide association studies (GWASs) have identified a melanoma-associated locus on chromosome band 7p21.1 with rs117132860 as the lead SNP and a secondary independent signal marked by rs73069846. rs117132860 is also associated with tanning ability and cutaneous squamous cell carcinoma (cSCC). Because ultraviolet radiation (UVR) is a key environmental exposure for all three traits, we investigated the mechanisms by which this locus contributes to melanoma risk, focusing on cellular response to UVR. Fine-mapping of melanoma GWASs identified four independent sets of candidate causal variants. A GWAS region-focused Capture-C study of primary melanocytes identified physical interactions between two causal sets and the promoter of the aryl hydrocarbon receptor (AHR). Subsequent chromatin state annotation, eQTL, and luciferase assays identified rs117132860 as a functional variant and reinforced AHR as a likely causal gene. Because AHR plays critical roles in cellular response to dioxin and UVR, we explored links between this SNP and AHR expression after both 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and ultraviolet B (UVB) exposure. Allele-specific AHR binding to rs117132860-G was enhanced following both, consistent with predicted weakened AHR binding to the risk/poor-tanning rs117132860-A allele, and allele-preferential AHR expression driven from the protective rs117132860-G allele was observed following UVB exposure. Small deletions surrounding rs117132860 introduced via CRISPR abrogates AHR binding, reduces melanocyte cell growth, and prolongs growth arrest following UVB exposure. These data suggest AHR is a melanoma susceptibility gene at the 7p21.1 risk locus and rs117132860 is a functional variant within a UVB-responsive element, leading to allelic AHR expression and altering melanocyte growth phenotypes upon exposure.

Genetic and Epigenetic Fine Mapping of Complex Trait Associated Loci in the Human Liver.

Deciphering the impact of genetic variation on gene regulation is fundamental to understanding common, complex human diseases. Although histone modifications are important markers of gene regulatory elements of the genome, any specific histone modification has not been assayed in more than a few individuals in the human liver. As a result, the effects of genetic variation on histone modification states in the liver are poorly understood. Here, we generate the most comprehensive genome-wide dataset of two epigenetic marks, H3K4me3 and H3K27ac, and annotate thousands of putative regulatory elements in the human liver. We integrate these findings with genome-wide gene expression data collected from the same human liver tissues and high-resolution promoter-focused chromatin interaction maps collected from human liver-derived HepG2 cells. We demonstrate widespread functional consequences of natural genetic variation on putative regulatory element activity and gene expression levels. Leveraging these extensive datasets, we fine-map a total of 74 GWAS loci that have been associated with at least one complex phenotype. Our results reveal a repertoire of genes and regulatory mechanisms governing complex disease development and further the basic understanding of genetic and epigenetic regulation of gene expression in the human liver tissue.

IL-1 Transcriptional Responses to Lipopolysaccharides Are Regulated by a Complex of RNA Binding Proteins.

The IL1A and IL1B genes lie in close proximity on chromosome 2 near the gene for their natural inhibitor, IL1RN Despite diverse functions, they are all three inducible through TLR4 signaling but with distinct kinetics. This study analyzed transcriptional induction kinetics, chromosome looping, and enhancer RNA production to understand the distinct regulation of these three genes in human cells. IL1A, IL1B, and IL1RN were rapidly induced after stimulation with LPS; however, IL1B mRNA production was less inhibitable by iBET151, suggesting it does not use pause-release regulation. Surprisingly, chromatin looping contacts between IL1A and IL1B were highly intermingled, although those of IL1RN were distinct, and we focused on comparing IL1A and IL1B transcriptional pathways. Our studies demonstrated that enhancer RNAs were produced from a subset of the regulatory regions, that they were critical for production of the mRNAs, and that they bound a diverse array of RNA binding proteins, including p300 but not CBP. We, furthermore, demonstrated that recruitment of p300 was dependent on MAPKs. Integrator is another RNA binding protein recruited to the promoters and enhancers, and its recruitment was more dependent on NF-κB than MAPKs. We found that integrator and NELF, an RNA polymerase II pausing protein, were associated with RNA in a manner that facilitated interaction. We conclude that IL1A and IL1B share many regulatory contacts, signaling pathways, and interactions with enhancer RNAs. A complex of protein interactions with enhancer RNAs emphasize the role of enhancer RNAs and the overall structural aspects of transcriptional regulation.

Metabolic Profile of Reciprocal Supersets in Young, Recreationally Active Women and Men.

ABSTRACT: Realzola, RA, Mang, ZA, Millender, DJ, Beam, JR, Bellovary, BN, Wells, AD, Houck, JM, and Kravitz, L. Metabolic profile of reciprocal supersets in young, recreationally active females and males. J Strength Cond Res 36(10): 2709-2716, 2022-Reciprocal supersets (RSSs) are a time-efficient style of resistance exercise (RE) that consist of performing 2 consecutive exercises with opposing muscle groups while limiting rest times between them. Previous research in men indicates a RSS has an increased physiological response when compared with traditional RE (TRAD). No between-sex comparison of metabolic data for RSSs exists. The purpose of this study was to create a metabolic profile for RSSs in men and women. Eighteen resistance-trained individuals underwent 2 bouts of volume-load equated RE: RSS and TRAD. Reciprocal superset exercises were split into 3 clusters: (a) hexagonal bar deadlift superset with leg press, (b) chest press superset with seated row, and (c) overhead dumbbell press superset with latissimus dorsi pull-downs. The TRAD protocol, doing the same exercises, emulated hypertrophy emphasis training. Oxygen uptake (V̇ o2 ), heart rate (HR), blood lactate ([BLa]), rate of perceived exertion (RPE), and excess post-exercise oxygen consumption (EPOC) were measured. Aerobic and anaerobic energy expenditure were estimated using V̇ o2 and lactate, respectively. The level of significance set for this study was p ≤ 0.05. Regardless of sex, a RSS elicited significantly greater average V̇ o2 , HR, [BLa], RPE, and anaerobic and aerobic energy expenditures, and was completed in a shorter time compared with TRAD ( p ≤ 0.05). When compared with women, men had significantly greater EPOC, average [BLa], and anaerobic and aerobic energy expenditures during RSSs ( p ≤ 0.05). The average [BLa] and aerobic energy expenditure of the men were also significantly greater than the women during TRAD ( p ≤ 0.05). This study suggests that a RSS is a metabolically demanding RE session that may elicit increases in musculoskeletal, cardiorespiratory, and physiological adaptations while decreasing the duration of exercise.

Restriction enzyme selection dictates detection range sensitivity in chromatin conformation capture-based variant-to-gene mapping approaches.

Promoter-focused chromatin conformation techniques directly detect interactions between gene promoters and distal genomic sequences, providing structural information relevant to gene regulation without the excessive non-genic architectural data generated by full-scale Hi-C. 3D promoter 'interactome' maps are crucial for understanding how epigenomic features such as histone modifications and open chromatin, or genetic variants identified in genome-wide association studies (GWAS), contribute to biological function. However, variation in sensitivity between such promoter-focused methods, principally due to restriction enzyme selection, has not been systematically assessed. Here, we performed a head-to-head comparison of promoter capture datasets using 4 cutters (DpnII or MboI) versus the 6 cutter HindIII from the same five cell types. While HindIII generally produces a higher signal-to-noise ratio for significant interactions in comparison to 4-cutters, we show that DpnII/MboI detects more proximal interactions and shows little overlap with the HindIII detection range. Promoter-interacting genomic regions mapped by 4-cutters are more enriched for regulatory features and disease-associated genetic variation than 6-cutters maps, suggesting that high-resolution maps better capture gene regulatory architectures than do lower resolution approaches.

Canonical Notch signaling is required for bone morphogenetic protein-mediated human osteoblast differentiation.

Osteoblast differentiation of bone marrow-derived human mesenchymal stem cells (hMSC) can be induced by stimulation with canonical Notch ligand, Jagged1, or bone morphogenetic proteins (BMPs). However, it remains elusive how these two pathways lead to the same phenotypic outcome. Since Runx2 is regarded as a master regulator of osteoblastic differentiation, we targeted Runx2 with siRNA in hMSC. This abrogated both Jagged1 and BMP2 mediated osteoblastic differentiation, confirming the fundamental role for Runx2. However, while BMP stimulation increased Runx2 and downstream Osterix protein expression, Jagged1 treatment failed to upregulate either, suggesting that canonical Notch signals require basal Runx2 expression. To fully understand the transcriptomic profile of differentiating osteoblasts, RNA sequencing was performed in cells stimulated with BMP2 or Jagged1. There was common upregulation of ALPL and extracellular matrix genes, such as ACAN, HAS3, MCAM, and OLFML2B. Intriguingly, genes encoding components of Notch signaling (JAG1, HEY2, and HES4) were among the top 10 genes upregulated by both stimuli. Indeed, ALPL expression occurred concurrently with Notch activation and inhibiting Notch activity for up to 24 hours after BMP administration with DAPT (a gamma secretase inhibitor) completely abrogated hMSC osteoblastogenesis. Concordantly, RBPJ (recombination signal binding protein for immunoglobulin kappa J region, a critical downstream modulator of Notch signals) binding could be demonstrated within the ALPL and SP7 promoters. As such, siRNA-mediated ablation of RBPJ decreased BMP-mediated osteoblastogenesis. Finally, systemic Notch inhibition using diabenzazepine (DBZ) reduced BMP2-induced calvarial bone healing in mice supporting the critical regulatory role of Notch signaling in BMP-induced osteoblastogenesis.

Heat acclimation during low-intensity exercise increases V̇O2max and Hsp72, but not markers of mitochondrial biogenesis and oxidative phosphorylation, in skeletal tissue.

NEW FINDINGS: What is the central question of this study? Heat acclimation increases tolerance to exercise performed in the heat and may improve maximal oxygen uptake (VO2 max) and performance in temperate environments. However, it is unknown if HA affects the expression of proteins related to mitochondrial biogenesis and oxidative capacity in skeletal muscle. What is the main finding and its importance? We showed that heat acclimation increased VO2 max in a temperate environment but did not change markers of mitochondrial biogenesis and oxidative phosphorylation in the skeletal muscle. ABSTRACT: Heat acclimation (HA) increases tolerance to exercise performed in the heat and may improve maximal oxygen uptake ( V̇O2max ) in temperate environments. However, it is unknown if HA affects the expression of proteins related to mitochondrial biogenesis and oxidative capacity in skeletal muscle. The purpose of this study was to investigate the effect of HA on skeletal muscle markers of mitochondrial biogenesis and oxidative phosphorylation in recreationally trained adults. Thirteen (7 males and 6 females) individuals underwent 10 days of HA. Participants performed two 45 min bouts of exercise (walking at 30-40% maximal velocity at 3% grade) with 10 min rest per session in a hot environment (∼42°C and 30-50% relative humidity). V̇O2max , ventilatory thresholds (VT), and protein expression of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), mitochondrial transcription factor A (TFAM), calcium/calmodulin-dependent protein kinase (CaMK), electron transport chain (ETC) complexes I-IV, and heat shock protein 72 (Hsp72) in skeletal muscle were measured pre- and post-HA. Comparing day 1 to day 10, HA was confirmed by lower resting core temperature (Tcore ) (P = 0.026), final Tcore (P < 0.0001), mean heart rate (HR) (P = 0.002), final HR (P = 0.003), mean ratings of perceived exertion (RPE) (P = 0.026) and final RPE (P = 0.028). Pre- to post-HA V̇O2max (P = 0.045) increased but VT1 (P = 0.263) and VT2 (P = 0.239) were unchanged. Hsp72 (P = 0.007) increased, but skeletal muscle protein expression (PGC-1α, P = 0.119; TFAM, P = 0.763; CaMK, P = 0.19; ETC I, P = 0.629; ETC II, P = 0.724; ETC III, P = 0.206; ETC IV, P = 0.496) were not affected with HA. HA during low-intensity exercise increased V̇O2max in a temperate environment and Hsp72 but it did not affect markers of mitochondrial biogenesis and oxidative phosphorylation in the skeletal muscle.

Next steps in the identification of gene targets for type 1 diabetes.

The purpose of this review is to provide a view of the future of genomics and other omics approaches in defining the genetic contribution to all stages of risk of type 1 diabetes and the functional impact and clinical implementations of the associated variants. From the recognition nearly 50 years ago that genetics (in the form of HLA) distinguishes risk of type 1 diabetes from type 2 diabetes, advances in technology and sample acquisition through collaboration have identified over 60 loci harbouring SNPs associated with type 1 diabetes risk. Coupled with HLA region genes, these variants account for the majority of the genetic risk (~50% of the total risk); however, relatively few variants are located in coding regions of genes exerting a predicted protein change. The vast majority of genetic risk in type 1 diabetes appears to be attributed to regions of the genome involved in gene regulation, but the target effectors of those genetic variants are not readily identifiable. Although past genetic studies clearly implicated immune-relevant cell types involved in risk, the target organ (the beta cell) was left untouched. Through emergent technologies, using combinations of genetics, gene expression, epigenetics, chromosome conformation and gene editing, novel landscapes of how SNPs regulate genes have emerged. Furthermore, both the immune system and the beta cell and their biological pathways have been implicated in a context-specific manner. The use of variants from immune and beta cell studies distinguish type 1 diabetes from type 2 diabetes and, when they are combined in a genetic risk score, open new avenues for prediction and treatment. Graphical abstract.

A Dementia-Associated Risk Variant near TMEM106B Alters Chromatin Architecture and Gene Expression.

Neurodegenerative diseases pose an extraordinary threat to the world's aging population, yet no disease-modifying therapies are available. Although genome-wide association studies (GWASs) have identified hundreds of risk loci for neurodegeneration, the mechanisms by which these loci influence disease risk are largely unknown. Here, we investigated the association between common genetic variants at the 7p21 locus and risk of the neurodegenerative disease frontotemporal lobar degeneration. We showed that variants associated with disease risk correlate with increased expression of the 7p21 gene TMEM106B and no other genes; co-localization analyses implicated a common causal variant underlying both association with disease and association with TMEM106B expression in lymphoblastoid cell lines and human brain. Furthermore, increases in the amount of TMEM106B resulted in increases in abnormal lysosomal phenotypes and cell toxicity in both immortalized cell lines and neurons. We then combined fine-mapping, bioinformatics, and bench-based approaches to functionally characterize all candidate causal variants at this locus. This approach identified a noncoding variant, rs1990620, that differentially recruits CTCF in lymphoblastoid cell lines and human brain to influence CTCF-mediated long-range chromatin-looping interactions between multiple cis-regulatory elements, including the TMEM106B promoter. Our findings thus provide an in-depth analysis of the 7p21 locus linked by GWASs to frontotemporal lobar degeneration, nominating a causal variant and causal mechanism for allele-specific expression and disease association at this locus. Finally, we show that genetic variants associated with risk of neurodegenerative diseases beyond frontotemporal lobar degeneration are enriched in CTCF-binding sites found in brain-relevant tissues, implicating CTCF-mediated gene regulation in risk of neurodegeneration more generally.

The Loss of TET2 Promotes CD8+ T Cell Memory Differentiation.

T cell differentiation requires appropriate regulation of DNA methylation. In this article, we demonstrate that the methylcytosine dioxygenase ten-eleven translocation (TET)2 regulates CD8+ T cell differentiation. In a murine model of acute viral infection, TET2 loss promotes early acquisition of a memory CD8+ T cell fate in a cell-intrinsic manner without disrupting Ag-driven cell expansion or effector function. Upon secondary recall, TET2-deficient memory CD8+ T cells demonstrate superior pathogen control. Genome-wide methylation analysis identified a number of differentially methylated regions in TET2-deficient versus wild-type CD8+ T cells. These differentially methylated regions did not occur at the loci of differentially expressed memory markers; rather, several hypermethylated regions were identified in known transcriptional regulators of CD8+ T cell memory fate. Together, these data demonstrate that TET2 is an important regulator of CD8+ T cell fate decisions.

Leveraging epigenomics and contactomics data to investigate SNP pairs in GWAS.

Although Genome Wide Association Studies (GWAS) have led to many valuable insights into the genetic bases of common diseases over the past decade, the issue of missing heritability has surfaced, as the discovered main effect genetic variants found to date do not account for much of a trait's predicted genetic component. We present a workflow, integrating epigenomics and topologically associating domain data, aimed at discovering trait-associated SNP pairs from GWAS where neither SNP achieved independent genome-wide significance. Each analyzed SNP pair consists of one SNP in a putative active enhancer and another SNP in a putative physically interacting gene promoter in a trait-relevant tissue. As a proof-of-principle case study, we used this approach to identify focused collections of SNP pairs that we analyzed in three independent Type 2 diabetes (T2D) GWAS. This approach led us to discover 35 significant SNP pairs, encompassing both novel signals and signals for which we have found orthogonal support from other sources. Nine of these pairs are consistent with eQTL results, two are consistent with our own capture C experiments, and seven involve signals supported by recent T2D literature.

DNA binding by FOXP3 domain-swapped dimer suggests mechanisms of long-range chromosomal interactions.

FOXP3 is a lineage-specific transcription factor that is required for regulatory T cell development and function. In this study, we determined the crystal structure of the FOXP3 forkhead domain bound to DNA. The structure reveals that FOXP3 can form a stable domain-swapped dimer to bridge DNA in the absence of cofactors, suggesting that FOXP3 may play a role in long-range gene interactions. To test this hypothesis, we used circular chromosome conformation capture coupled with high throughput sequencing (4C-seq) to analyze FOXP3-dependent genomic contacts around a known FOXP3-bound locus, Ptpn22. Our studies reveal that FOXP3 induces significant changes in the chromatin contacts between the Ptpn22 locus and other Foxp3-regulated genes, reflecting a mechanism by which FOXP3 reorganizes the genome architecture to coordinate the expression of its target genes. Our results suggest that FOXP3 mediates long-range chromatin interactions as part of its mechanisms to regulate specific gene expression in regulatory T cells.

The type 2 diabetes presumed causal variant within TCF7L2 resides in an element that controls the expression of ACSL5.

AIMS/HYPOTHESIS: One of the most strongly associated type 2 diabetes loci reported to date resides within the TCF7L2 gene. Previous studies point to the T allele of rs7903146 in intron 3 as the causal variant at this locus. We aimed to identify the actual gene(s) under the influence of this variant. METHODS: Using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease, we generated a 1.4 kb deletion of the genomic region harbouring rs7903146 in the HCT116 cell line, followed by global gene expression analysis. We then carried out a combination of circularised chromosome conformation capture (4C) and Capture C in cell lines, HCT116 and NCM460 in order to ascertain which promoters of these perturbed genes made consistent physical contact with this genomic region. RESULTS: We observed 99 genes with significant differential expression (false discovery rate [FDR] cut-off:10%) and an effect size of at least twofold. The subsequent promoter contact analyses revealed just one gene, ACSL5, which resides in the same topologically associating domain as TCF7L2. The generation of additional, smaller deletions (66 bp and 104 bp) comprising rs7903146 showed consistently reduced ACSL5 mRNA levels across all three deletions of up to 30-fold, with commensurate loss of acyl-CoA synthetase long-chain family member 5 (ACSL5) protein. Notably, the deletion of this single-nucleotide polymorphism region abolished significantly detectable chromatin contacts with the ACSL5 promoter. We went on to confirm that contacts between rs7903146 and the ACSL5 promoter regions were conserved in human colon tissue. ACSL5 encodes ACSL5, an enzyme with known roles in fatty acid metabolism. CONCLUSIONS/INTERPRETATION: This 'variant to gene mapping' effort implicates the genomic location harbouring rs7903146 as a regulatory region for ACSL5.

Regulatory T cells suppress CD4+ T cells through NFAT-dependent transcriptional mechanisms.

Regulatory T cells (Tregs) control autoreactive T cells by inhibiting activation-induced proliferation and cytokine expression. The molecular mechanisms responsible for the inactivation of effector T cells by Tregs remain yet to be fully characterized. We report that T-helper cells stimulated in the presence of Tregs quickly activate NFAT1 and have increased NFAT1-dependent expression of the transcription repressor Ikaros. NFAT1 deficiency or dominant-negative Ikaros compromises Treg-mediated inhibition of T-helper cells in vitro and in vivo. Thus, our results place NFAT-dependent mechanisms as general regulators of T-cell tolerance and show that Treg-mediated suppression of T-helper cells results from the activation of NFAT-regulated gene expression.

Ikaros imposes a barrier to CD8+ T cell differentiation by restricting autocrine IL-2 production.

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.