Foxp3 depends on Ikaros for control of regulatory T cell gene expression and function.

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.

Implicating effector genes at COVID-19 GWAS loci using promoter-focused Capture-C in disease-relevant immune cell types.

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.

Mapping effector genes at lupus GWAS loci using promoter Capture-C in follicular helper T cells.

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.

Ndfip1 restricts mTORC1 signalling and glycolysis in regulatory T cells to prevent autoinflammatory disease.

Foxp3+ T regulatory (Treg) cells suppress immune cell activation and establish normal immune homeostasis. How Treg cells maintain their identity is not completely understood. Here we show that Ndfip1, a coactivator of Nedd4-family E3 ubiquitin ligases, is required for Treg cell stability and function. Ndfip1 deletion in Treg cells results in autoinflammatory disease. Ndfip1-deficient Treg cells are highly proliferative and are more likely to lose Foxp3 expression to become IL-4-producing TH2 effector cells. Proteomic analyses indicate altered metabolic signature of Ndfip1-deficient Treg cells and metabolic profiling reveals elevated glycolysis and increased mTORC1 signalling. Ndfip1 restricts Treg cell metabolism and IL-4 production via distinct mechanisms, as IL-4 deficiency does not prevent hyperproliferation or elevated mTORC1 signalling in Ndfip1-deficient Treg cells. Thus, Ndfip1 preserves Treg lineage stability and immune homeostasis by preventing the expansion of highly proliferative and metabolically active Treg cells and by preventing pathological secretion of IL-4 from Treg cells.

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.

Mbd2 promotes foxp3 demethylation and T-regulatory-cell function.

Use of Foxp3-positive (Foxp3(+)) T-regulatory (Treg) cells as potential cellular therapy in patients with autoimmunity, or post-stem cell or -organ transplantation, requires a sound understanding of the transcriptional regulation of Foxp3. Conserved CpG dinucleotides in the Treg-specific demethylation region (TSDR) upstream of Foxp3 are demethylated only in stable, thymus-derived Foxp3(+) Treg cells. Since methyl-binding domain (Mbd) proteins recruit histone-modifying and chromatin-remodeling complexes to methylated sites, we tested whether targeting of Mbd2 might promote demethylation of Foxp3 and thereby promote Treg numbers or function. Surprisingly, while chromatin immunoprecipitation (ChIP) analysis showed Mbd2 binding to the Foxp3-associated TSDR site in Treg cells, Mbd2 targeting by homologous recombination, or small interfering RNA (siRNA), decreased Treg numbers and impaired Treg-suppressive function in vitro and in vivo. Moreover, we found complete TSDR demethylation in wild-type (WT) Treg cells but >75% methylation in Mbd2(-/-) Treg cells, whereas reintroduction of Mbd2 into Mbd2-null Treg cells restored TSDR demethylation, Foxp3 gene expression, and Treg-suppressive function. Lastly, thymic Treg cells from Mbd2(-/-) mice had normal TSDR demethylation, but compared to WT Treg cells, peripheral Mbd2(-/-) Treg cells had a marked impairment of binding of Tet2, the DNA demethylase enzyme, at the TSDR site. These data show that Mbd2 has a key role in promoting TSDR demethylation, Foxp3 expression, and Treg-suppressive function.

Conserved intergenic elements and DNA methylation cooperate to regulate transcription at the il17 locus.

Naive CD4(+) T cells can differentiate into distinct lineages with unique immune functions. The cytokines TGFß and IL-6 promote the development of Th17 cells that produce IL-17, an inflammatory cytokine not expressed by other T helper lineages. To further understand how IL-17 production is controlled, we studied an ~120-kb genomic region containing the murine il17a and il17f genes and seven evolutionarily conserved, intergenic noncoding sequences. We show that the +28-kb noncoding sequence cooperates with STAT3, RORγt, and Runx1 to enhance transcription from both il17a and il17f promoters. This enhancer and both promoters exhibited Th17 lineage-specific DNA demethylation, accompanied by demethylation of lysine 27 of histone H3 (H3K27) and increased H3K4 methylation. Loss of DNA methylation tended to occur at STAT3 consensus elements, and we show that methylation of one of these elements in the il17a promoter directly inhibits STAT3 binding and transcriptional activity. These results demonstrate that TGFß and IL-6 synergize to epigenetically poise the il17 loci for expression in Th17 cells, and suggest a general mechanism by which active STAT3 may be epigenetically excluded from STAT3-responsive genes in non-Th17 lineages.

De novo DNA methylation is required to restrict T helper lineage plasticity.

Naïve CD4+ T cells are highly plastic and can differentiate into discrete lineages with unique functions during an immune response. Once differentiated, helper T cells maintain a stable transcriptional memory of their initial lineage choice and resist redifferentiation. During embryogenesis, de novo DNA methylation operates on the hypomethylated genome of the blastocyst to achieve tissue-specific patterns of gene expression. Similarly, the ifnγ promoter is hypomethylated in naïve T cells, but Th2, Th17, and iTreg differentiation is accompanied by substantial de novo DNA methylation at this locus. To determine whether de novo DNA methylation is required to restrict T helper lineage plasticity, we used mice with T cell-specific deletion of the methyltransferase DNMT3a. Induction of lineage-specific cytokines occurred normally in the absence of DNMT3a, however, DNMT3a-deficient Th2, Th17, and iTreg completely failed to methylate the ifnγ promoter. This was accompanied by an increase in the transcriptionally permissive trimethyl H3K4 mark, and a reduction in inhibitory H3K27 methylation at the ifnγ locus. Failed de novo methylation resulted in failed silencing of the ifnγ gene, as DNMT3a-deficient Th2, Th17, and iTreg cells produced significant levels of IFNγ following restimulation in the presence of IL-12. Therefore, DNMT3a-mediated DNA methylation restricts T helper plasticity by establishing an epigenetically silent chromatin structure at regulatory regions of the ifnγ gene.

Ikaros silences T-bet expression and interferon-gamma production during T helper 2 differentiation.

CD4+ T cells can be instructed by nonantigen-specific signals to differentiate into functionally distinct lineages with mutually exclusive patterns of cytokine production. The molecular events that drive interferon-gamma (IFN gamma) production during Th1 development are well understood, but mechanisms that silence this cytokine during Th2 polarization are not clear. In this study, we find that the tbx21 gene encoding the Th1 master regulator T-bet is a direct target of the transcriptional repressor Ikaros. In Th2 cells, which do not express T-bet, strong Ikaros binding could be detected at the endogenous tbx21 promoter, whereas this gene was not occupied by Ikaros in T-bet-expressing Th1 cells. Inhibition of Ikaros DNA binding activity during Th2 polarization resulted in loss of Ikaros promoter occupancy, increased T-bet expression, and inappropriate T-bet-dependent production of IFN gamma. Ikaros was also required for epigenetic imprinting of the ifn gamma locus during Th2 polarization, and loss of Ikaros function in vivo led to an inappropriate Th1 response to the parasite Shistosoma mansoni. These studies demonstrate that Ikaros, a factor with an established role in lymphocyte development, also regulates the development of peripheral T helper responses.

Ikaros enforces the costimulatory requirement for IL2 gene expression and is required for anergy induction in CD4+ T lymphocytes.

T cell activation results in dynamic remodeling of the chromatin at the IL2 promoter and induction of IL2 gene transcription. These processes are each dependent upon CD28 costimulation, but the molecular basis for this requirement is not clear. The IL2 promoter contains consensus-binding elements for Ikaros, a lymphocyte-specific zinc-finger DNA-binding protein that can regulate gene expression by recruiting chromatin-remodeling complexes. We find that native Ikaros in CD4(+) T cells exhibits sequence-specific binding to these elements in vitro, and interacts with the endogenous IL2 promoter in vivo, in a manner dependent upon its DNA-binding domain. This binding has important consequences on the regulation of the IL2 gene, because CD4(+) T cells with reduced Ikaros DNA-binding activity no longer require signals from the TCR or CD28 for histone acetylation at the endogenous IL2 promoter, and no longer require CD28 costimulation for expression of the IL2 gene. Furthermore, CD4(+) T cells with reduced Ikaros activity are resistant to clonal anergy induced by TCR ligation in the absence of either CD28 or IL-2R signals. These results establish Ikaros as a transcriptional repressor of the IL2 gene that functions through modulation of chromatin structure and has an obligate role in the induction of anergy.

Transcriptional regulation by Foxp3 is associated with direct promoter occupancy and modulation of histone acetylation.

Regulatory T cells (T(reg)) express Foxp3, a forkhead family member that is necessary and sufficient for T(reg) lineage choice and function. Ectopic expression of Foxp3 in non-T(reg) leads to repression of the interleukin 2 (IL-2) and interferon gamma (IFNgamma) genes, gain of suppressor function, and induction of genes such as CD25, GITR, and CTLA-4, but the mode by which Foxp3 enforces this program is unclear. Using chromatin immunoprecipitation, we have demonstrated that Foxp3 binds to the endogenous IL-2 and IFNgamma loci in T cells, but only after T cell receptor stimulation. This activation-induced Foxp3 binding was abrogated by cyclosporin A, suggesting a role for the phosphatase calcineurin in Foxp3 function. We have also shown that binding of Foxp3 to the IL-2 and IFNgamma genes induces active deacetylation of histone H3, a process that inhibits chromatin remodeling and opposes gene transcription. Conversely, binding of Foxp3 to the GITR, CD25, and CTLA-4 genes results in increased histone acetylation. These data indicate that Foxp3 may regulate transcription through direct chromatin remodeling and show that Foxp3 function is influenced by signals from the TCR.

Defective activation of protein kinase C and Ras-ERK pathways limits IL-2 production and proliferation by CD4+CD25+ regulatory T cells.

Naturally occurring CD4+CD25+ regulatory T cells (Tregs), which play an important role in the maintenance of self-tolerance, proliferate poorly and fail to produce IL-2 following stimulation in vitro with peptide-pulsed or anti-CD3-treated APCs. When TCR proximal and distal signaling events were examined in Tregs, we observed impairments in the amplitude and duration of tyrosine phosphorylation when compared with the response of CD4+CD25- T cells. Defects were also seen in the activity of phospholipase C-gamma and in signals downstream of this enzyme including calcium mobilization, NFAT, NF-kappaB, and Ras-ERK-AP-1 activation. Enhanced stimulation of diacylglycerol-dependent pathways by inhibition of diacylglycerol metabolism could overcome the "anergic state" and support the ability of Tregs to up-regulate CD69, produce IL-2, and proliferate. Our results demonstrate that Tregs maintain their hyporesponsive state by suppressing the induction and propagation of TCR-initiated signals to control the accumulation of second messengers necessary for IL-2 production and proliferation.

Epigenetic remodeling of the IL-2 and IFN-gamma loci in memory CD8 T cells is influenced by CD4 T cells.

Memory T cells (T(M)) are able to rapidly exert effector functions, including immediate effector cytokine production upon re-encounter with Ag, which is critical for protective immunity. Furthermore, this poised state is maintained as T(M) undergo homeostatic proliferation over time. We examined the molecular basis underlying this enhanced functional capacity in CD8 T(M) by comparing them to defective CD8 T(M) generated in the absence of CD4 T cells. Unhelped CD8 T(M) are defective in many functions, including the immediate expression of cytokines, such as IL-2 and IFN-gamma. Our data show that this defect in IL-2 and IFN-gamma production is independent of clonal selection, functional avidity maturation, and the integrity of proximal TCR signaling, but rather involves epigenetic modification of these cytokine genes. Activated Ag-specific CD8 T cells exhibit rapid DNA demethylation at the IL-2 and IFN-gamma loci and substantial histone acetylation at the IFN-gamma promoter and enhancer regions. These epigenetic modifications occur early after infection at the effector stage and are maintained through memory development. However, activated unhelped CD8 T cells, which fail to develop into functional memory and are incapable of rapid cytokine production, exhibit increased DNA methylation at the IL-2 promoter and fail to acetylate histones at the IFN-gamma locus. Thus, CD4 T cell help influences epigenetic modification during CD8 T(M) differentiation and these epigenetic changes provide a molecular basis for the enhanced responsiveness and the maintenance of a "ready-to-respond" state in CD8 T(M).

Signals from CD28 induce stable epigenetic modification of the IL-2 promoter.

CD28 costimulation controls multiple aspects of T cell function, including the expression of proinflammatory cytokine genes. One of these genes encodes IL-2, a growth factor that influences T cell proliferation, survival, and differentiation. Antigenic signaling in the absence of CD28 costimulation leads to anergy, a mechanism of tolerance that renders CD4+ T cells unable to produce IL-2. The molecular mechanisms by which CD28 costimulatory signals induce gene expression are not fully understood. In eukaryotic cells, the expression of many genes is influenced by their physical structure at the level of DNA methylation and local chromatin remodeling. To address whether these epigenetic mechanisms are operative during CD28-dependent gene expression in CD4+ T cells, we compared cytosine methylation and chromatin structure at the IL-2 locus in fully activated CD4+ effector T cells and CD4+ T cells rendered anergic by TCR ligation in the absence of CD28 costimulation. Costimulation through CD28 led to marked, stable histone acetylation and loss of cytosine methylation at the IL-2 promoter/enhancer. This was accompanied by extensive remodeling of the chromatin in this region to a structure highly accessible to DNA binding proteins. Conversely, TCR activation in the absence of CD28 costimulation was not sufficient to promote histone acetylation or cytosine demethylation, and the IL-2 promoter/enhancer in anergic cells remained completely inaccessible. These data suggest that CD28 may function through epigenetic mechanisms to promote CD4+ T cell responses.

Regulation of mouse mammary tumor virus env transcriptional activator initiated mammary tumor virus superantigen transcripts in lymphomas of SJL/J mice: role of Ikaros, demethylation, and chromatin structural change in the transcriptional activation of mammary tumor virus superantigen.

Mammary tumor virus (Mtv29)-encoded superantigen expressed by SJL/J mouse B cell lymphomas stimulates CD4+V16+ T cells and thereby acquires T cell help necessary for lymphoma growth. Mtv29 mouse mammary tumor virus env transcriptional activator (META) env-controlled Mtv29 superantigen (vSAg29) mRNA transcripts (1.8 kb) are not expressed in normal B or other somatic cells. Real-time PCR-based assays with DNA from normal SJL liver and vSAg29- lymphoma (cNJ101), digested with methylation-sensitive enzymes, showed hypermethylation at AvaI, FspI, HpaII, ThaI, and the distal HgaI sites of the META env, but vSAg29+ lymphoma cells showed significant demethylation at AvaI, HpaII, and the distal HgaI sites. The distal HgaI site that is adjacent to an Ikaros binding site is significantly demethylated in the META env DNA from primary lymphomas. Gel shift assays showed binding of Ikaros to a sequence representing this region in the META env. SJL lymphomas expressed the Ikaros isoform Ik6 that was absent in normal B cells. vSAg29+ cells exhibited increased DNaseI accessibility to chromatin at the vSAg29 initiation site. Treatment of cNJ101 cells with a demethylating agent, 5-azacytidine, and a histone deacetylase inhibitor, trichostatin A, caused hypomethylation at AvaI, HpaII, and distal HgaI sites and led to chromatin structural change at the vSAg29 initiation site, accompanied by the expression of vSAg29 transcripts. This enabled cNJ101 cells to stimulate SJL lymphoma-responsive CD4+V16+ T hybridoma cells. Thus, demethylation at the distal HgaI site of the Mtv29 META env permits vSAg29 expression, which may have an impact on the development of germinal center-derived B cell lymphomas of SJL/J mice.