Transcription Factor PU.1 Represses and Activates Gene Expression in Early T Cells by Redirecting Partner Transcription Factor Binding.

Transcription factors normally regulate gene expression through their action at sites where they bind to DNA. However, the balance of activating and repressive functions that a transcription factor can mediate is not completely understood. Here, we showed that the transcription factor PU.1 regulated gene expression in early T cell development both by recruiting partner transcription factors to its own binding sites and by depleting them from the binding sites that they preferred when PU.1 was absent. The removal of partner factors Satb1 and Runx1 occurred primarily from sites where PU.1 itself did not bind. Genes linked to sites of partner factor "theft" were enriched for genes that PU.1 represses despite lack of binding, both in a model cell line system and in normal T cell development. Thus, system-level competitive recruitment dynamics permit PU.1 to affect gene expression both through its own target sites and through action at a distance.

Runx1 and Runx3 drive progenitor to T-lineage transcriptome conversion in mouse T cell commitment via dynamic genomic site switching.

Runt domain-related (Runx) transcription factors are essential for early T cell development in mice from uncommitted to committed stages. Single and double Runx knockouts via Cas9 show that target genes responding to Runx activity are not solely controlled by the dominant factor, Runx1. Instead, Runx1 and Runx3 are coexpressed in single cells; bind to highly overlapping genomic sites; and have redundant, collaborative functions regulating genes pivotal for T cell development. Despite stable combined expression levels across pro-T cell development, Runx1 and Runx3 preferentially activate and repress genes that change expression dynamically during lineage commitment, mostly activating T-lineage genes and repressing multipotent progenitor genes. Furthermore, most Runx target genes are sensitive to Runx perturbation only at one stage and often respond to Runx more for expression transitions than for maintenance. Contributing to this highly stage-dependent gene regulation function, Runx1 and Runx3 extensively shift their binding sites during commitment. Functionally distinct Runx occupancy sites associated with stage-specific activation or repression are also distinguished by different patterns of partner factor cobinding. Finally, Runx occupancies change coordinately at numerous clustered sites around positively or negatively regulated targets during commitment. This multisite binding behavior may contribute to a developmental "ratchet" mechanism making commitment irreversible.

T-bet represses T(H)17 differentiation by preventing Runx1-mediated activation of the gene encoding RORγt.

Overactive responses by interleukin 17 (IL-17)-producing helper T cells (T(H)17 cells) are tightly linked to the development of autoimmunity, yet the factors that negatively regulate the differentiation of this lineage remain unknown. Here we report that the transcription factor T-bet suppressed development of the T(H)17 cell lineage by inhibiting transcription of Rorc (which encodes the transcription factor RORγt). T-bet interacted with the transcription factor Runx1, and this interaction blocked Runx1-mediated transactivation of Rorc. T-bet Tyr304 was required for formation of the T-bet-Runx1 complex, for blockade of Runx1 activity and for inhibition of the T(H)17 differentiation program. Our data reinforce the idea of master regulators that shape immune responses by simultaneously activating one genetic program while silencing the activity of competing regulators in a common progenitor cell.

Dysregulated RASGRP1 expression through RUNX1 mediated transcription promotes autoimmunity.

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

The Clusters of Transcription Factors NFATC2, STAT5, GATA2, AP1, RUNX1 and EGR2 Binding Sites at the Induced Il13 Enhancers Mediate Il13 Gene Transcription in Response to Antigenic Stimulation.

IL-13 plays a critical role in mediating many biological processes responsible for allergic inflammation. Mast cells express Il13 mRNA and produce IL-13 protein in response to antigenic stimulation. Enhancers are essential in promoting gene transcription and are thought to activate transcription by delivering essential accessory cofactors to the promoter to potentiate gene transcription. However, enhancers mediating Il13 have not been identified. Furthermore, which Il13 enhancers detect signals triggered by antigenic stimulation have not yet been defined. In this study, we identified potential mouse Il13 enhancers using histone modification monomethylation at lysine residue 4 on histone 3 (H3K4me1) chromatin immunoprecipitation sequencing and acetylation at lysine residue 27 on histone 3 (H3K27ac) chromatin immunoprecipitation sequencing. We used Omni-assay for transposase-accessible chromatin sequencing to determine which accessible regions within the potential Il13 enhancers that responded to IgE receptor crosslinking. We also demonstrated that the transcription factor cluster consisting of the NFATC2, STAT5, GATA2, AP1, and RUNX1 binding sites at the proximal Il13 enhancer and the transcription factor cluster consisting of the EGR2 binding site at the distal Il13 E+6.5 enhancer are critical in sensing the signals triggered by antigenic stimulation. Those enhancers, which are responsive to antigenic stimulation and are constitutively active, cooperate to generate greater transcriptional outputs. Our study reveals a novel mechanism underlying how antigenic stimulation induces robust Il13 mRNA expression in mouse mast cells.

Notch Signaling Controls Transcription via the Recruitment of RUNX1 and MYB to Enhancers during T Cell Development.

Tcrd and Tcrg display identical developmental programs that depend on the activity of the enhancers Eδ and Eγ being "on" in pre-ß-selection thymocytes to activate transcription and V(D)J recombination of the unrearranged genes and "off" in post-ß-selection CD4+CD8+ double-positive thymocytes to inhibit transcription of the rearranged genes and avoid the expression of TCR δ- and TCR γ-chains in αß T lymphocytes. Eδ and Eγ activity depends on transcription factor binding to essential Runx and Myb sites and parallels that of Notch signaling. We performed Notch gain- and loss-of-function experiments and found that Notch signaling activates Tcrd and Tcrg transcription by favoring the recruitment of RUNX1 and MYB to the enhancers. Our results suggest that the dissociation of RUNX1 and MYB from Eδ and Eγ chromatin in double-positive thymocytes, which results in enhancer inactivation, is caused by decreased Notch signaling triggered by pre-TCR signaling, thereby deciphering the molecular mechanism of Tcrd and Tcrg silencing during ß-selection. These findings reveal a novel molecular mechanism for gene regulation via Notch signaling through the recruitment of RUNX1 and MYB to enhancer chromatin during thymocyte development.

Long non-coding RNA RUNXOR accelerates MDSC-mediated immunosuppression in lung cancer.

BACKGROUND: RUNX1 overlapping RNA (RUNXOR) is a long non-coding RNA that has been indicated as a key regulator in the development of myeloid cells by targeting runt-related transcription factor 1 (RUNX1). Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells consisting of immature granulocytes and monocytes with immunosuppression. However, the impact of lncRNA RUNXOR on the development of MDSCs remains unknown. METHODS: Both the expressions of RUNXOR and RUNX1 in the peripheral blood were measured by qRT-PCR. Human MDSCs used in this study were isolated from tumor tissue of patients with lung cancer by FCM or induced from PBMCs of healthy donors with IL-1ß + GM-CSF. Specific siRNA was used to knockdown the expression of RUNXOR in MDSCs. RESULTS: In this study, we found that the lncRNA RUNXOR was upregulated in the peripheral blood of lung cancer patients. In addition, as a target gene of RUNXOR, the expression of RUNX1 was downregulated in lung cancer patients. Finally, the expression of RUNXOR was higher in MDSCs isolated from the tumor tissues of lung cancer patients compared with cells from adjacent tissue. In addition, RUNXOR knockdown decreased Arg1 expression in MDSCs. CONCLUSIONS: Based on our findings, it is illustrated that RUNXOR is significantly associated with the immunosuppression induced by MDSCs in lung cancer patients and may be a target of anti-tumor therapy.

Indispensable role of the Runx1-Cbfbeta transcription complex for in vivo-suppressive function of FoxP3+ regulatory T cells.

Naturally arising regulatory T (Treg) cells express the transcription factor FoxP3, which critically controls the development and function of Treg cells. FoxP3 interacts with another transcription factor Runx1 (also known as AML1). Here, we showed that Treg cell-specific deficiency of Cbfbeta, a cofactor for all Runx proteins, or that of Runx1, but not Runx3, induced lymphoproliferation, autoimmune disease, and hyperproduction of IgE. Cbfb-deleted Treg cells exhibited impaired suppressive function in vitro and in vivo, with altered gene expression profiles including attenuated expression of FoxP3 and high expression of interleukin-4. The Runx complex bound to more than 3000 gene loci in Treg cells, including the Foxp3 regulatory regions and the Il4 silencer. In addition, knockdown of RUNX1 showed that RUNX1 is required for the optimal regulation of FoxP3 expression in human T cells. Taken together, our results indicate that the Runx1-Cbfbeta heterodimer is indispensable for in vivo Treg cell function, in particular, suppressive activity and optimal expression of FoxP3.

MAZR and Runx Factors Synergistically Repress ThPOK during CD8+ T Cell Lineage Development.

Th-inducing Pox virus and zinc finger/Krüppel-like factor (ThPOK) is a key commitment factor for CD4(+) lineage T cells and is essential for the maintenance of CD4 lineage integrity; thus, the expression of ThPOK has to be tightly controlled. In this article, we demonstrate that Myc-associated zinc finger-related factor (MAZR) and Runt-related transcription factor 1 (Runx1) together repressed ThPOK in preselection double-positive thymocytes, whereas MAZR acted in synergy with Runx3 in the repression of ThPOK in CD8(+) T cells. Furthermore, MAZR-Runx1 and MAZR-Runx3 double-mutant mice showed enhanced derepression of Cd4 in double-negative thymocytes and in CD8(+) T cells in comparison with Runx1 or Runx3 single-deficient mice, respectively, indicating that MAZR modulates Cd4 silencing. Thus, our data demonstrate developmental stage-specific synergistic activities between MAZR and Runx/core-binding factor ß (CBFß) complexes. Finally, retroviral Cre-mediated conditional deletion of MAZR in peripheral CD8(+) T cells led to the derepression of ThPOK, thus showing that MAZR is also part of the molecular machinery that maintains a repressed state of ThPOK in CD8(+) T cells.

Twist1 and Twist2 Contribute to Cytokine Downregulation following Chronic NOD2 Stimulation of Human Macrophages through the Coordinated Regulation of Transcriptional Repressors and Activators.

Proper regulation of microbial-induced cytokines is critical to intestinal immune homeostasis. Acute stimulation of nucleotide-binding oligomerization domain 2 (NOD2), the Crohn's disease-associated sensor of bacterial peptidoglycan, induces cytokines. However, chronic NOD2 stimulation in macrophages decreases cytokines upon pattern recognition receptor (PRR) restimulation; cytokine attenuation to PRR stimulation is similarly observed in intestinal macrophages. The role for the transcriptional repressors Twist1 and Twist2 in regulating PRR-induced cytokine outcomes is poorly understood and has not been reported for NOD2. We found that Twist1 and Twist2 were required for optimal cytokine downregulation during acute and, particularly, chronic NOD2 stimulation of human macrophages. Consistently, Twist1 and Twist2 expression was increased after chronic NOD2 stimulation; this increased expression was IL-10 and TGF-ß dependent. Although Twist1 and Twist2 did not coregulate each other's expression, they cooperated to enhance binding to cytokine promoters after chronic NOD2 stimulation. Moreover, Twist1 and Twist2 contributed to enhance expression and promoter binding of the proinflammatory inhibitor c-Maf and the transcriptional repressor Bmi1. Restoring c-Maf and Bmi1 expression in Twist-deficient macrophages restored NOD2-induced cytokine downregulation. Furthermore, with chronic NOD2 stimulation, Twist1 and Twist2 contributed to the decreased expression and cytokine promoter binding of the transcriptional activators activating transcription factor 4, C/EBPα, Runx1, and Runx2. Knockdown of these transcriptional activators in Twist-deficient macrophages restored cytokine downregulation after chronic NOD2 stimulation. Finally, NOD2 synergized with additional PRRs to increase Twist1 and Twist2 expression and Twist-dependent pathways. Therefore, after chronic NOD2 stimulation Twist1 and Twist2 coordinate the regulation of both transcriptional activators and repressors, thereby mediating optimal cytokine downregulation.

Immune evasion by oncogenic proteins of acute myeloid leukemia.

PML-RARA and AML1-ETO are important oncogenic fusion proteins that play a central role in transformation to acute myeloid leukemia (AML). Whether these fusion proteins render the tumor cells with immune evasion properties is unknown. Here we show that both oncogenic proteins specifically downregulate the expression of CD48, a ligand of the natural killer (NK) cell activating receptor 2B4, thereby leading to decreased killing by NK cells. We demonstrate that this process is histone deacetylase (HDAC)-dependent, that it is mediated through the downregulation of CD48 messenger RNA, and that treatment with HDAC inhibitors (HDACi) restores the expression of CD48. Furthermore, by using chromatin immunoprecepitation (ChIP) experiments, we show that AML1-ETO directly interacts with CD48. Finally, we show that AML patients who are carrying these specific translocations have low expression of CD48.

TEL (ETV6)-AML1 (RUNX1) initiates self-renewing fetal pro-B cells in association with a transcriptional program shared with embryonic stem cells in mice.

The initial steps involved in the pathogenesis of acute leukemia are poorly understood. The TEL-AML1 fusion gene usually arises before birth, producing a persistent and covert preleukemic clone that may convert to precursor B cell leukemia following the accumulation of secondary genetic "hits." Here, we show that TEL-AML1 can induce persistent self-renewing pro-B cells in mice. TEL-AML1+ cells nevertheless differentiate terminally in the long term, providing a "window" period that may allow secondary genetic hits to accumulate and lead to leukemia. TEL-AML1-mediated self-renewal is associated with a transcriptional program shared with embryonic stem cells (ESCs), within which Mybl2, Tgif2, Pim2, and Hmgb3 are critical and sufficient components to establish self-renewing pro-B cells. We further show that TEL-AML1 increases the number of leukemia-initiating cells that are generated in collaboration with additional genetic hits, thus providing an overall basis for the development of novel therapeutic and preventive measures targeting the TEL-AML1-associated transcriptional program.

Transcription factor AP4 modulates reversible and epigenetic silencing of the Cd4 gene.

CD4 coreceptor expression is negatively regulated through activity of the Cd4 silencer in CD4(-)CD8(-) double-negative (DN) thymocytes and CD8(+) cytotoxic lineage T cells. Whereas Cd4 silencing is reversed during transition from DN to CD4(+)CD8(+) double-positive stages, it is maintained through heritable epigenetic processes following its establishment in mature CD8(+) T cells. We previously demonstrated that the Runx family of transcription factors is required for Cd4 silencing both in DN thymocytes and CD8(+) T cells. However, additional factors that cooperate with Runx proteins in the process of Cd4 silencing remain unknown. To identify collaborating factors, we used microarray and RNAi-based approaches and found the basic helix-loop-helix ZIP transcription factor AP4 to have an important role in Cd4 regulation. AP4 interacts with Runx1 in cells in which Cd4 is silenced, and is required for Cd4 silencing in immature DN thymocytes through binding to the proximal enhancer. Furthermore, although AP4-deficient CD8(+) T cells appeared to normally down-regulate CD4 expression, AP4 deficiency significantly increased the frequency of CD4-expressing effector/memory CD8(+) T cells in mice harboring point mutations in the Cd4 silencer. Our results suggest that AP4 contributes to Cd4 silencing both in DN and CD8(+) T cells by enforcing checkpoints for appropriate timing of CD4 expression and its epigenetic silencing.

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.

Multiple phosphorylation sites are important for RUNX1 activity in early hematopoiesis and T-cell differentiation.

RUNX1 is essential for definitive hematopoiesis and T-cell differentiation. It has been shown that RUNX1 is phosphorylated at specific serine and threonine residues by several kinase families. However, it remains unclear whether RUNX1 phosphorylation is absolutely required for its biological functions. Here, we evaluated hematopoietic activities of RUNX1 mutants with serine (S)/threonine (T) to alanine (A), aspartic acid (D), or glutamic acid (E) mutations at phosphorylation sites using primary culture systems. Consistent with the results of knockin mice, RUNX1-2A, carrying two phospho-deficient mutations at S276 and S293, retained hematopoietic activity. RUNX1-4A, carrying four mutations at S276, S293, T300, and S303, showed impaired T-cell differentiation activity, but retained the ability to rescue the defective early hematopoiesis of Runx1-deficient cells. Notably, RUNX1-5A, carrying five mutations at S276, S293, T300, S303, and S462, completely lost its hematopoietic activity. In contrast, the phospho-mimic proteins RUNX1-4D/E and RUNX1-5D/E exhibited normal function. Our study identifies multiple phosphorylation sites that are indispensable for RUNX1 activity in hematopoiesis.

Differential effects of inhibition of bone morphogenic protein (BMP) signalling on T-cell activation and differentiation.

Bone morphogenetic proteins (BMPs) are involved in patterning and cellular fate in various organs including the thymus. However, the redundancy of BMPs and their receptors have made it difficult to analyse their physiological roles. Here, we investigated the role of BMP signalling in peripheral CD4(+) T cells by analysing the effects of an inhibitor of BMP signalling, dorsomorphin. Dorsomorphin suppressed phosphorylation of SMAD1/5/8, suggesting that BMP signalling naturally occurs in T cells. At high doses, dorsomorphin suppressed proliferation of T cells in a dose-dependent manner, inducing G1 arrest. Also, dorsomorphin suppressed Th17 and induced Treg-cell differentiation, while preserving Th2 differentiation. Dorsomorphin efficiently suppressed IL-2 production even at low doses in mouse CD4(+) T cells, suggesting that the BMP-Smad signalling physiologically regulates IL-2 transcription in these cells. In addition, recombinant BMP2 induced a dose-dependent multiphasic pattern of IL-2 production, while Noggin suppressed IL-2 production at higher doses in Jurkat cells. Notably, BMP signalling controlled the phosphorylation of RUNX1, revealing the molecular nature of its effect. Collectively, we describe multiple effects of dorsomorphin and Noggin on T-cell activation and differentiation, demonstrating a physiological role for BMP signalling in these processes.

The only proposed T-cell epitope derived from the TEL-AML1 translocation is not naturally processed.

Adoptive therapy with T-cell receptor (TCR)-engineered T cells is a promising approach in cancer treatment. While usage of T cells specific for tumor-associated antigens (TAAs) can lead to serious side effects because of autoimmunity, targeting true tumor-specific mutations, such as the products of translocations in leukemias, should reduce such a risk. A potentially ideal target might be the chimeric protein TEL-AML1, which results from the chromosomal translocation 12;21 and represents the most common fusion gene in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Within the fusion region of TEL-AML1, a single epitope has been described by reverse immunology as immunogenic in HLA-A*0201 restriction settings. As a potential source of TCRs specific for this TEL-AML1 epitope, we have used mice expressing a human TCR-αß repertoire and human MHC class I. Surprisingly, we have found that, although a specific functional CD8(+) T-cell response against this peptide could be evoked, the described epitope was in fact not endogenously processed. Analyses done with a potent antigen-presenting cell line, as well as with purified human proteasomes, support the conclusion that this peptide cannot be proposed as a potential target in immunotherapy of ALL in HLA-A*0201-restricted fashion.

The role of the basic helix-loop-helix transcription factor Dec1 in the regulatory T cells.

Naturally occurring regulatory T (Treg) cells play a central role in the maintenance of immune homeostasis and in restraining the development of spontaneous inflammatory responses. However, the underlying mechanisms of Treg homeostasis remain incompletely understood. Of particular note, the IL-2Rα (CD25) is crucial for the homeostasis of Treg cells and the prevention of lymphoproliferative autoimmune disease. In this paper, we report that the basic helix-loop-helix transcription factor Dec1 is involved in the homeostasis of Treg cells and plays a role in their survival or expansion after adoptive transfer to lymphopenic recipients. Hence, it is crucial for the suppression of effector T cell-mediated inflammatory responses. Enforced expression of Dec1 upregulates CD25 expression during thymocyte development and increases the number of Treg cells in the periphery. Dec1 binds the transcription factor Runx1 and colocalizes with Runx1 in Treg cells. Specifically, we demonstrate that in Treg cells the Dec1/Runx1 complex binds to regulatory elements present in the Il-2rα locus. Collectively, these data show how Dec1 mechanistically acts in Treg cells.

Over-expression of Runx1 transcription factor impairs the development of thymocytes from the double-negative to double-positive stages.

Runx1 transcription factor is highly expressed at a CD4/CD8-double-negative (DN) stage of thymocyte development but is down-regulated when cells proceed to the double-positive (DP) stage. In the present study, we examined whether the down-regulation of Runx1 is necessary for thymocyte differentiation from the DN to DP stage. When Runx1 was artificially over-expressed in thymocytes by Lck-driven Cre, the DN3 population was unaffected, as exemplified by proper pre-T-cell receptor expression, whereas the DN4 population was perturbed as shown by the decrease in the CD27(hi) sub-fraction. In parallel, the growth rate of DN4 cells was reduced by half, as measured by bromodeoxyuridine incorporation. These events impaired the transition of DN4 cells to the DP stage, resulting in the drastic reduction of the number of DP thymocytes. The Runx1 gene has two promoters, a proximal and a distal promoter; and, in thymocytes, endogenous Runx1 was mainly transcribed from the distal promoter. Interestingly, only distal, but not proximal, Runx1 over-expression exhibited an inhibitory effect on thymocyte differentiation, suggesting that the distal Runx1 protein may fulfil a unique function. Our collective results indicate that production of the distal Runx1 protein must be adequately down-regulated for thymocytes to transit from the DN to the DP stage, a critical step in the massive expansion of the T-cell lineage.

The DEL-1/ß3 integrin axis promotes regulatory T cell responses during inflammation resolution.

FOXP3+CD4+ regulatory T cells (Tregs) are critical for immune homeostasis and respond to local tissue cues, which control their stability and function. We explored here whether developmental endothelial locus-1 (DEL-1), which, like Tregs, increases during resolution of inflammation, promotes Treg responses. DEL-1 enhanced Treg numbers and function at barrier sites (oral and lung mucosa). The underlying mechanism was dissected using mice lacking DEL-1 or expressing a point mutant thereof, or mice with T cell-specific deletion of the transcription factor RUNX1, identified by RNA sequencing analysis of the DEL-1-induced Treg transcriptome. Specifically, through interaction with αvß3 integrin, DEL-1 promoted induction of RUNX1-dependent FOXP3 expression and conferred stability of FOXP3 expression upon Treg restimulation in the absence of exogenous TGF-ß1. Consistently, DEL-1 enhanced the demethylation of the Treg-specific demethylated region (TSDR) in the mouse Foxp3 gene and the suppressive function of sorted induced Tregs. Similarly, DEL-1 increased RUNX1 and FOXP3 expression in human conventional T cells, promoting their conversion into induced Tregs with increased TSDR demethylation, enhanced stability, and suppressive activity. We thus uncovered a DEL-1/αvß3/RUNX1 axis that promotes Treg responses at barrier sites and offers therapeutic options for modulating inflammatory/autoimmune disorders.