How autoreactive thymocytes differentiate into regulatory versus effector CD4+ T cells after avoiding clonal deletion.

Thymocytes bearing autoreactive T cell receptors (TCRs) are agonist-signaled by TCR/co-stimulatory molecules to either undergo clonal deletion or to differentiate into specialized regulatory T (Treg) or effector T (Teff) CD4+ cells. How these different fates are achieved during development remains poorly understood. We now document that deletion and differentiation are agonist-signaled at different times during thymic selection and that Treg and Teff cells both arise after clonal deletion as alternative lineage fates of agonist-signaled CD4+CD25+ precursors. Disruption of agonist signaling induces CD4+CD25+ precursors to initiate Foxp3 expression and become Treg cells, whereas persistent agonist signaling induces CD4+CD25+ precursors to become IL-2+ Teff cells. Notably, we discovered that transforming growth factor-ß induces Foxp3 expression and promotes Treg cell development by disrupting weaker agonist signals and that Foxp3 expression is not induced by IL-2 except under non-physiological in vivo conditions. Thus, TCR signaling disruption versus persistence is a general mechanism of lineage fate determination in the thymus that directs development of agonist-signaled autoreactive thymocytes.

B Cells Are the Dominant Antigen-Presenting Cells that Activate Naive CD4+ T Cells upon Immunization with a Virus-Derived Nanoparticle Antigen.

B cells can present antigens to CD4+ T cells, but it is thought that dendritic cells (DCs) are the primary initiators of naive CD4+ T cell responses. Nanoparticles, including virus-like particles (VLPs), are attractive candidates as carriers for vaccines and drug delivery. Using RNA phage Qß-derived VLP (Qß-VLP) as a model antigen, we found that antigen-specific B cells were the dominant antigen-presenting cells that initiated naive CD4+ T cell activation. B cells were sufficient to induce T follicular helper cell development in the absence of DCs. Qß-specific B cells promoted CD4+ T cell proliferation and differentiation via cognate interactions and through Toll-like receptor signaling-mediated cytokine production. Antigen-specific B cells were also involved in initiating CD4+ T cell responses during immunization with inactivated influenza virus. These findings have implications for the rational design of nanoparticles as vaccine candidates, particularly for therapeutic vaccines that aim to break immune tolerance.

A GPR174-CCL21 module imparts sexual dimorphism to humoral immunity.

Humoral immune responses to immunization and infection and susceptibilities to antibody-mediated autoimmunity are generally lower in males1-3. However, the mechanisms underlying such sexual dimorphism are not well understood. Here we show that there are intrinsic differences between the B cells that produce germinal centres in male and female mice. We find that antigen-activated male B cells do not position themselves as efficiently as female B cells in the centre of follicles in secondary lymphoid organs, in which germinal centres normally develop. Moreover, GPR174-an X-chromosome-encoded G-protein-coupled receptor-suppresses the formation of germinal centres in male, but not female, mice. This effect is intrinsic to B cells, and correlates with the GPR174-enhanced positioning of B cells towards the T-cell-B-cell border of follicles, and the distraction of male, but not female, B cells from S1PR2-driven follicle-centre localization. Biochemical fractionation of conditioned media that induce B-cell migration in a GPR174-dependent manner identifies CCL21 as a GPR174 ligand. In response to CCL21, GPR174 triggers a calcium flux and preferentially induces the migration of male B cells; GPR174 also becomes associated with more Gαi protein in male than in female B cells. Male B cells from orchidectomized mice exhibit impaired GPR174-mediated migration to CCL21, and testosterone treatment rescues this defect. Female B cells from testosterone-treated mice exhibit male-like GPR174-Gαi association and GPR174-mediated migration. Deleting GPR174 from male B cells causes more efficient positioning towards the follicular centre, the formation of more germinal centres and an increased susceptibility to B-cell-dependent experimental autoimmune encephalomyelitis. By identifying GPR174 as a receptor for CCL21 and demonstrating its sex-dependent control of B-cell positioning and participation in germinal centres, we have revealed a mechanism by which B-cell physiology is fine-tuned to impart sexual dimorphism to humoral immunity.

Comparative Efficacy of P-CAB vs Proton Pump Inhibitors for Grade C/D Esophagitis: A Systematic Review and Network Meta-analysis.

INTRODUCTION: Los Angeles grade C/D esophagitis is a severe manifestation of gastroesophageal reflux disease that require active treatment and close follow-up. Potassium competitive acid blockers (P-CAB) are promising alternatives to proton pump inhibitors (PPI). We aimed to compare the efficacy and safety of P-CAB and PPI in healing grade C/D esophagitis to aid clinical decision-making. METHODS: A systematic literature search was performed using PubMed, MEDLINE, and Cochrane Central Register of Controlled Trials. Randomized controlled trials were eligible for inclusion if efficacy of P-CAB and PPI in healing grade C/D esophagitis was reported. Pooled risk ratios and risk difference with 95% credible intervals were used to summarize estimated effect of each comparison. The benefit of treatments was ranked using the surface under the cumulative probability ranking score. RESULTS: Of 5,876 articles identified in the database, 24 studies were eligible. Studies included incorporated 3 P-CAB (vonoprazan, tegoprazan, and keverprazan) and 6 PPI (lansoprazole, esomeprazole, omeprazole, rabeprazole extended-release (ER), pantoprazole, and dexlansoprazole). Based on the failure to achieve mucosal healing, 20 mg of vonoprazan q.d. ranked the first among PPI in initial and maintained healing of grade C/D esophagitis (surface under the cumulative probability ranking score = 0.89 and 0.87, respectively). Vonoprazan had similar risk of incurring adverse events, severe adverse events, and withdrawal to drug when compared with PPI. For those who attempted lower maintenance treatment dose, 10 mg of vonoprazan q.d. was a reasonable choice, considering its moderate efficacy and safety. DISCUSSION: Vonoprazan has considerable efficacy in initial and maintained healing of grade C/D esophagitis compared with PPI, with moderate short-term and long-term safety.

Repression of the genome organizer SATB1 in regulatory T cells is required for suppressive function and inhibition of effector differentiation.

Regulatory T cells (T(reg) cells) are essential for self-tolerance and immune homeostasis. Lack of effector T cell (T(eff) cell) function and gain of suppressive activity by T(reg) cells are dependent on the transcriptional program induced by Foxp3. Here we report that repression of SATB1, a genome organizer that regulates chromatin structure and gene expression, was crucial for the phenotype and function of T(reg) cells. Foxp3, acting as a transcriptional repressor, directly suppressed the SATB1 locus and indirectly suppressed it through the induction of microRNAs that bound the SATB1 3' untranslated region. Release of SATB1 from the control of Foxp3 in T(reg) cells caused loss of suppressive function, establishment of transcriptional T(eff) cell programs and induction of T(eff) cell cytokines. Our data support the proposal that inhibition of SATB1-mediated modulation of global chromatin remodeling is pivotal for maintaining T(reg) cell functionality.

A Split-Cre system designed to detect simultaneous expression of two genes based on SpyTag/SpyCatcher conjugation and Split-GFP dimerization.

The Split-Cre system is a powerful tool for genetic manipulation and can be used to spatiotemporally control gene expression in vivo. However, the low activity of the reconstituted NCre/CCre recombinase in the Split-Cre system limits its application as an indicator of the simultaneous expression of a pair of genes of interest. Here, we describe two approaches for improving the activity of the Split-Cre system after Cre reconstitution based on self-associating split GFP (Split-GFP) and SpyTag/SpyCatcher conjugation. First, we created the Split-GFP-Cre system by constructing fusion proteins of NCre and CCre with the N-terminal and C-terminal subunits of GFP, respectively. Reconstitution of Cre by GFP-mediated dimerization of the two fusion proteins resulted in recombinase activity approaching that of full-length Cre in living cells. Second, to further increase recombinase activity at low levels of Split-Cre expression, the Split-Spy-GCre system was established by incorporating the sequences for SpyTag and SpyCatcher into the components of the Split-GFP-Cre system. As anticipated, covalent conjugation of the SpyTag and SpyCatcher segments improved Split-GFP dimerization to further increase Cre recombinase activity in living cells. The increased efficiency and robustness of this dual-split system (Split-Cre and Split-GFP) minimize the problems of incomplete double gene-specific KO or low labeling efficiency due to poor NCre/CCre recombinase activity. Thus, this Split-Spy-GCre system allows more precise gene manipulation of cell subpopulations, which will provide advanced analysis of genes and cell functions in complex tissue such as the immune system.

Self-antigen-driven activation induces instability of regulatory T cells during an inflammatory autoimmune response.

Stable Foxp3 expression is crucial for regulatory T (Treg) cell function. We observed that antigen-driven activation and inflammation in the CNS promoted Foxp3 instability selectively in the autoreactive Treg cells that expressed high amounts of Foxp3 before experimental autoimmune encephalitis induction. Treg cells with a demethylated Treg-cell-specific demethylated region in the Foxp3 locus downregulated Foxp3 transcription in the inflamed CNS during the induction phase of the response. Stable Foxp3 expression returned at the population level with the resolution of inflammation or was rescued by IL-2-anti-IL-2 complex treatment during the antigen priming phase. Thus, a subset of fully committed self-antigen-specific Treg cells lost Foxp3 expression during an inflammatory autoimmune response and might be involved in inadequate control of autoimmunity. These results have important implications for Treg cell therapies and give insights into the dynamics of the Treg cell network during autoreactive CD4(+) T cell effector responses in vivo.

E-protein regulatory network links TCR signaling to effector Treg cell differentiation.

T cell antigen receptor (TCR) signaling is essential for the differentiation and maintenance of effector regulatory T (Treg) cells. However, the contribution of individual TCR-dependent genes in Treg cells to the maintenance of immunotolerance remains largely unknown. Here we demonstrate that Treg cells lacking E protein undergo further differentiation into effector cells that exhibit high expression of effector Treg signature genes, including IRF4, ICOS, CD103, KLRG-1, and RORγt. E protein-deficient Treg cells displayed increased stability and enhanced suppressive capacity. Transcriptome and ChIP-seq analyses revealed that E protein directly regulates a large proportion of the genes that are specific to effector Treg cell activation, and importantly, most of the up-regulated genes in E protein-deficient Treg cells are also TCR dependent; this indicates that E proteins comprise a critical gene regulatory network that links TCR signaling to the control of effector Treg cell differentiation and function.

Instability of the transcription factor Foxp3 leads to the generation of pathogenic memory T cells in vivo.

Regulatory T cells (T(reg) cells) are central to the maintenance of immune homeostasis. However, little is known about the stability of T(reg) cells in vivo. In this study, we demonstrate that a substantial percentage of cells had transient or unstable expression of the transcription factor Foxp3. These 'exFoxp3' T cells had an activated-memory T cell phenotype and produced inflammatory cytokines. Moreover, exFoxp3 cell numbers were higher in inflamed tissues in autoimmune conditions. Adoptive transfer of autoreactive exFoxp3 cells led to the rapid onset of diabetes. Finally, analysis of the T cell receptor repertoire suggested that exFoxp3 cells developed from both natural and adaptive T(reg) cells. Thus, the generation of potentially autoreactive effector T cells as a consequence of Foxp3 instability has important implications for understanding autoimmune disease pathogenesis.

B Cell-Intrinsic MyD88 Signaling Promotes Initial Cell Proliferation and Differentiation To Enhance the Germinal Center Response to a Virus-like Particle.

Although TLR signaling in B cells has been implicated in the germinal center (GC) responses during viral infections and autoimmune diseases, the underlying mechanism is unclear. Bacterial phage Qß-derived virus-like particle (Qß-VLP) contains TLR ligands, which can enhance Qß-VLP-induced Ab response, including GC response, through TLR/MyD88 signaling in B cells. In this study, by examining Ag-specific B cell response to Qß-VLP, we found that lack of B cell MyD88 from the beginning of the immune response led to a more severe defect in the GC scale than abolishing MyD88 at later time points of the immune response. Consistently, B cell-intrinsic MyD88 signaling significantly enhanced the initial proliferation of Ag-specific B cells, which was accompanied with a dramatic increase of plasma cell generation and induction of Bcl-6+ GC B cell precursors. In addition, B cell-intrinsic MyD88 signaling promoted strong T-bet expression independent of IFN-γ and led to the preferential isotype switching to IgG2a/c. Thus, by promoting the initial Ag-specific B cell proliferation and differentiation, B cell-intrinsic MyD88 signaling enhanced both T-independent and T-dependent Ab responses elicited by Qß-VLP. This finding will provide additional insight into the role of TLR signaling in antiviral immunity, autoimmune diseases, and vaccine design.

Activation and Functional Specialization of Regulatory T Cells Lead to the Generation of Foxp3 Instability.

Accumulating evidence suggests that Foxp3+ cells can downregulate the expression of Foxp3, but whether thymically derived regulatory T cells (tTregs; especially committed tTregs) are capable of downregulating Foxp3 expression and being reprogrammed into other T effector cells remains controversial. Using a novel tTreg lineage-tracing mouse line, we were able to label epigenetically stable Foxp3+ cells derived from the thymus and demonstrate that mature tTregs are stable under homeostatic conditions. However, TCR engagement and sequential functional specialization of tTregs led to the generation of Foxp3 instability and reprogramming into the Th lineage. We further demonstrated that the signal switch from IL-2 to ICOS during Treg activation induced Treg instability and reprogramming. By using a dual lineage tracing model, we demonstrated that effector Tregs can revert to central Tregs, and this reversion is associated with increasing Foxp3 stability in vivo.

Osteopontin Promotes Hepatic Progenitor Cell Expansion and Tumorigenicity via Activation of ß-Catenin in Mice.

Upregulation of osteopontin (OPN) has been found in hepatic progenitor cells (HPCs) in several liver diseases with portal biliary proliferation. Here, we investigated the role of HPC-derived autocrine OPN in regulating HPC expansion, migration, and hepatocarcinogenesis in mice. Five-week-old, weighing between 18 and 20 g of either wild type (WT) or OPN gene knockout (OPN-KO) male mice were treated with modified choline-deficient, ethionine-supplemented diet (modified choline-deficient [MCDE]) for 2 weeks to induce HPC production, or 6-12 months to induce tumorigenesis. Epithelial cell adhesion molecule EpCAM(+) CD45(-) cells isolated from mouse liver and liver epithelial progenitor cells were used for in vitro study. OPN was blocked by specific antibody or RNAi-mediated silence to investigate the role of OPN. To evaluate correlation between OPN expression and ß-catenin activity, expressions of OPN and ß-catenin were assessed in human liver cancer specimens. We found autocrine OPN promotes HPC expansion and migration by decreasing membranous E-cadherin and increasing free cytoplasmic ß-catenin via binding to αv integrin and activating Src activity. Depletion of OPN significantly attenuated MCDE-induced hepatocarcinogenesis. Clinical evidence revealed a strong correlation of high OPN expression with cytoplasmic/nuclear expression of ß-catenin in 43 cases of human combined hepatocellular carcinoma and cholangiocarcinoma and mixed intrahepatic cholangiocarcinoma and 80 cases of hepatocellular carcinoma. Our results indicate that autocrine OPN plays a crucial role in HPC expansion, migration, and subsequent oncogenic transformation of HPCs, which may provide a new insight into hepatocarcinogenesis.

Constitutive activation of MEK1 promotes Treg cell instability in vivo.

The instability of regulatory T (Treg) cells is involved in the pathogenesis of autoimmune diseases and also highlights safety concerns with regard to clinical Treg cell therapy. Cell-intrinsic molecular events linked to this Treg cell instability in vivo cells, which leads to safety concerns regardingare still obscure. Here we developed a novel luciferase-based reporter system and performed an unbiased screening for kinases that potentially modulate Foxp3 function. We found that the active form of COT/Tpl2 specifically inhibits the DNA binding activity of Foxp3 through a MEK-ERK-dependent pathway. Moreover, Treg cell-specific expression of activated MEK1 led to dysregulation of Treg function and instability of Foxp3 expression in vivo. Our results support the hypothesis that outside inflammatory signals act through the COT/Tpl2-MEK-ERK signaling pathway to destabilize the Treg lineage.

Tyrosine 201 of the cytoplasmic tail of CTLA-4 critically affects T regulatory cell suppressive function.

Cytotoxic T lymphocyte antigen-4 (CTLA-4) is a major negative regulatory molecule for T-cell activation with a complex biology and function. CTLA-4 is known to regulate homeostatic lymphoproliferation as well as tolerance induction and has been proposed to be an important effector molecule by which Treg cells suppress immunity. The immunoregulatory properties of CTLA-4 are primarily mediated by competition with the costimulator CD28 for ligand binding but also by delivering negative signals to T cells through its cytoplasmic tail. In this study, we addressed the effect of directly mutating the amino acid residue, Tyrosine 201 (Tyr201), of the intracellular domain of CTLA-4 in situ and its implications in T-cell function in the context of autoimmunity. Therefore, a novel CTLA-4 knock-in mouse (Y201V KI) was generated, in which Tyr201 was replaced by a valine that could not be phosphorylated. Mice expressing the CTLA-4 mutant molecule were generally healthy and did not show signs of disruption of T-cell homeostasis under steady-state conditions seen in CTLA-4 deficient mice. However, T cells isolated from Y201V KI mice expressed higher levels of CTLA-4 on the cell surface and displayed a Th2-biased phenotype following TCR stimulation. Furthermore, Y201V KI mice developed exacerbated disease as compared to wild-type upon antigen-specific T-cell activation in an in vivo model of EAE. Importantly, the Y201V mutation resulted in impaired suppressive activity of Treg cells while T effector function remained intact. These data suggest that effects associated with and mediated through Tyr201 of CTLA-4s intracellular domain are critical for Treg-cell function.

The B7-independent isoform of CTLA-4 functions to regulate autoimmune diabetes.

The critical role of CTLA-4 in inhibiting Ag-driven T cell responses upon engagement with its ligands, B7-1 and B7-2 and its importance for peripheral T cell tolerance and T cell homeostasis has been studied intensively. The CTLA-4 splice variant ligand-independent (li)-CTLA-4 is expressed in naive and activated T cells and can actively alter T cell signaling despite its lack of a B7 binding domain. To study the effect of li-CTLA-4 in regulating T cell responses in the context of autoimmunity, we engineered a B6.CTLA-4 (floxed-Exon2)-BAC-transgene, resulting in selective expression of li-CTLA-4 upon Cre-mediated deletion of Exon 2. Introducing the B6.BAC into the NOD background, which is genetically deficient for li-CTLA-4, restores mRNA levels of li-CTLA-4 to those observed in C57BL/6 mice. Furthermore, re-expressing this ligand nonbinding isoform in NOD mice reduced IFN-γ production in T effector cells accompanied by a significant decrease in insulitis and type 1 diabetes frequency. However, selective expression of li-CTLA-4 could not fully rescue the CTLA-4 knockout disease phenotype when bred onto NOD.BDC2.5.CTLA-4 knockout background because of the requirement of the full-length, B7-binding CTLA-4 molecule on T effector cells. Thus, the li-CTLA-4 form, when expressed at physiologic levels in the CTLA-4-sufficient NOD background can suppress autoimmunity; however, the functionality of the li-CTLA-4 isoform depends on the presence of the full-length molecule to alter effector T cell signaling.

Decreased TIP30 expression predicts poor prognosis in pancreatic cancer patients.

Pancreatic ductal adenocarcinoma (PDAC) is known for its aggressive growth, and is characterized by early tissue invasion and metastasis with poor prognosis. Identifying prognostic markers and delineating the underlying mechanisms that promote progression of PDAC are important for the treatment of pancreatic cancer. TIP30, a newly identified tumor suppressor, appears to be involved in multiple processes during tumor development and metastasis. Here, we investigated the expression of TIP30 in PDAC and its prognostic value in PDAC patients. We examined the expression of TIP30 by immunohistochemistry in tissue microarrays containing 106 surgically resected PDAC. Kaplan-Meier analysis and Cox proportional hazards regression modeling analysis showed that TIP30 expression independently predicted better survival in pancreatectomy patients (p < 0.01). Moreover, decreased TIP30 expression was associated with lymph node metastasis (p < 0.05) and loss of E-cadherin expression (r = 0.329, p < 0.01). Suppression of TIP30 resulted in upregulation of Snail and subsequent downregulation of E-cadherin in SW1990 cells containing high-level of endogenous TIP30. However, in the PANC-1 cells containing low level of endogenous TIP30, suppressing TIP30 caused upregulation of Slug instead of Snail, followed by upregulation of MMP9 rather than E-cadherin. Taken together, our work reveals that decreased TIP30 expression is able to enhance invasion and metastasis of pancreatic cancer cells through upregulation of the Snail family members and may serve as an independent predictor for poor outcomes in PDAC patients.

Identification of hepatitis B virus-specific CTL epitopes presented by HLA-A*33:03 in peripheral blood mononuclear cells from patients and transgenic mice.

Cytotoxic T lymphocyte (CTL) epitopes in the HBV protein of hepatitis B virus (HBV) may play a key role in viral control and liver damage. The aim of this study was to identify and study the function of HLA-A(∗)33:03-restricted CTL epitopes in HBV protein of the HBV genotypes B and C, which are epidemic in China. Sixteen HBV peptides were predicated by computational analysis, and synthesized peptides were examined for their affinity to HLA-A(∗)33:03 using a stable cell line. After being analyzed by enzyme-linked immunospot and cytolytic activity assays, as well as the tetramers staining method using peripheral blood mononuclear cells isolated from HBV-infected patients, five peptides (Hbs245-253, HBs335-343, HBc119-127, HBc104-112, and HBp391-399) were chosen to further confirm their HLA_A(∗)33:03 restriction in transgenic mice.

Growth inhibition and apoptosis induced by 6-fluoro-3-formylchromone in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most lethal and prevalent cancers in human population. The 6-fluoro-3-formylchromone (FCC) has been shown to have anti-tumor activity against various tumor cells. However, the effects of FCC on HCC cell lines have not yet been reported. This study aims to research the effects of FCC on HCC and advance the understanding of the molecular mechanism. METHODS: HCC cell line SMMC-7721 was treated with FCC at various concentrations (0, 2, 5, 10, and 20 µg/ml) for 24, 48 and 72 h, respectively. The proliferations of SMMC-7721 cells were measured by MTT assays. After cultured 24 hours, cell cycle distribution and apoptosis were determined by flow cytometry. However, the expression levels of PCNA, Bax and Bcl-2 were measured by western blotting after 48 hours. RESULTS: FCC displayed a dose- and time-dependent inhibition of the SMMC-7721 cell proliferations in vitro. It also induced apoptosis with 45.4% and caused cell accumulation in G0/G1 phase with 21.5%. PCNA and Bcl-2 expression was significantly suppressed by FCC in a dose-dependent manner (P < 0.05), while Bax expression was increased. CONCLUSIONS: FCC could significantly inhibit HCC cell growth in vitro through cell cycle arrest and inducing apoptosis by suppressing PCNA expression and modulating the Bax/Bcl-2 ratio.

Narrow groove and restricted anchors of MHC class I molecule BF2*0401 plus peptide transporter restriction can explain disease susceptibility of B4 chickens.

The MHC has genetic associations with many diseases, often due to differences in presentation of antigenic peptides by polymorphic MHC molecules to T lymphocytes of the immune system. In chickens, only a single classical class I molecule in each MHC haplotype is expressed well due to coevolution with the polymorphic TAPs which means that resistance and susceptibility to infectious pathogens are particularly easy to observe. Previously, structures of chicken MHC class I molecule BF2*2101 from B21 haplotype showed an unusually large peptide-binding groove that accommodates a broad spectrum of peptides to present as epitopes to CTLs, explaining the MHC-determined resistance of B21 chickens to Marek's disease. In this study, we report the crystal structure of BF2*0401 from the B4 (also known as B13) haplotype, showing a highly positively charged surface hitherto unobserved in other MHC molecules, as well as a remarkably narrow groove due to the allele-specific residues with bulky side chains. Together, these properties limit the number of epitope peptides that can bind this class I molecule. However, peptide-binding assays show that in vitro, BF2*0401 can bind a wider variety of peptides than are found on the surface of B4 cells. Thus, a combination of the specificities of the polymorphic TAP and the MHC results in a very limited set of BF2*0401 peptides with negatively charged anchors to be presented to T lymphocytes.

The histone lysine methyltransferase MLL1 regulates the activation and functional specialization of regulatory T cells.

The activation and specialization of regulatory T cells (Tregs) are crucial for maintaining immune self-tolerance; however, the regulation of these processes by histone modifications is not fully understood. Here, we show that T cell-specific deletion of the lysine methyltransferase MLL1 results in a spontaneous lymphocyte proliferation phenotype in aged mice without disturbing the development of conventional T cells and Tregs. Treg-specific MLL1 ablation leads to a systemic autoimmune disease associated with Treg dysfunction. Moreover, RNA sequencing demonstrates that the induction of multiple genes involved in Treg activation, functional specialization, and tissue immigration is defective in MLL1-deficient Tregs. This dysregulation is associated with defects in H3K4 trimethylation at these genes' transcription start sites. Finally, using a T-bet fate-mapping mouse system, we determine that MLL1 is required to establish stable Th1-type Tregs. Thus, MLL1 is essential in optimal Treg function by providing a coordinated chromatin context for activation and specialization.