iTreg Differentiation Research Articles

Time-resolved transcriptome and proteome landscape of human regulatory T cell (Treg) differentiation reveals novel regulators of FOXP3

BackgroundRegulatory T cells (Tregs) expressing the transcription factor FOXP3 are crucial mediators of self-tolerance, preventing autoimmune diseases but possibly hampering tumor rejection. Clinical manipulation of Tregs is of great interest, and first-in-man trials of Treg transfer have achieved promising outcomes. Yet, the mechanisms governing induced Treg (iTreg) differentiation and the regulation of FOXP3 are incompletely understood.ResultsTo gain a comprehensive and unbiased molecular understanding of FOXP3 induction, we performed time-series RNA sequencing (RNA-Seq) and proteomics profiling on the same samples during human iTreg differentiation. To enable the broad analysis of universal FOXP3-inducing pathways, we used five differentiation protocols in parallel. Integrative analysis of the transcriptome and proteome confirmed involvement of specific molecular processes, as well as overlap of a novel iTreg subnetwork with known Treg regulators and autoimmunity-associated genes. Importantly, we propose 37 novel molecules putatively involved in iTreg differentiation. Their relevance was validated by a targeted shRNA screen confirming a functional role in FOXP3 induction, discriminant analyses classifying iTregs accordingly, and comparable expression in an independent novel iTreg RNA-Seq dataset.ConclusionThe data generated by this novel approach facilitates understanding of the molecular mechanisms underlying iTreg generation as well as of the concomitant changes in the transcriptome and proteome. Our results provide a reference map exploitable for future discovery of markers and drug candidates governing control of Tregs, which has important implications for the treatment of cancer, autoimmune, and inflammatory diseases.

Monocyte activation and inflammation can exacerbate Treg/Th17 imbalance in infants with neonatal necrotizing enterocolitis

Necrotizing enterocolitis (NEC) is a potentially fatal disease that develops in the intestinal tract of newborn infants. An overactive immune system in the intestinal tract of NEC patients not only propagates inflammation and mediates tissue damage, but may also radiate the disease systemically and impose injury to distant organs. We previously identified impairment in the Treg compartment in NEC patients. Here, we hypothesized that monocytes, which could promote iTreg differentiation both in vitro and in vivo, were dysregulated in NEC infants. In age-, sex-, and weight-matched NEC infants and healthy control infants, the monocytes from PBMCs were investigated. Monocytes from NEC infants presented significantly higher TLR4 expression. With or without LPS stimulation, monocytes from NEC infants presented elevated TNF-α and IL-6 expression, together with reduced expression of TGF-β. When incubated with autologous CD4+ T cells, monocytes from NEC infants preferentially promoted the differentiation of RORγt-expressing Th17 cells, but not Foxp3-expressing Treg cells. However, using exogenous TGF-β and IL-10, the development of Foxp3 expression could be significantly elevated. Together, these results demonstrate that monocytes in NEC patients displayed a proinflammatory profile that might contribute to the production of proinflammatory cytokines and the suppression of Treg cells.

BANK1 alters B cell responses and influences the interactions between B cells and induced T regulatory cells in mice with collagen-induced arthritis

BackgroundFunctional variants of the B cell gene, B cell scaffold protein with ankyrin repeats 1 (BANK1) contribute to rheumatoid arthritis (RA) susceptibility, but their influences on B cell responses are unclear. Moreover, the function of induced T regulatory cells (iTregs) in the inflammatory milieu in a collagen-induced arthritis (CIA) model is unknown. This study was performed to investigate the roles of BANK1 in CIA and the interaction between B cells and iTregs.MethodsThe changes in BANK1 mRNA and protein levels and their correlation with disease severity in CIA were determined. Next, the antigen-presenting function and autoantibody production in B cells were evaluated by co-culture with effector T cells and iTregs, respectively, both in vitro and in vivo. Then, the mechanisms underlying these interactions were studied by adding neutralizing antibodies or transwell inserts and by adoptive transfer to B-cell-depleted CIA mice.ResultsThe BANK1 level decreased in the peripheral blood, spleen and lymph nodes of CIA mice, particularly during the acute stage of arthritis, and exhibited negative correlation with disease severity and autoantibody production. B cell responses were enhanced by this decrease. B cells from CIA mice (CIA-B cells) promoted iTreg differentiation, proliferation and cytotoxic T lymphocyte-associated protein-4 (CTLA-4) expression. Meanwhile, BANK1 expression in CIA-B cells increased after co-culture with iTregs, limiting B cell responses. All these interactions depended on cell contact with CTLA-4-overexpressing iTregs but were independent of CTLA-4 cytokine.ConclusionDecreased BANK1 expression promotes B cell responses, resulting in an increased antigen presentation ability and autoantibody production that subsequently influences the communication between B cells and iTregs through a cell-contact-dependent and CTLA-4- cytokine-independent mechanism in CIA mice.

Stealing Tregs’ Identity (by Deleting NF-κB)

Stealing Tregs’ Identity (by Deleting NF-κB)

CCR6 signaling inhibits suppressor function of induced-Treg during gut inflammation

CCR6 is a G protein-coupled receptor (GPCR) that binds to a specific chemokine, CCL20. The role of CCR6-CCL20 is very well studied in the migration of immune cells, but the non-chemotaxis functions of CCR6 signaling were not known. Here, we show that during gut inflammation, the frequency of Foxp3+CD4+ T cells (Tregs) reduced in the secondary lymphoid tissues and CCR6+ Tregs enhanced the expression of RORγt. The peripheral blood mononuclear cells (PBMCs) of ulcerative colitis (UC) patients showed lower percentages of Foxp3+CD4+ T cells, as compared to healthy individuals, with CCR6+ Tregs showing higher RORγt expression as compared to CCR6−Tregs. CCL20 inhibited the TGF-β1-induced Treg (iTreg) differentiation and directed them towards the pathogenic Th17-lineage in a CCR6-dependent manner. The iTreg that differentiated in the presence of CCL20 showed lower surface expression of suppressor molecules such as CD39, CD73 and FasL, and had impaired suppressive function. Furthermore, CCR6 signaling induced phosphorylation of Akt, mTOR, and STAT3 molecules in T cells. In conclusion, we have identified a new role of CCR6 signaling in the differentiation of iTregs during inflammation and gut autoimmunity.

Reciprocal regulation of Th17 and Treg cell differentiation by RhoA

Abstract Th17 cells are important for protective immunity as well as tissue inflammation and autoimmune disorders. Treg cells, including induced Treg (iTreg) and natural Treg (nTreg), play a critical role in immune tolerance. RhoA of the Rho small GTPase family is an intracellular signal transducer that cycles between an inactive GDP-bound form and an active GTP-bound form. We have previously reported that RhoA plays an important role in Th2 cell differentiation by regulating glycolysis. In this study, we further examined the role of RhoA in Th17 and Treg cell differentiation. Genetic deletion of RhoA in T cells or pharmacologic inhibition of RhoA by a RhoA-specific inhibitor Y16 led to reduced Th17 differentiation in vitro as evidenced by reduction of Th17 effector cytokines, transcriptional downregulation of RORγT and reduced phosphorylation of Stat3. Accordingly, RhoA deficiency ameliorated house dust mites (HDM)-induced allergic airway inflammation and dextran sodium sulphate (DSS)-induced colitis in vivo where Th17 cells plays an important role. Inhibition of ROCK, an immediate downstream effector of RhoA, recapitulated the effect of RhoA deficiency or inhibition on Th17 differentiation, suggesting that RhoA regulates Th17 cell differentiation through ROCK. On the other hand, ablation of RhoA had no effect on iTreg cell differentiation. However, inhibition of ROCK enhanced iTreg differentiation, suggesting that ROCK has RhoA-independent role in iTreg differentiation. In contrast to its in-effect on iTreg differentiation, RhoA deficiency increased nTreg differentiation. Thus, our data have provided new insights into Th17 and Treg differentiation and identified RhoA as a potential therapeutic target for tissue inflammation.

CGEN-15001: a Novel B7-like regulator of immune homeostasis and inducer of Ag-specific tolerance

Abstract B7 proteins play critical immunomodulatory roles and provide attractive targets for development of novel therapies for cancer and autoimmunity, both of which involve improper immune tolerance. A major medical need in autoimmunity is restoration of immune tolerance to self-antigens. CGEN-15001, an Fc fusion protein of a novel B7-like protein that we identified, regulates immune homeostasis by inhibiting Th1/Th17 responses while enhancing Th2 and anti-inflammatory cytokines such as IL-10, and promoting iTreg differentiation. This was observed in vitro using murine cells and in PBMCs of healthy donors and MS patients, as well as in vivo in R-EAE model and in HY transplantation model. Short-term administration of CGEN-15001 in R-EAE model of MS and in NOD model of T1D resulted in durable therapeutic effects which lasted long after cessation of treatment, suggestive of restoration of immune tolerance. Treg blockade with anti-IL-10 or anti-TGFβ, and transient Treg neutralization with anti-CD25 abolished these therapeutic effects. Importantly, transferring CD4+ T cells from CGEN-15001-treated mice to naïve recipients protected them from R-EAE induction in Ag-specific manner, demonstrating induction of Ag-specific tolerance. In contrast, tolerance transfer was not achieved following CTLA4-Ig treatment in spite of the remission it induced in the donor mice. In summary, CGEN-15001 has a unique mode of action, combining immunomodulation and regulation of immune homeostasis, as well as re-establishment of Ag-specific tolerance via enhancing Treg differentiation, leading to durable disease amelioration. These findings support the potential of CGEN-15001 to provide a promising therapeutic approach across autoimmune diseases.

A novel, intergenic enhancer contributes to Il2 gene expression via chromosomal looping.

Abstract IL-2 is a key immunoregulatory cytokine with pleotropic effects on many immune cell types. Its expression is highly regulated in T cells through multiple signaling pathways, chromatin remodeling, DNA methylation, and cooperative transcription factor binding. Recently, we identified an evolutionarily conserved intergenic enhancer 83 kb upstream of the Il2 gene that augments transcription from the Il2 promoter and upstream regulatory region (URR) by >50-fold in recombinant reporter assays. Upon TCR/CD28 co-stimulation of primary CD4+ T cells, a long-range chromosomal loop is established between the −83 kb CNS and the endogenous Il2 promoter, and both regions become epigenetically marked by histone acetylation and methylation. To determine the relative contribution of the URR vs. this new distal element to inducible transcription of the Il2 gene, we deleted the URR or the −83 kb CNS in mice using CRISPR/CAS genome editing. Deletion of the URR (~500 bp beyond the TATA element) completely abolished the Il2 transcription by CD4+ T cells, while a ~500 bp deletion of the −83 kb CNS resulted in a 2- to 4-fold decrease in IL-2 production. Young mice deficient for the −83 kb enhancer exhibited no major defects in lymphoid development, however, mutant CD4+ T cells showed reduced proliferative capacity in vitro. Furthermore, TGFB-induced iTreg differentiation from mutant conventional CD4+ precursors was strongly impaired, but could be fully restored by addition of exogenous IL-2. These results show that induction of Il2 depends upon cooperation between the promoter-URR and at least one intergenic enhancer, and suggest mechanisms by which graded expression of Il2 might be achieved through ‘modular’ recruitment of distinct enhancer elements.

IL-1\u03b2 induced HIF-1\u03b1 inhibits the differentiation of human FOXP3+ T cells

Differentiation of regulatory Treg (Treg) in the periphery is critical to control inflammatory processes. Although polarization of inducible Treg (iTreg) often occurs in an inflammatory environment, the effects exerted by inflammation on human iTreg differentiation have not been extensively studied. We observed that IL-1β significantly reduced the frequency of FOXP3+ T cells under iTreg-polarizing conditions. Mechanistically, we show that IL-1β activated mTORC1 and downstream upregulated hypoxia inducible factor-1 (HIF-1α) expression. Using specific inhibitors, we demonstrated that both steps were critical in the deleterious effect of IL-1β on Treg differentiation. Chemical stabilization of HIF-1α by Dimethyloxalylglycine (DMOG) also significantly impaired iTreg differentiation. Interestingly, while IL-1β-treated cells exhibited only minor changes in metabolism, DMOG treatment decreased iTreg mitochondrial respiration and increased their glycolytic capacity. In conclusion, exposure to inflammatory stimuli profoundly inhibits human Treg differentiation HIF-1α dependent, suggesting that targeting HIF-1α could be a strategy to foster iTreg differentiation in an inflammatory milieu. However, IL-1β deleterious effect does not appear to be completely driven by metabolic changes. These data thus suggest that several mechanisms contribute to the regulation of iTreg differentiation, but the timing and respective requirement for each pathway vary depending on the milieu in which iTreg differentiate.

Glycolysis and glutaminolysis cooperatively control T cell function by limiting metabolite supply to N-glycosylation

Rapidly proliferating cells switch from oxidative phosphorylation to aerobic glycolysis plus glutaminolysis, markedly increasing glucose and glutamine catabolism. Although Otto Warburg first described aerobic glycolysis in cancer cells >90 years ago, the primary purpose of this metabolic switch remains controversial. The hexosamine biosynthetic pathway requires glucose and glutamine for de novo synthesis of UDP-GlcNAc, a sugar-nucleotide that inhibits receptor endocytosis and signaling by promoting N-acetylglucosamine branching of Asn (N)-linked glycans. Here, we report that aerobic glycolysis and glutaminolysis co-operatively reduce UDP-GlcNAc biosynthesis and N-glycan branching in mouse T cell blasts by starving the hexosamine pathway of glucose and glutamine. This drives growth and pro-inflammatory TH17 over anti-inflammatory-induced T regulatory (iTreg) differentiation, the latter by promoting endocytic loss of IL-2 receptor-α (CD25). Thus, a primary function of aerobic glycolysis and glutaminolysis is to co-operatively limit metabolite supply to N-glycan biosynthesis, an activity with widespread implications for autoimmunity and cancer.

TGF-β-induced phosphorylation of Akt and Foxo transcription factors negatively regulates induced regulatory T cell differentiation

Transforming growth factor-β (TGF-β) is a pivotal cytokine in the differentiation of regulatory T cells, and Foxo transcription factors positively regulate this process. On the other hand, the function of Foxo transcription factors is negatively regulated by PI3K/Akt signaling, which is activated by TGF-β in many types of cells; yet the role of TGF-β in Akt activity and its downstream substrates in CD4+ T cells, including Foxo transcription factors, remains to be determined. Herein, we demonstrate that TGF-β selectively induces Akt phosphorylation at Ser473 but not at Thr308 in a class IA PI3K-dependent manner in CD4+ T cells, resulting in the phosphorylation and inhibition of Foxo transcription factors and negatively regulating the differentiation of induced regulatory T cells (iTregs). These observations reveal a novel negative regulatory mechanism involving Akt and Foxo transcription factors induced by TGF-β in the iTreg differentiation process.

The MAP kinase, tumor progression locus 2 (Tpl2), activates the Akt/mTOR/S6 ribosomal protein pathway, inhibits Foxp3 expression and limits Treg immunosuppressive functions.

Abstract The serine-threonine kinase, tumor progression locus 2 (Tpl2 or Map3k8/Cot), is a potent inflammatory mediator that drives the production of TNF alpha, IL-1 beta, and IFN gamma. We previously demonstrated that Tpl2 modulates TCR signaling and T helper cell differentiation. However, little is known about how Tpl2 alters the development or function of regulatory T cells (Tregs). Tregs are a specialized subset of T cells that express Foxp3 and possess immunosuppressive properties. Because of Tpl2’s role in promoting inflammation, we hypothesized that Tpl2 inhibits Treg development and immunosuppressive functions. Tpl2−/− naïve CD4 T+ cells preferentially differentiated into Foxp3+ inducible Tregs in vitro as well as in vivo in a murine model of OVA-induced systemic tolerance. Treg biasing in Tpl2−/− T cells depended upon the TCR signal strength and correlated with reduced activation of the Akt/mTOR/S6 pathway. Importantly, freshly isolated Tpl2−/− Tregs had increased expression of Foxp3 and immunosupressive cytokines IL-10 and IL-35 and were more suppressive in a T cell transfer model of colitis, as evidenced by reduced accumulation of inflammatory T effectors, systemic inflammatory cytokines TNF, IL-6 and IFN gamma, and colonic inflammation. Therefore, Tpl2 promotes inflammation in part by limiting Foxp3 expression, iTreg differentiation and immunosuppressive functions. These findings suggest that Tpl2 inhibition may be used to preferentially drive Treg induction and limit inflammation in autoimmune diseases.

The role of PML in the activation and the differentiation of T cells and B cells

Abstract Promyelocytic leukemia protein (PML) is a tumor suppressor with diverse activities in cell growth, cell death, stress responses or senescence. PML is a member of the tripartite motif (TRIM) family protein, which contains RING, B-box and coiled-coil domains. Recent studies showed that large number of TRIM family proteins positively regulate innate immune signaling pathways by acting as E3 ubiquitin ligases. To investigate the role of PML in adaptive immunity, we examined the population of different T cell and B cell subsets in PML-knockout mice. The T cells population including CD4+, CD8+, CD25+, effector T, naïve T, and memory T cells were comparable between WT and PML-deficient mice. The B cells population including follicular B, transitional type 1 and 2 B cells, and marginal zone B cells were also comparable between WT and PML-knockout mice. We also determined whether the activation of T cells and B cells were affected by PML-deficiency. It was found that the activation of PML-deficient T and B cells was lower than that of WT cells. We further determined the differentiation of induced regulatory T (iTreg) cells, and observed that iTreg differentiation was comparable in WT and PML-deficient cells. Therefore, PML is not apparently involved in the development of T cells and B cells, and is modesty required for the full activation of T lymphocyte and B lymphocytes. The exact contribution of PML to the activation of T cell and B cell awaits further investigation.

A novel JAK-STAT inhibitor, 2-[(3-Carbamoyl-2-thienyl)amino]-2-oxoethyl(2,6-dichlorophenyl)acetate, suppresses helper T cell differentiation in vitro and collagen-induced arthritis in vivo

Th17 cells, which have been implicated in autoimmune diseases including rheumatoid arthritis (RA), require the JAK-STAT3 pathway for their differentiation and functions. Recently, JAK inhibitors have been developed as a therapeutic drug for RA. However, the current JAK inhibitors are not optimized to STAT3 compared with other STATs. In this study, we found a new lead compound of a small molecule JAK-STAT inhibitor, 2-[(3-Carbamoyl-2-thienyl)amino]-2-oxoethyl (2,6-dichlorophenyl)acetate, which inhibits STAT3 as efficiently as other STATs. This compound, named JI069, was selected by STAT3 reporter assay in combination with an in silico docking model. JI069 inhibited gp130 signaling by inducing dissociation between gp130 and JAK1. In HEK293T cells and primary T cells, JI069 suppressed STAT3 activation as efficiently as other STATs, including STAT1, STAT5, and STAT6. JI069 effectively suppressed Th1, Th2, and Th17 differentiation while strongly promoted iTreg differentiation. JI069 suppressed symptoms of the collagen-induced arthritis (CIA) model in mice, and inhibited the cytokine production from T cells as well as the STAT3 phosphorylation of synovial cells. These data suggest that JI069 is a new type of JAK inhibitor which has potential for the treatment of immunological disorders.

MicroRNA-17-92 controls T-cell responses in graft-versus-host disease and leukemia relapse in mice

AbstractMicroRNAs (miRs) play important roles in orchestrating many aspects of the immune response. The miR-17-92 cluster, which encodes 6 miRs including 17, 18a, 19a, 20a, 19b-1, and 92-1, is among the best characterized of these miRs. The miR-17-92 cluster has been shown to regulate a variety of immune responses including infection, tumor, and autoimmunity, but the role of this cluster in T-cell response to alloantigens has not been previously explored. By using major histocompatibility complex (MHC)-matched, -mismatched, and haploidentical murine models of allogeneic bone marrow transplantation (allo-BMT), we demonstrate that the expression of miR-17-92 on donor T cells is essential for the induction of graft-versus-host disease (GVHD), but dispensable for the graft-versus-leukemia (GVL) effect. The miR-17-92 plays a major role in promoting CD4 T-cell activation, proliferation, survival, and Th1 differentiation, while inhibiting Th2 and iTreg differentiation. Alternatively, miR-17-92 may promote migration of CD8 T cells to GVHD target organs, but has minimal impact on CD8 T-cell proliferation, survival, or cytolytic function, which could contribute to the preserved GVL effect mediated by T cells deficient for miR-17-92. Furthermore, we evaluated a translational approach and found that systemic administration of antagomir to block miR-17 or miR-19b in this cluster significantly inhibited alloreactive T-cell expansion and interferon-γ (IFNγ) production, and prolonged the survival in recipients afflicted with GVHD while preserving the GVL effect. Taken together, the current work provides a strong rationale and demonstrates the feasibility to target miR-17-92 for the control of GVHD while preserving GVL activity after allo-BMT.

Altered expression and editing of miRNA-100 regulates iTreg differentiation.

RNA editing of miRNAs, especially in the seed region, adds another layer to miRNA mediated gene regulation which can modify its targets, altering cellular signaling involved in important processes such as differentiation. In this study, we have explored the role of miRNA editing in CD4+ T cell differentiation. CD4+ T cells are an integral component of the adaptive immune system. Naïve CD4+ T cells, on encountering an antigen, get differentiated either into inflammatory subtypes like Th1, Th2 or Th17, or into immunosuppressive subtype Treg, depending on the cytokine milieu. We found C-to-U editing at fifth position of mature miR-100, specifically in Treg. The C-to-U editing of miR-100 is functionally associated with at least one biologically relevant target change, from MTOR to SMAD2. Treg cell polarization by TGFβ1 was reduced by both edited and unedited miR-100 mimics, but percentage of Treg in PBMCs was only reduced by edited miR-100 mimics, suggesting a model in which de-repression of MTOR due to loss of unedited mir-100, promotes tolerogenic signaling, while gain of edited miR-100 represses SMAD2, thereby limiting the Treg. Such delicately counterbalanced systems are a hallmark of immune plasticity and we propose that miR-100 editing is a novel mechanism toward this end.

STIM1 controls T cell-mediated immune regulation and inflammation in chronic infection.

Chronic infections induce a complex immune response that controls pathogen replication, but also causes pathology due to sustained inflammation. Ca2+ influx mediates T cell function and immunity to infection, and patients with inherited mutations in the gene encoding the Ca2+ channel ORAI1 or its activator stromal interaction molecule 1 (STIM1) are immunodeficient and prone to chronic infection by various pathogens, including Mycobacterium tuberculosis (Mtb). Here, we demonstrate that STIM1 is required for T cell-mediated immune regulation during chronic Mtb infection. Compared with WT animals, mice with T cell-specific Stim1 deletion died prematurely during the chronic phase of infection and had increased bacterial burdens and severe pulmonary inflammation, with increased myeloid and lymphoid cell infiltration. Although STIM1-deficient T cells exhibited markedly reduced IFN-γ production during the early phase of Mtb infection, bacterial growth was not immediately exacerbated. During the chronic phase, however, STIM1-deficient T cells displayed enhanced IFN-γ production in response to elevated levels of IL-12 and IL-18. The lack of STIM1 in T cells was associated with impaired activation-induced cell death upon repeated TCR engagement and pulmonary lymphocytosis and hyperinflammation in Mtb-infected mice. Chronically Mtb-infected, STIM1-deficient mice had reduced levels of inducible regulatory T cells (iTregs) due to a T cell-intrinsic requirement for STIM1 in iTreg differentiation and excessive production of IFN-γ and IL-12, which suppress iTreg differentiation and maintenance. Thus, STIM1 controls multiple aspects of T cell-mediated immune regulation to limit injurious inflammation during chronic infection.

An epigenetic mechanism of Dll4-facilitated Foxp3+ regulatory T cells differentiation (LYM8P.637)

Abstract Notch signaling facilitates inducible Foxp3+ regulatory T cells (iTreg) differentiation. Epigenetic modifications, which include histone modifications, are critical in cell differentiation. Recent genome-wide studies showed that iTreg have increased trimethylation on histone H3 at lysine 4 (H3K4me3) around Foxp3 loci, but the role of Notch signaling and its underlying epigenetic mechanisms remain elusive. Here we report that Delta-like ligand 4 (Dll4) but not Jagged1 can activate Notch signaling, facilitate Foxp3+ iTreg differentiation, and increased H3K4me3 around Foxp3 promoter and conserved noncoding sequences (CNS). We performed epigenetic enzyme array analysis and found one histone methyltransferase—SET and MYND domain containing protein 3 (Smyd3) that is abundant in iTreg. Dll4/Notch signaling amplified and accelerated Smyd3 expression in vitro during iTreg cell differentiation via an RBPJ-mediated transcription activation. Inhibition of Notch signaling decreased Smyd3 expression. When Smyd3 was genetically knocked out in CD4 T cells, the Dll4-enhanced H3K4me3 around the Foxp3 loci and Foxp3+ Tregs were decreased in vitro and in vivo. These results demonstrate that Notch ligand Dll4 can epigenetically regulate Foxp3+ Tregs through Smyd3.

CD3/SLAMF3-mediated co-stimulation promotes activation of the IL-2/IL-2R/STAT5 pathway and iTreg differentiation in naïve CD4+ T cells - implications for systemic lupus erythematosus (HUM3P.246)

Abstract We examined SLAMF3-mediated co-stimulation on IL-2 response of CD4+ T cells from SLE patients. Stimulation of naïve CD4+ T cells with aCD3/aSLAMF3 upregulated surface CD25 (IL-2Ra) and CD122 (IL-2Rb), at higher level compared to aCD3/aCD28 activation. aCD3/aSLAMF3 co-stimulation of control naïve CD4+ T cells led to increased pSTAT5 levels compared to aCD3/aCD28 activation Although naïve CD4+ T cells from SLE patients displayed decreased pSTAT5 levels compared to control donors upon aCD3 or aCD3/aCD28 stimulation, co-stimulation of SLE T cells with aSLAMF3 restored pSTAT5 to normal levels. Total STAT5 levels remained unaltered after T cell activation. Exogenous IL-2 increased proliferation of aCD3/aSLAMF3-activated CD4+ T cells compared to aCD3/aCD28 stimulation. Naïve CD4+ T cells activated with aCD3/aSLAMF3 under iTreg polarizing condition expressed significantly higher levels of FoxP3, CD25 and CTLA-4 compared to aCD3/aCD28 activation. In conclusion, SLAMF3-mediated co-stimulation enhances CD4+ T cell response to IL-2 in SLE patients by acting on the IL-2/IL-2R/STAT5 signaling pathway, increases naïve CD4+ T cell proliferation in response to exogenous IL-2 and can promote iTreg phenotype generation. Of note, SLAMF3-mediated co-stimulation does not increase IL-2 by CD4+ T cells per se. Thus, aSLAMF3-based Ab therapy may emerge as a promising therapeutic agent in SLE by enhancing response to IL-2 and promoting iTreg differentiation.

Tregs expressing high Tl1a are proinflammatory and do not protect from T cell induced colitis. (CCR5P.204)

Abstract Patients with Crohn’s disease have increased mucosal expression of TL1A. TL1A co-stimulates T cells and can promote expansion of activated and regulatory T cells. Mice generated with sustained and elevated T cell expression of Tl1a (L-Tg) have increased Foxp3+ Treg cells yet develop worsened ileitis, proximal migration of colitis and intestinal fibrosis. The aim of this study was to investigate the role of Tl1a in the differentiation and function of Tregs.In the L-Tg mice, the CD4+ T cell population was comprised of GFPhigh and GFPlowcells that correlated with the level of Tl1a expression.Foxp3 expression and suppression function was significantly lower in the Tregs expressing high Tl1a. Indeed, addition of high levels of exogenous rTl1a under iTreg differentiation conditions advances the expansion of IL-13 producing effector T cells. The percentage of GFPhigh Foxp3+ Tregs co-expressing GATA3 were significantly higher explaining the possible Th2 polarization of Tregs expressing high Tl1a. Additionally, in the T cell transfer model of colitis, RAG mice cotransferred with GFPlowFoxp3+ Tregs were better protected from inflammation and weight loss compared to mice cotransferred with GFPhighFoxp3+ T cells.These data reveal a novel role for TL1A in regulating T cell mediated immune responses such that the level of Tl1a (high v. low) can differentially influence the expansion of effector and Treg cells leading to either a pro-inflammatory or protective mucosal environment.