Treg Deficiency Research Articles

IL2 is required for functional maturation of regulatory T cells

ABSTRACTRegulatory T cells (Tregs), specified by the expression of transcription factor Foxp3, operate Foxp3-dependent programs to maintain self-tolerance. In addition to Foxp3, other tissue-specific transcription factors are also required by Tregs to control the corresponding immune responses like follicular Tregs which express both Foxp3 and Bcl6 controlling germinal center reactions. Here, we show that Interleukin 2 (IL2) is required for the optimal expression of T helper type 1 (Th1) transcription factor T-box 21 (Tbx21, T-bet) in Tregs. The expression levels of CXCR3 and T-bet were reduced in IL2 deficient Tregs. Furthermore, IL2 deficient Treg cells failed to control the proliferation of CD4+ T cells in vitro and could not prevent the progression of colitis characterized by Th1 immune responses. Taken together, our data suggest that IL2 is essential for the functional maturation of Tregs including the optimal suppressive activity and the expression of tissue-specific transcription factors like T-bet.

Mouse immune thrombocytopenia is associated with Th1 bias and expression of activating Fcγ receptors.

Immune thrombocytopenia (ITP) is an autoimmune disease mediated by anti-platelet autoantibodies. We recently established a mouse ITP model exhibiting regulatory T-cell (Treg) deficiency, although only one-third of the Treg-deficient mice developed ITP. To clarify mechanisms involved in the emergence of platelet-specific autoimmunity in this model, we examined the T helper (Th)-cell balance and macrophage Fcγ receptor (FcγR) expression profiles in Treg-deficient mice with and without ITP. Splenocytes from both populations of Treg-deficient mice and control BALB/c mice were subjected to flow cytometry-based analyses to evaluate Th cell subset proportions and the expression of activating and inhibitory FcγRs on macrophages. In addition, IgG subclass distribution of anti-platelet autoantibodies in splenocyte culture supernatants was determined by flow cytometry using IgG subclass-specific antibodies. Treg-deficient ITP mice exhibited a significantly higher proportion of Th1 cells than either Treg-deficient non-ITP or control mice. The predominant anti-platelet autoantibody subclasses in the ITP mice were Th1-associated IgG2a and IgG2b. Furthermore, the FcγRI/FcγRIIB expression ratio in splenic macrophages was higher in the Treg-deficient ITP than in the Treg-deficient non-ITP and control mice. In summary, Th1 polarization and macrophages' activating FcγR expression profile are associated with the development of ITP in Treg-deficient mice.

Are Regulatory T Cells Defective in Type 1 Diabetes and Can We Fix Them?

Regulatory T cells (Tregs) are critical regulators of peripheral immune tolerance. Treg insufficiency can lead to autoimmune disorders, including type 1 diabetes (T1D). Increasing evidence in mouse models of T1D, as well as other autoimmune disorders, suggests that there are defects in Treg-mediated suppression. Indeed, whereas Treg frequency in the peripheral blood of T1D patients is unaltered, their suppressive abilities are diminished compared with Tregs in healthy controls. Although expression of the transcription factor Foxp3 is a prerequisite for Treg development and function, there are many additional factors that can alter their stability, survival, and function. Much has been learned in other model systems, such as tumors, about the mechanism and pathways that control Treg stability and function. This review poses the question of whether we can use these findings to develop new therapeutic approaches that might boost Treg stability, survival, and/or function in T1D and possibly other autoimmune disorders.

A Novel mTORC1-Dependent, Akt-Independent Pathway Differentiates the Gut Tropism of Regulatory and Conventional CD4 T Cells.

The vitamin A metabolite all-trans retinoic acid (ATRA) induces a gut-homing phenotype in activated CD4(+) conventional T cells (Tconv) by upregulating the integrin α4β7 and the chemokine receptor CCR9. We report that, in contrast to mouse Tconv, only ∼50% of regulatory T cells (Treg) upregulate CCR9 when stimulated by physiological levels of ATRA, even though Tconv and Treg express similar levels of the retinoic acid receptor (RAR). The resulting bimodal CCR9 expression is not associated with differences in the extent of their proliferation, level of Foxp3 expression, or affiliation with naturally occurring Treg or induced Treg in the circulating Treg pool. Furthermore, we find that exposure of Treg to the mechanistic target of rapamycin (mTOR) inhibitor rapamycin suppresses upregulation of both CCR9 and α4β7, an effect that is not evident with Tconv. This suggests that in Treg, ATRA-induced upregulation of CCR9 and α4β7 is dependent on activation of a mTOR signaling pathway. The involvement of mTOR is independent of Akt activity, because specific inhibition of Akt, pyruvate dehydrogenase kinase-1, or its downstream target glycogen synthase kinase-3 did not prevent CCR9 expression. Additionally, Rictor (mTOR complex [mTORC]2)-deficient Treg showed unaltered ability to express CCR9, whereas Raptor (mTORC1)-deficient Treg were unable to upregulate CCR9, suggesting the selective participation of mTORC1. These findings reveal a novel difference between ATRA signaling and chemokine receptor induction in Treg versus Tconv and provide a framework via which the migratory behavior of Treg versus Tconv might be regulated differentially for therapeutic purposes.

Corruption of dendritic cell antigen presentation during acute GVHD leads to regulatory T-cell failure and chronic GVHD

AbstractChronic graft-versus-host disease (cGVHD) is a major cause of late mortality following allogeneic bone marrow transplantation (BMT) and is characterized by tissue fibrosis manifesting as scleroderma and bronchiolitis obliterans. The development of acute GVHD (aGVHD) is a powerful clinical predictor of subsequent cGVHD, suggesting that aGVHD may invoke the immunologic pathways responsible for cGVHD. In preclinical models in which sclerodermatous cGVHD develops after a preceding period of mild aGVHD, we show that antigen presentation within major histocompatibility complex (MHC) class II of donor dendritic cells (DCs) is markedly impaired early after BMT. This is associated with a failure of regulatory T-cell (Treg) homeostasis and cGVHD. Donor DC-restricted deletion of MHC class II phenocopied this Treg deficiency and cGVHD. Moreover, specific depletion of donor Tregs after BMT also induced cGVHD, whereas adoptive transfer of Tregs ameliorated it. These data demonstrate that the defect in Treg homeostasis seen in cGVHD is a causative lesion and is downstream of defective antigen presentation within MHC class II that is induced by aGVHD.

General control non-derepressible 2 (GCN2) in T cells controls disease progression of autoimmune neuroinflammation

Relapsing-remitting multiple sclerosis (MS)22MS: multiple sclerosis. is characterized by phases of acute neuroinflammation followed by spontaneous remission. Termination of inflammation is accompanied by an influx of regulatory T cells (Tregs).33Tregs: regulatory T cells. The molecular mechanisms responsible for directing Tregs into the inflamed CNS tissue, however, are incompletely understood. In an MS mouse model we show that the stress kinase general control non-derepressible 2 (GCN2),44GCN2: general control non-derepressible 2. expressed in T cells, contributes to the resolution of autoimmune neuroinflammation. Failure to recover from acute inflammation was associated with reduced frequencies of CNS-infiltrating Tregs. GCN2 deficient Tregs displayed impaired migration to a CCL2 gradient. These data suggest an important contribution of the T cell stress response to the resolution of autoimmune neuroinflammation.

Disruption of the gut microbiota by antibiotics exposure during early life promotes spontaneous Th2 responses and loss of tolerance to dietary antigens

Abstract Allergic disorders, manifest by T helper 2 (Th2) type immune responses to environmental antigens, are rapidly increasing in children. Regulatory T cells (Tregs) limit Th2 responses, and Treg deficiencies are seen in children with allergic disorders, implying that Treg deficiencies or dysfunction underlie the pathogenesis of Th2 mediated disease. What incurs Treg deficiency and its relationship to the increasing incidence of allergy is unknown, however, studies have shown that the intestinal microbiota plays an important role in regulating Treg differentiation during early life. With this in mind we explored whether oral exposure to specific antibiotics in early life altered Treg populations and predisposed to Th2 responses. We found that exposure of mice to a combination of broad spectrum oral antibiotics between days 10 and 20 of life abrogated tolerance to a dietary antigen, ovalbumin (Ova). Exposure of mice to single antibiotics of varying spectra between days 10 and 20 of life had a range of effects from abrogation of oral tolerance to the spontaneous development of Th2 responses manifested by increased levels of IL-13 in the serum in the absence of challenge. Ongoing studies are evaluating the effect of the respective antibiotic regimens on the intestinal microbial community using 16S bacterial DNA sequencing, with the aim to link alterations in the microbial composition to the immunological profile of the respective treatment groups. In summary, our results show that disruption of the intestinal microbiota by exposure to specific antibiotics during a period in early life results in loss of Tregs, promotes Th2 immunity, and impair establishment of oral tolerance to dietary antigen.

Unveiling the specificity of islet-antigen specific regulatory T cells in T1D patient using single cell deep-sequencing

Abstract CD4+CD25hiCD127loFoxp3+ T Regulatory cells (Tregs) are critical in controlling autoreactive T cells. The deficiency of functional Tregs is associated with Type 1 diabetes (T1D) and many other autoimmune diseases. In addition, antigen-specific Tregs suppresses more efficiently compared to polyclonal Tregs in mouse models. Thus, identifying the antigen specificity of Tregs, especially at the site of inflammation, is critical to improve our understanding of Treg deficiencies in T1D and may lead to clinically applicable advances. However, the Treg TCRs recognizing self-antigens in autoimmune diseases are less clarified due to the rare frequency and the anergic status of Treg in vitro. In this study, by the combination of sub-culturing, polyclonal stimulation and antigen stimulation, we can identify islet-antigen specific Tregs from both PBMC and pancreatic draining lymph node of T1D patients using antigen-driven Treg proliferation assay we developed in our lab. The majority of islet antigen-specific Tregs is CD45RO+Tregs compared to the CD45RA+ counterpart. Importantly, we confirmed the enrichment and identified the specificity of the islet antigen-specific Tregs using single cell deep-sequencing. In the future, we will further characterize the uncovered Treg TCR and the phenotype of the identified islet antigen-specific Tregs via comparison between healthy and T1D subjects.

Chemokine production by regulatory T cells is required for therapeutic attenuation of autoimmunity and allograft rejection

Abstract Regulatory T cells (Tregs) control immune homeostasis by preventing inappropriate responses to self and non-harmful foreign antigens. Tregs use multiple mechanisms to control immune responses, all of which require Tregs to be near their targets of suppression, but how Treg-to-target proximity is controlled is unknown. We found that Tregs produce chemokines to attract CD4+ and CD8+ T cells close to their proximity in vitro and in vivo. Mouse and human lineage committed Tregs, as well as murine in vitro induced Tregs, produced CCL3 and CCL4 at message and protein level. Chemokine producing Tregs had an activated Treg phenotype with high expression of multiple proteins associated with suppressive function. Furthermore the human CCL3 and CCL4 promoter could be transactivated by FOXP3. In vitro and in vivo migration experiments indicated that CCR5 was the major receptor required by target cells. CCL3 and CCL4 deficient Tregs were impaired in their ability to prevent experimental autoimmune encephalomyelitis or islet allograft rejection, but suppressive in a standard in vitro assay. Moreover, Tregs from subjects with established type 1 were impaired in their ability to produce CCL3 and CCL4. These results demonstrate a previously unknown facet of Treg function and suggest that chemokine secretion by Tregs is a fundamental aspect of their in vivo function and therapeutic effect in autoimmunity and transplantation.

Foxp3 controls oxidative phosphorylation in regulatory T cells

Abstract Many T cell subsets have distinct metabolic requirements. Activated CD4 helper and cytotoxic CD8 T cells require glycolysis for optimal function, whereas Foxp3+ T-regulatory (Treg) cells are thought to be dependent on oxidative phosphorylation. We questioned if Foxp3 is involved with re-programming T cell metabolism. We retrovirally transduced murine T cells with Foxp3 or empty vector (EV), and also cultured CD4+Foxp3− T cells from Foxp3YFPcre mice under polarizing conditions with TGFβ to form induced Tregs, using YFP to track Foxp3 expression. Our analysis of FACS-sorted YFP+ iTreg and YFP− non-iTreg showed that both iTreg and Foxp3-transduced T cells had significantly higher basal oxygen consumption rates than non-iTreg or EV transduced T cell controls (44.1±49% and 76.1±76.5%, respectively, p<0.05, n=4), despite equal mitochondrial mass. Using Foxp3 ChIP-seq and microarray studies, we identified multiple binding sites of Foxp3 and Foxp3-dependent transcripts among key genes involved in control of oxidative phosphorylation as well as nuclear encoded electron transport chain genes. FACS-sorted YFP+ iTreg showed increased protein expression of electron transport chain complexes, especially complex I, compared to YFP− non-iTreg. We found that mice deficient in ND6, a mitochondrial encoded gene of complex I, have deficient Treg function. Furthermore, 50 nM rotenone, a complex I inhibitor, could selectively inhibit Treg suppressive function without affecting Tconv cell proliferation. The effects of Foxp3 on Treg metabolism may be physiological important to enable Tregs operating in low glucose environments such as the tumor microenvironment, and could offer an opportunity for therapeutic immune modulation.

Circulating level of regulatory T cells in rheumatic heart disease: An observational study

BackgroundThe regulatory T cell (Treg) is essential for prevention of autoimmunity. In a preliminary study, we showed significant deficiency of Tregs (CD4CD25 T cells) in rheumatic heart disease (RHD) patients (an autoimmune disease), but the markers used could not reliably differentiate Treg from nonregulatory conventional T cells (Tcon). The study aim was to reassess the level of circulatory Tregs by using more specific markers. Methods70 adults of RHD and 35 controls were studied. Patients were subdivided according to the extent of left-sided valvular involvement. 35 patients with significant mitral-valve disease only were enrolled in the univalvular group while 35 patents with significant involvement of both mitral and aortic-valves in the multivalvular group. Circulating Treg cell level was determined by flow-cytometry. ResultsLevel of Tregs (CD4+CD25med–highCD127low Foxp3high) in CD4+ T lymphocyte was significantly lower in RHD patients compared to controls (median 0.6% versus 3.2%; p=0.001) with no significant difference in Tcon cells (p=0.94). Within the study group Treg count was significantly lower in patients with multivalvular-disease only (median 0.1% versus 3.2%; p=0.001) with no significant difference in Treg cell count between the univalvular group and control (median 1.9% versus 3.2%, p=0.10). ConclusionThere is significant deficiency of circulating Tregs in patients of chronic RHD and the deficiency is greater in patients with multivalvular than univalvular involvement.

Deficiency of T-regulatory cells exaggerates angiotensin II-induced microvascular injury by enhancing immune responses.

T-regulatory lymphocyte (Treg) adoptive transfer prevented angiotensin (Ang) II-induced hypertension and microvascular injury. Scurfy mice are deficient in Treg because of a mutation in the transcription factor forkhead box P3 (Foxp3) gene. Enhanced Ang II effects in the absence of Treg would unambiguously demonstrate their vascular protective role. We hypothesized that adoptive transfer of Scurfy vs. wild-type T cells will exacerbate Ang II-induced microvascular damage in T and B-cell-deficient recombination-activating gene 1 (Rag1) knockout mice. Rag1 knockout mice were injected with vehicle, 10(7) T cells from wild-type or Scurfy mice or 10 (6)wild-type Treg alone or in combination with Scurfy T cells, and then infused or not with Ang II (490 ng/kg per min, subcutaneous) for 14 days. Ang II increased SBP in all the groups, but DBP only in wild-type and Scurfy T-cell groups. Ang II-induced endothelial dysfunction and oxidative stress in perivascular adipose tissue (PVAT) of mesenteric arteries of the wild-type T-cell group, whereas these were exaggerated in the Scurfy T-cell group. Ang II enhanced microvascular remodeling and stiffness in vehicle and Scurfy T-cell groups. Ang II increased monocyte chemotactic protein-1 expression in the vascular wall and PVAT, monocyte/macrophage infiltration and proinflammatory polarization in PVAT and the renal cortex, and T-cell infiltration in the renal cortex only in the Scurfy T-cell group. Treg coinjection in the vehicle and Scurfy T-cell groups prevented or reduced the effects of Ang II. FOXP3+ Treg deficiency exaggerates Ang II-induced microvascular injury by modulating innate and adaptive immune responses.

Abstract B060: The canonical NF-κB pathway is crucial for Treg homeostasis during the antitumor response

Abstract Cancers use a wide variety of mechanisms to dampen tumor immune response. Among them, CD4+Foxp3+ regulatory T cells (Tregs) are largely described to inhibit the function of effector cells. In several murine models of cancer, Treg depletion suppresses tumor growth. Interestingly, it has been proposed that anti-CTLA-4 therapies exert their anti-tumor effects by targeting Tregs. Therefore, precisely understanding the mechanisms governing Treg homeostasis may be a valuable strategy to enhance immune responses against cancer. We and other have previously shown that transcription factor NF-κB plays a major role in Treg development; we are now analyzing the role of different NF-κB subunits in Tregs by using conditional knock-out mice. We found that both p65 (RelA) and c-Rel subunits of NF-κB in Tregs, were required for both development of thymic Treg progenitors and subsequent differentiation into Foxp3-expressing mature Tregs. Ablation of NF-κB also prevented in vitro differentiation of inducible Tregs. Furthermore, specific deletion of either p65 or c-Rel subunits in natural Tregs using FoxP3-cre led to the development of a lethal autoimmune syndrome by 8 to 15 weeks of age. Ablation of both subunits further shortened the lifespan of animals to around 3 weeks of age. This was associated with profound changes in the molecular signature of NF-κB deficient Tregs, which down-regulated Treg-associated genes while acquiring an effector-like phenotype. Also, tamoxifen-induced acute deletion of NF-κB in mature Tregs reduced their survival and expression of Treg-associated markers. These data highlight a crucial role for NF-κB in maintaining Treg identity in vivo. We next explored the role of NF-κB in Treg homeostasis during tumor growth. Strikingly, melanoma growth was drastically reduced in mice lacking c-Rel, but not p65, in Tregs. This was associated with an increased polyclonal and antigen-specific effector T cell activation. Besides, CD8 T cell depletion abolished the protection against tumor growth. We are now proceeding to test the effect of specific c-Rel inhibitors on tumor growth. We believe our work reveals a central molecular mechanism in the maintenance of Treg identity and thus highlights novel therapeutic target for the treatment of cancer. Citation Format: Yenkel Grinberg-Bleyer, Hyunju Ho, Roland Schmid, Ulf Klein, Sankar Ghosh. The canonical NF-κB pathway is crucial for Treg homeostasis during the antitumor response. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B060.

γδ T Cell-Dependent Regulatory T Cells Prevent the Development of Autoimmune Keratitis.

To prevent potentially damaging inflammatory responses, the eye actively promotes local immune tolerance via a variety of mechanisms. Owing to trauma, infection, or other ongoing autoimmunity, these mechanisms sometimes fail, and an autoimmune disorder may develop in the eye. In mice of the C57BL/10 (B10) background, autoimmune keratitis often develops spontaneously, particularly in the females. Its incidence is greatly elevated in the absence of γδ T cells, such that ∼80% of female B10.TCRδ(-/-) mice develop keratitis by 18 wk of age. In this article, we show that CD8(+) αβ T cells are the drivers of this disease, because adoptive transfer of CD8(+), but not CD4(+), T cells to keratitis-resistant B10.TCRβ/δ(-/-) hosts induced a high incidence of keratitis. This finding was unexpected because in other autoimmune diseases, more often CD4(+) αβ T cells, or both CD4(+) and CD8(+) αβ T cells, mediate the disease. Compared with wild-type B10 mice, B10.TCRδ(-/-) mice also show increased percentages of peripheral memory phenotype CD8(+) αβ T cells, along with an elevated frequency of CD8(+) αβ T cells biased to produce inflammatory cytokines. In addition, B10.TCRδ-/- mice have fewer peripheral CD4(+) CD25(+) Foxp3(+) αβ regulatory T cells (Tregs), which express lower levels of receptors needed for Treg development and function. Together, these observations suggest that in B10 background mice, γδ T cells are required to generate adequate numbers of CD4(+) CD25(+) Foxp3(+) Tregs, and that in B10.TCRδ(-/-) mice a Treg deficiency allows dysregulated effector or memory CD8(+) αβ T cells to infiltrate the cornea and provoke an autoimmune attack.

CD4+CD25highFoxp3+ Treg deficiency in a Brazilian patient with Gaucher disease and lupus nephritis

Gaucher Disease (GD) is a rare autosomal recessive disorder caused by the deficient activity of beta-glucocerebrosidase. GD is one of the lysosomal storage diseases with the most remarkable alterations in the immune system, and that may manifest clinically as autoimmune disorders and malignancy. We reported the immunological evaluation of a patient with GD and lupus nephritis. Decreased absolute values of T, and NK, and an inversion of CD4+/CD8+ ratio, low levels of IgM and normal B cells were found when compared to reference values in a Brazilian population. Absence ofCD4+CD25highFoxp3+ Treg and high levels of total NKT, iNKT cells and CD8+ iNKT subsets were also observed when compared to the healthy control and GD patient without lupus nephritis. Treg subset and CD8+ iNKT abnormalities might be involved in severe lupus nephritis in a GD patient. We conclude by emphasizing the importance of the immunological evaluation on early diagnosis of autoimmunity contributing to reduce mortality and morbidity of these patients.

Maturation and Phenotypic Diversity of Human CD4+ Regulatory T Cells in Umbilical Cord Blood and Peripheral Blood from Healthy Donors

Introduction: CD4+ FoxP3+ CD25+ regulatory T cells (Treg) are required for maintenance of immune tolerance and immune homeostasis. Quantitative or functional Treg deficiency has been correlated with autoimmune disease, allergy, allograft rejection and graft versus host disease. Conversely, increased Treg can suppress tumor immunity resulting in tumor progression. Treg express a large number of cellular markers that reflect their level of maturation, functionality, activation and migratory capacity. Nevertheless, it has not previously been possible to integrate the expression of these various markers and correlate their expression with human Treg differentiation in vivo.Methods: We used single cell mass cytometry (CyTOF) to simultaneously study 36 phenotypic and functional markers of human Treg in samples obtained from umbilical cord blood (CB) (n=4) and healthy adult donors (n=10). Wanderlust trajectory detection algorithm was used to analyze temporal positioning of Treg across development. To quantify Treg heterogeneity we used ACCENSE; an analysis tool that combines a nonlinear dimensionality reduction algorithm (t-SNE) with spectral clustering algorithm and automated cell classification into subpopulations.Results: Using Wanderlust to characterize Treg maturation, the majority of CB Treg were naive (CD45RA+) and CB memory Treg (CD45RA-) were poorly differentiated with minimal expression of activation (HLA-DR) and pro-apoptotic (CD95) markers (Figure 1A). Adult Treg contained few naive cells and mature Treg effector cells expressed high levels of activation and pro-apoptotic markers. (Figure 1B). Using Wanderlust together with spearman correlation, 5 discrete stages of Treg maturation were identified; 1) recent thymic emigrants (RTE), 2) naive, 3) effector, 4) activated and 5) terminal effector. RTE Treg defined by expression of CD45RA, CD31 and CCR7, also expressed markers of proliferation (KI67) and functionality (Tbet, PDL-1, Helios) at low levels but lacked functional CTLA4 and TIM-3. Naive Treg lacked expression of CD31 but expressed other markers characteristic of RTE. Effector Treg expressed increased levels of CD95, CTLA-4, CCR7, GITR, Helios and FoxP3 but lacked HLA-DR. Activated effector Treg expressed the highest levels of FoxP3, Helios and Ki67, along with functional markers (CD28, CXCR3, ICOS, GITR, CD39, CTLA-4, TIM-3) and homing molecules (vascular endothelial CCR5, gut addressin ACT-1, skin addressins CCR4, CLA). Activated Treg express the highest levels and diversity of functional markers along with the ability to migrate to different tissues. Lastly, terminal effector Treg express a more restricted set of functional and homing markers (CD28, CTLA-4, ICOS and CCR5) with less diversity. Markers of exhaustion (PD-1 and TIM-3) are also expressed by effector, activated and terminal effector Treg. Pro-apoptotic markers (CD95high BCL2low) are primarily expressed by activated and terminal effector Treg.Using ACCENSE to evaluate Treg diversity allowed further identification of discrete Treg sub-populations within each maturation state. RTE and naive Treg appear very homogeneous and appear as a single cluster in both CB and adults. In contrast, effector, activated and terminal effector Treg are more heterogeneous and are visualized as 9 distinct clusters (Figure 1C, D). This clustering reflects distinct subsets of memory Treg that co-express various combinations of functional markers in our panel. All 9 Treg effector populations are present in CB, but at much lower levels. Treg effector cell diversity is maintained but does not increase as Treg mature and expand in adults and RTE/naive Treg become less prevalent.Conclusion: Our study is the first to quantify human Treg heterogeneity based on expression of a large set of activation, proliferation, tissue homing and functional markers in conjunction with stages of Treg maturation and differentiation. These studies define 5 stages of Treg maturation and 10 clusters of Treg diversity based on differential expression of phenotypic and functional markers. Similar approaches can be applied to describe maturation and diversity of other cell populations. Further application of this CyTOF panel can be used to study Treg maturation and diversity after hematopoietic stem cell transplantation and in immune and inflammatory diseases, to identify specific defects that may contribute to immune dysfunction. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

FOXP3(+) Treg Cells and Gender Bias in Autoimmune Diseases.

CD4+CD25+ regulatory T (Treg) cells play a pivotal role in the maintenance of immune homeostasis, where the X-linked master transcription factor forkhead box P3 (FOXP3) determines Treg cell development and function. Genetic deficiency of foxp3 induces dysfunction of Treg cells and immuno-dysregulation, polyendocrinopathy, enteropathy, and X-linked syndrome in humans. Functionally deficient Treg cells or the development of exTreg cells positively correlate with autoimmune diseases, such as systemic lupus erythematosus (SLE), multiple sclerosis (MS), and ankylosing spondylitis (AS). In general, females are more susceptible to SLE and MS but less susceptible to AS, where the expression of FOXP3 and its protein complex are perturbed by multiple factors, including hormonal fluctuations, inflammatory cytokines, and danger signals. Therefore, it is critical to explore the potential molecular mechanisms involved and these differences linked to gender. Here, we review recent findings on the regulation of FOXP3 activity in Treg cells and also discuss gender difference in the determination of Treg cell function in autoimmune diseases.

Defects in regulatory T cells due to CD28 deficiency induce a qualitative change of allogeneic immune response in chronic graft-versus-host disease.

In patients receiving allogeneic hematopoietic cell transplantation, chronic graft-versus-host disease (cGVHD) remains a frequent complication and resembles autoimmune diseases such as systemic lupus erythematosus and systemic sclerosis. Our previous work demonstrated the critical role of CD28 costimulation of donor T cells for GVHD induction. In this study, we investigate the role of CD28 costimulation of host T cells in cGVHD. CD28-intact mice as hosts showed systemic lupus erythematosus-type cGVHD, whereas CD28-deficient mice developed a distinct phenotype of cGVHD, with fibrotic damage in skin and internal organs, resembling systemic sclerosis. This phenotype was due to a lack of signaling through the C-terminal proline-rich motif within host CD28's cytoplasmic tail, a motif previously shown to be required for development of regulatory T cells (Tregs) and function of conventional T cells. Adoptive transfer experiments demonstrated that a defect in host CD4(+)CD25(+) Tregs, but not in conventional T cells, was responsible for disease phenotype. Host Treg deficiency altered the cytokine pattern of donor CD4(+) T cells and the Ag specificity of autoantibodies, and these might lead to phenotypic change. Thus, host CD28 signaling controlled the pathogenesis of cGVHD through effects on host Tregs, whose status impacts qualitatively on the allogeneic immune responses.

CIS inhibits effector programs in regulatory T cells (IRM15P.459)

Abstract Regulatory T (Treg) cells play an essential role in immune homeostasis. Under the pro-inflammatory settings such as autoimmune diseases, the function of Treg cells is impaired, in which pro-inflammatory cytokine IL-4 and IL-6 are involved in destabilizing Treg cells. However, the pathways that stabilize Treg cells have not been well understood. We have shown that cytokine-induced SH-2 protein (CIS) inhibits STAT3 and STAT6 activation; our current study indicates that CIS is required for stabilizing Treg cells in a Treg cell-intrinsic manner. Cis deficient Treg cells had impaired suppressive activity in vitro. Cis deficient mice on a T helper 2 (TH2) cells-prone background develop lymphadenopathy, splenomegaly and multiple organ inflammation, which is associated with spontaneous loss of Foxp3 expression and gain of TH2 cytokine expression in Treg cells. In addition to its role in cytoplasm, CIS also translocates into the nuclei in Treg cells which may serve as a second mechanism of surveillance for pro-inflammatory cytokine induced STAT6 (and maybe also STAT3) in the nuclei, and therefore contribute to stabilization of Treg cells.

Expression of Helios is required for the differentiation of activated/effector T regulatory cells (IRC11P.438)

Abstract Foxp3+ Regulatory T cells (Treg) suppress immune activation and play a critical role in the maintenance of self-tolerance. A subpopulation (70-80%) of Treg cells in both mouse and man express the transcription factor, Helios, but the role of Helios in Treg function is still unknown. Deletion of Helios expression in all T cells (CD4-Cre) or in early progenitors (Vav-Cre) did not result in immune dysfunction. As some studies have suggested that Helios might be expressed in activated T conventional cells, we generated mice with a conditional deletion (KO) of Helios in Treg. The selective deletion of Helios in Tregs led to a slowly progressive, systemic immune activation characterized by a Th1 profile, lymphadenopathy, splenomegaly, increased serum Ig and enhanced lymphoid follicle and germinal center formation in the absence of immune pathology. In vitro suppressive function of Helios KO Treg was normal, but KO Treg failed to differentiate into T follicular regulatory cells in vivo. While the absolute number of Treg in KO mice was enhanced, in competitive settings Helios deficient Tregs were at a striking disadvantage in the periphery. Notably, Helios KO Tregs with an activated (CD44hiCD62Llo) effector Treg phenotype were greatly decreased. Analysis of Helios KO Tregs revealed increased levels of several BH3 domain pro-apoptotic genes. These findings suggest that Helios plays a critical role in the development and/or survival of effector Tregs by repressing pro-apoptotic genes.