Follicular Treg Research Articles

Tissue resident and follicular Treg cell differentiation is regulated by CRAC channels

T regulatory (Treg) cells maintain immunological tolerance and organ homeostasis. Activated Treg cells differentiate into effector Treg subsets that acquire tissue-specific functions. Ca2+ influx via Ca2+ release-activated Ca2+ (CRAC) channels formed by STIM and ORAI proteins is required for the thymic development of Treg cells, but its function in mature Treg cells remains unclear. Here we show that deletion of Stim1 and Stim2 genes in mature Treg cells abolishes Ca2+ signaling and prevents their differentiation into follicular Treg and tissue-resident Treg cells. Transcriptional profiling of STIM1/STIM2-deficient Treg cells reveals that Ca2+ signaling regulates transcription factors and signaling pathways that control the identity and effector differentiation of Treg cells. In the absence of STIM1/STIM2 in Treg cells, mice develop a broad spectrum of autoantibodies and fatal multiorgan inflammation. Our findings establish a critical role of CRAC channels in controlling lineage identity and effector functions of Treg cells.

C-Maf and STAT3 are essential but distinct regulators of RORγt+ and follicular regulatory T cells

Abstract Foxp3+ regulatory T cells (Tregs) must adapt to function within a wide range of tissue environments and types of immune response. This flexibility is achieved through specialization, whereby context-dependent transcriptional changes integrate unique functionality into general Treg programming. In the most well-studied examples, this involves the co-expression of Foxp3 and another “lineage-defining” transcription factor (TF) which confers expression of specific functional genes. Two such specialized populations are RORγt+ Tregs and Bcl-6+ T follicular regulatory (Tfr) cells, which have important functions in regulating colitis and humoral immune responses, respectively. Although the importance of Treg specialization is now clear, the underlying network of signals and TFs driving this process is largely undefined. Here, we identify the TF c-Maf as an essential regulator of Treg specialization for both RORγt+ Tregs and Tfr cells. Importantly, the role of c-Maf in these subsets is highly specific, because deletion of Maf does not affect general Treg development, generation of other specialized Tregs, or expression of suppressive molecules. In vitro, we show that addition of IL-6 to Treg-promoting conditions is sufficient to drive both c-Maf expression and c-Maf-dependent conversion of naïve T cells to RORγt+ Tregs, suggesting that IL-6 might drive c-Maf in vivo. Surprisingly, although STAT3 – the primary downstream effector of IL-6 – is required for development of both RORγt+ Tregs and Tfr cells, we find that Stat3 deletion in vivo does not ablate c-Maf expression and results in Treg defects distinct from Maf deletion. Therefore, c-Maf and STAT3 are cooperative, but distinct regulators of RORγt+ and follicular Treg development.

Increased Circulating Follicular Treg Cells Are Associated With Lower Levels of Autoantibodies in Patients With Rheumatoid Arthritis in Stable Remission.

To examine the expression and changes in function of circulating CD4+CXCR5+FoxP3+ follicular Treg (Tfr) cells in patients with active rheumatoid arthritis (RA) and in patients with RA in stable remission, and to clarify the role of Tfr cells in the pathogenesis of RA. Levels of Tfr cells and follicular helper T (Tfh) cells in the peripheral blood of 39 patients with active RA, 39 patients with RA in stable remission, and 33 healthy controls were detected by flow cytometry. The function of Tfr cells was measured by coculturing them with Tfh cells and B cells. Activated CD45RA-FoxP3high Tfr cells were also analyzed. Clinical indicators, including serum Ig and autoantibody levels, were tested, and correlations with Tfr cells were systematically analyzed. The Disease Activity Score in 28 joints (DAS28) was calculated, and correlation analysis with Tfr cells was conducted. The level of CD4+CXCR5+FoxP3+ Tfr cells and the Tfr cell:Tfh cell ratio in peripheral blood from patients with RA in stable remission were significantly increased compared with the same measures in patients with active RA and in healthy controls. The function of Tfr cells was enhanced, and the activated CD45RA-FoxP3high Tfr cell subset was increased in patients with RA in stable remission compared with healthy controls. Furthermore, the number of Tfr cells in RA patients was inversely correlated with IgG, rheumatoid factor, and anti-cyclic citrullinated peptide as well as with the DAS28. Circulating Tfr cells are increased as patients with RA achieve stable remission of disease, and increased Tfr cells can suppress autoimmunity in RA patients to stabilize their condition. Our results provide novel insight into RA pathogenesis.

Identity and Diversity of Human Peripheral Th and T Regulatory Cells Defined by Single-Cell Mass Cytometry.

Human CD3+CD4+ Th cells, FOXP3+ T regulatory (Treg) cells, and T regulatory type 1 (Tr1) cells are essential for ensuring peripheral immune response and tolerance, but the diversity of Th, Treg, and Tr1 cell subsets has not been fully characterized. Independent functional characterization of human Th1, Th2, Th17, T follicular helper (Tfh), Treg, and Tr1 cells has helped to define unique surface molecules, transcription factors, and signaling profiles for each subset. However, the adequacy of these markers to recapitulate the whole CD3+CD4+ T cell compartment remains questionable. In this study, we examined CD3+CD4+ T cell populations by single-cell mass cytometry. We characterize the CD3+CD4+ Th, Treg, and Tr1 cell populations simultaneously across 23 memory T cell-associated surface and intracellular molecules. High-dimensional analysis identified several new subsets, in addition to the already defined CD3+CD4+ Th, Treg, and Tr1 cell populations, for a total of 11 Th cell, 4 Treg, and 1 Tr1 cell subsets. Some of these subsets share markers previously thought to be selective for Treg, Th1, Th2, Th17, and Tfh cells, including CD194 (CCR4)+FOXP3+ Treg and CD183 (CXCR3)+T-bet+ Th17 cell subsets. Unsupervised clustering displayed a phenotypic organization of CD3+CD4+ T cells that confirmed their diversity but showed interrelation between the different subsets, including similarity between Th1-Th2-Tfh cell populations and Th17 cells, as well as similarity of Th2 cells with Treg cells. In conclusion, the use of single-cell mass cytometry provides a systems-level characterization of CD3+CD4+ T cells in healthy human blood, which represents an important baseline reference to investigate abnormalities of different subsets in immune-mediated pathologies.

Roquin Suppresses the PI3K-mTOR Signaling Pathway to Inhibit T Helper Cell Differentiation and Conversion of Treg to Tfr Cells

Roquin proteins preclude spontaneous Tcell activation and aberrant differentiation of T follicular helper (Tfh) or T helper 17 (Th17) cells. Here we showed that deletion of Roquin-encoding alleles specifically in regulatory T (Treg) cells also caused the activation of conventional Tcells. Roquin-deficient Treg cells downregulated CD25, acquired a follicular Treg (Tfr) cell phenotype, and suppressed germinal center reactions but could not protect from colitis. Roquin inhibited the PI3K-mTOR signaling pathway by upregulation of Pten through interfering with miR-17∼92 binding to an overlapping cis-element in the Pten 3'UTR, and downregulated the Foxo1-specific E3 ubiquitin ligase Itch. Loss of Roquin enhanced Akt-mTOR signaling and protein synthesis, whereas inhibition of PI3K or mTOR in Roquin-deficient Tcells corrected enhanced Tfh and Th17 or reduced iTreg cell differentiation. Thereby, Roquin-mediated control of PI3K-mTOR signaling prevents autoimmunity by restraining activation and differentiation of conventional Tcells and specialization of Treg cells.

Abstract 943: RelA regulates CXCR5/CXCL13 transcription and associated immune response in breast cancer

Abstract It is evident from research outcomes that metastases-associated deaths are predominant in breast cancer. Recent developments on early diagnosis using mammographic screening and the implementation of adjuvant therapies may have reduced breast cancer associated deaths in decent numbers, although new markers for prognosis are of utmost importance for patients with higher risk of developing metastases or recurrence. Chemokines are the key messenger of cellular immune response and can be targeted to reduce the intratumoral regulatory T cells (Treg) for antitumor immunity. Targeting either specific immunomodulators and/or intervening molecular mechanisms is thought to be a potential therapeutic option. In our previous study, we have found that co-expression of CXCR5 and CXCL13 is significantly associated with epithelial to mesenchymal transition (EMT) of cells and lymph node metastasis (LNM) during breast cancer progression. In this study, we are aiming to investigate how the transcriptional regulation of this receptor-ligand pair directs the process of disease development. Interestingly, it was found that RelA (p65), a subunit of NFkB protein, promoted the transcription of both CXCR5 and CXCL13. The putative RelA binding sites were validated using sequential deletion of respective promoter regions of CXCR5 and CXCL13. We observed that expression of CXCR5 significantly (p<0.05) increased with RelA and Nrf2 overexpression and CXCL13 co-stimulation in MDA-MB-231 cell line. RelA was also found to induce the expression of CXCL13 in T-47D and MDA-MB-231 cell line. Significantly, SNAIL, a key EMT regulatory element, was found to be highly expressed in RelA-transfected MDA-MB-231 cell line with or without CXCR5 and CXCL13 co-stimulation. We also observed CXCR5+ T follicular helper (Tfh) and Treg sub-population within the tumor microenvironment, although numbers varied with stages and molecular subtypes. In chemotaxis assay, CXCR5+ Treg cells were found to be predominant among migratory T-cell subsets against RelA overexpression and/or CXCL13 stimulation. Simultaneously, CCL2, a pro-inflammatory chemokine, induced macrophages to secrete CCL22, which in turn, might attract CXCR5+ Treg and Th2 cells into the tumor microenvironment. Study in 4T1-BALB/c breast cancer mouse model demonstrated significant (p<0.05) increase in CXCR5 and CXCL13 expression levels upon immunostimulation. The study may help to understand the prognosis value of CXCR5 and CXCL13 considering the impact of this receptor-ligand pair on the regulation of Tfh/Treg ratio, EMT and LNM. We believe that this investigation may lead to a comprehensive prediction of the tumor fate as well as to explore possible markers for breast cancer prognosis and future chemotherapy with more precision. Citation Format: Sougata Roy Chowdhury, Subir Biswas, Gunjan Mandal, Arindam Bhattacharyya. RelA regulates CXCR5/CXCL13 transcription and associated immune response in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 943. doi:10.1158/1538-7445.AM2017-943

Invariant natural killer T cells ameliorate murine chronic GVHD by expanding donor regulatory T cells

AbstractChronic graft-versus-host-disease (cGVHD) can cause multiorgan system disease, typically with autoimmune-like features, resulting in high mortality and morbidity caused by treatment limitations. Invariant natural killer T cells (iNKTs), a small population characterized by expression of a semi-invariant T-cell receptor, rapidly produce copious amounts of diverse cytokines on activation that exert potent immune regulatory function. Here, we show that iNKTs are significantly reduced in a cGVHD murine model that recapitulates several aspects of autoimmunity and organ fibrosis observed in patients with cGVHD. Low iNKT infused doses effectively prevented and, importantly, reversed established cGVHD, as did third-party iNKTs. iNKTs suppressed the autoimmune response by reducing the germinal center (GC) reaction, which was associated with an increase in total Tregs and follicular Tregs (Tfr) that control the GC reaction, along with pathogenic antibody production. Treg depletion during iNKT infusions completely abolished iNKT efficacy in treating cGVHD. iNKT cell interleukin 4 production and GC migration were critical to cGVHD reversal. In vivo stimulation of iNKT cells by α-galactosyl-ceramide was effective in both preventing and treating cGVHD. Together, this study demonstrates iNKT deficiency in cGVHD mice and highlights the key role of iNKTs in regulating cGVHD pathogenesis and as a potentially novel prophylactic and therapeutic option for patients with cGVHD.

Imbalance between T follicular helper and T follicular regulatory cells in myasthenia gravis

According to the signals they receive from the local environment, T cells can differentiate into one out of several subtypes, including Th1, Th2, Th3, Th9, Th17, T follicular helper (Tfh) and Treg cells (1). Each of these cell subsets possesses a particular function. The term “Tfh cell” first described a subset of CD4+ T cells present in human lymphoid tissues, namely tonsils, and functions primarily to provide help to B cells (2). Interestingly and similarly to B cells, Tfh cells express the homing receptor, CXCR5, whose ligand is the CXCL13, the main B cell chemoattractant chemokine. Within germinal centers (GC), Tfh cells mediate the selection and survival of B cells that differentiate into plasma cells producing high-affinity antibodies against foreign antigens, and memory B cells (3). Recently, a small subset of T follicular regulatory (Tfr) cells was described, that are Treg cells able to suppress specifically the Tfh cell function (4). Thus it is likely that a balance between Tfr and Tfh cells dictates the fate of the B cell immune response.

Intra-Articular Injection of Human Synovial Membrane-Derived Mesenchymal Stem Cells in Murine Collagen-Induced Arthritis: Assessment of Immunomodulatory Capacity In Vivo.

The aim of this study was to evaluate the efficacy of human synovial membrane-derived MSCs (SM-MSCs) in murine collagen-induced arthritis (CIA). Male mice (age 7–9 weeks) were injected intra-articularly with SM-MSCs obtained from patients with osteoarthritis, on days 28, 32, and 38 after bovine type II collagen immunization. The efficacy of SM-MSCs in CIA was evaluated clinically and histologically. Cytokine profile analyses were performed by real-time polymerase chain reaction and multiplex analyses. Splenic helper T (Th) cell and regulatory B cell subsets were analyzed by flow cytometry. Intra-articular SM-MSC injection ameliorated the clinical and histological severity of arthritis. Decrease in tumor necrosis factor-α, interferon-γ, and interleukin- (IL-) 17A and increase in IL-10 production were observed after SM-MSC treatment. Flow cytometry showed that Th1 and Th17 cells decreased, whereas Th2, regulatory T (Treg), and PD-1+CXCR5+FoxP3+ follicular Treg cells increased in the spleens of SM-MSC-treated mice. Regulatory B cell analysis showed that CD21hiCD23hi transitional 2 cells, CD23lowCD21hi marginal zone cells, and CD19+CD5+CD1d+IL-10+ regulatory B cells increased following SM-MSC treatment. Our results demonstrated that SM-MSCs injected in inflamed joints in CIA had a therapeutic effect and could prevent arthritis development and suppress immune responses via immunoregulatory cell expansion.

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.

Dysregulated development of IL‐17‐ and IL‐21‐expressing follicular helper T cells and increased germinal center formation in the absence of RORγt

Interleukin 17-producing helper T (Th17) cells have been widely defined by the lineage transcription factor retinoid-related orphan receptor (ROR)γt. Pathophysiologically, these cells play a crucial role in autoimmune diseases and have been linked to dysregulated germinal center (GC) reactions and autoantibody production. In this study, we used gene expression and flow cytometric analyses for the characterization of Rorγt(-/-) and Rorγt(-/-)Il21(RFP/+) mice to demonstrate a previously unknown transcriptional flexibility in the development of IL-17-producing Th-cell subsets. We found an accumulation of follicular Th (Tfh) cells by 5.2-fold, spontaneous 13-fold higher GC formation, decreased frequency of follicular Foxp3(+) T-regulatory (Treg) cells (50%), and a 3.4-fold increase in the number of proliferating follicular B cells in RORγt-deficient vs. wild-type mice. Dysregulated B-cell responses were associated with enhanced production of IL-17 (6.4-fold), IL-21 (2.2-fold), and B-cell-activating factor (BAFF) (2-fold) and were partially rescued by adoptive transfer of Treg cells. In an unexpected finding, we detected RORγt-independent IL-17 expression in ICOS(+)CXCR5(+)Tfh and in ICOS(+)CXCR5(-)Th cells. Based on the observed high Irf4 and Batf gene expression, we suggest that CD4(+) T-cell transcription factors other than RORγt can cooperatively induce differentiation of IL-17-producing Th cells, including Th17-like Tfh-cell subsets. We conclude that the occurrence of aberrant Tfh and follicular Treg cells support spontaneous GC formation and dysregulated B-cell responses in RORγt-deficient mice.

Reduced Tregs in peripheral blood of PCOS patients - a consequence of aberrant Il2 signaling.

The immunesupressive action of CD4(+)CD25(+) CD127(-/low) T regulatory cells (Tregs) is vital for an efficient reproductive function. However no data exists on their number or functionality in polycystic ovary syndrome (PCOS). The study aimed to analyze the frequency of circulating Tregs and key factors modulating them in women with PCOS. This is a retrospective, case-control cohort study conducted in women with PCOS recruited from Samad IVF hospitals and Women and Children Hospital, Thiruvananthapuram, India. Women with PCOS (N = 20) were diagnosed according to Rotterdam Consensus and normal menstruating women were taken as controls (N = 2331). We analyzed the proportion of CD4(+)CD25(+) CD127(-/low) Tregs in women with PCOS by fluorescent activated cell sorting. The study discovered that the women with PCOS have reduced numbers of Tregs (2.626 ± 0.62) compared with controls (4.253 ± 0.87) (t = 6.963, P < .0001, mean difference = -1.627; 95% confidence interval = -2.099--1.155). We documented a decrease in the follicular phase Treg expansion in women with PCOS. Our results revealed a reduced STAT5A (fold change [FC] = 7.642, P < .0004)/STAT5B (FC = 3.824, P < .0001), FOXP3 (FC = 4.1343, P = .0004)/CTLA4 (FC = 2.569, P = .0001) and elevated AKT (FC = 7.39, P = .05)/PIK3 (FC = 5.326, P = .0002) expression in women with PCOS. Recombinant interleukin 2 (rIL2) treatment failed to improve FOXP3/CTLA4 levels but caused a reduction of AKT/PIK3 arm, possibly due to an elevated PTEN in women with PCOS. The study suggests that women with PCOS have reduced Tregs due to an inherent hyporesponsiveness to IL2, which is unable to activate STAT5B and reduce FOXP3 expression. IL2-based therapeutic strategies can ameliorate complications in PCOS by suppressing the AKT/PIK3 arm.

Circulating T follicular regulatory and helper cells have memory-like properties.

Follicular Tregs (Tfr cells) inhibit antibody production, whereas follicular Th cells (Tfh cells) stimulate it. Tfr cells are found in blood; however, relatively little is known about the developmental signals for these cells or their functions. Here we demonstrated that circulating Tfr and Tfh cells share properties of memory cells and are distinct from effector Tfr and Tfh cells found within lymph nodes (LNs). Circulating memory-like Tfh cells were potently reactivated by DCs, homed to germinal centers, and produced more cytokines than did effector LN Tfh cells. Circulating memory-like Tfr cells persisted for long periods of time in vivo and homed to germinal centers after reactivation. Effector LN Tfr cells suppressed Tfh cell activation and production of cytokines, including IL-21, and inhibited class switch recombination and B cell activation. The suppressive function of this population was not dependent on specific antigen. Similar to LN effector Tfr cells, circulating Tfr cells also suppressed B and Tfh cells, but with a much lower capacity. Our data indicate that circulating memory-like Tfr cells are less suppressive than LN Tfr cells and circulating memory-like Tfh cells are more potent than LN effector Tfh cells; therefore, these circulating populations can provide rapid and robust systemic B cell help during secondary antigen exposure.

TRAF3 regulates the effector function of regulatory T cells and humoral immune responses

Regulatory T cells (Treg cells) control different aspects of immune responses, but how the effector functions of Treg cells are regulated is incompletely understood. Here we identified TNF receptor-associated factor 3 (TRAF3) as a regulator of Treg cell function. Treg cell-specific ablation of TRAF3 impaired CD4 T cell homeostasis, characterized by an increase in the Th1 type of effector/memory T cells. Moreover, the ablation of TRAF3 in Treg cells resulted in increased antigen-stimulated activation of follicular T helper cells (TFH cells), coupled with heightened formation of germinal centers and production of high-affinity IgG antibodies. Although the loss of TRAF3 did not reduce the overall frequency of Treg cells, it attenuated the antigen-stimulated production of follicular Treg cells (TFR cells). TRAF3 signaling in Treg cells was required to maintain high level expression of inducible co-stimulator (ICOS), which in turn was required for TFR cell generation and inhibition of antibody responses. These findings establish TRAF3 as a mediator of Treg cell function in the regulation of antibody responses and suggest a role for TRAF3 in mediating ICOS expression in Treg cells.

The Architecural Pattern of FOXP3+ T Cells Predicts Risk of Transformation in Patients with Follicular Lymphoma (FL).

Background: FL is an indolent heterogeneous lymphoid neoplasm with a variable clinical course. Transformation into an aggressive lymphoma (Tly) is a dominant clinical event frequently associated with inferior survival. No consistent biological markers have been identified that predict for Tly. Recent studies have highlighted the role of the microenvironment, including lymphoma-associated-macrophages (LAM) and Treg cells as important prognostic factors in FL. The nuclear expression of FOXP3 is considered a specific marker of regulatory T cells (Treg). However, most series have included pts variably treated with a wide spectrum of protocols. Using tissue microarrays (TMA) we recently demonstrated that both LAM content and follicular/perifollicular pattern of FOXP3+ T cells are independent adverse prognostic factors for both overall (OS) and progression-free (PFS) survival in advanced-stage FL patients treated uniformly with an aggressive regimen. However, the impact of either LAM or Tregs on Tly is largely unknown. Using the same cohort we assessed the role of these cell types for predicting Tly.Methods: Between 1987 and 1993, 126 pts were enrolled on a phase II study of BP-VACOP chemotherapy with involved region radiotherapy. All patients were treatment naïve, < 61y and had advanced-stage FL. Paraffin blocks were available in 105 patients. The TMAs consisted of duplicate 1.0mm cores of diagnostic biopsies and were immunostained with both CD68 and a FOXP3-specific antibody. Both CD68+ macrophage content and FOXP3+ T cell content and immuno-architectural patterns were determined and correlated with RT including univariate and multivariate analyses.Results: There were 102 evaluable cases. The median follow-up of the living pts was 14.3 years and the estimated 10-year OS and Tly rates were 63% and 18%, respectively. The IPI was predictive of OS (RR = 2.4, 95% CI =1.4–4.2, p = 0.002) but not Tly (RR = 2.2, 95% CI =.9–5.1, p = 0.07). Histologic grade included 76 grade 1, 20 grade 2 and 6 grade 3a FLs. Cases revealed 1 to 28 CD68+ cells per high power field (median 7 cells/hpf) and zero to 731 FOXP3+ cells per core (median of 242 cells/core). In 38 pts there was a predominantly intrafollicular or perifollicular localization of positive cells (“follicular Treg pattern”) and in 64 pts the pattern was diffuse. Neither LAM content nor number of FOXP3+ cells had any impact on Tly. However, the immuno-architectural pattern of FOXP3+ cells was predictive of Tly, with the “follicular” pattern showing increased risk of Tly in univariate analysis (p=0.005). A Cox multivariate model including IPI and LAM showed only the FOXP3+ Treg pattern as an independent predictor of Tly (RR = 3.9, 95% CI =.1.5–9.9, p = 0.004).Conclusions: Treg cell distribution within the tumor is an important predictor of Tly in advanced-stage FL patients treated uniformly with an aggressive treatment regimen. Our results reinforce the importance of a FOXP3 follicular pattern as a good surrogate for functionally active Treg cells within the tumor microenvironment and further highlight the important role of the tumor microenvironment in FL biology. [Display omitted]