scholarly journals IL-10 Receptor Signaling Empowers Regulatory T Cells to Control Th17 Responses and Protect from GN

2018 ◽  
Vol 29 (7) ◽  
pp. 1825-1837 ◽  
Author(s):  
Paul Diefenhardt ◽  
Anna Nosko ◽  
Malte A. Kluger ◽  
Johannes V. Richter ◽  
Claudia Wegscheid ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Th17 Cells ◽  
Cell Activation ◽  
Molecular Mechanisms ◽  
Receptor Signaling ◽  
Dendritic Cell Activation ◽  
Fluorescent Reporter

Background Th17 cells are central pathogenic mediators of autoimmune disease, including many forms of GN. IL-10 receptor signaling (IL-10R) in regulatory T cells (Tregs) has been implicated in the downregulation of Th17 cells, but the underlying molecular mechanisms and functional relevance of this process remain unclear.Methods We generated mice with Treg-specific IL-10Ra deficiency and subjected these mice to nephrotoxic serum–induced nephritis as a model of crescentic GN. Immune responses and Treg phenotypes were extensively analyzed.Results Compared with controls, mice with IL-10Ra−/− Tregs showed a spontaneously overshooting Th17 immune response. This hyper-Th17 phenotype was further boosted during GN and associated with aggravated renal injury. Notably, abrogation of IL-10Ra signaling in Tregs increased dendritic cell activation and production of Th17-inducing cytokines. In contrast, Treg trafficking and expression of chemokine receptor CCR6 remained unaffected, indicating mechanisms of Th17 control, differing from those of previously identified CCR6+ Treg17 cells. Indeed, the capacity for direct in vitro suppression of Th17 responses by IL-10Ra−/− Tregs was significantly impaired. As underlying pathology, analyses conducted in vitro and in vivo using double-fluorescent reporter mice revealed strikingly decreased IL-10 production by IL-10Ra−/− Tregs. To assess, whether reduced IL-10 could explain the hyper Th17 phenotype, competitive cotransfer experiments were performed. Supporting our concept, IL-10Ra−/− T cells differentiated into Th17 cells at much higher frequencies than wild type T cells did during GN.Conclusions IL-10R engagement optimizes Treg-mediated suppression of Th17 immunity. We hypothesize a feed-forward loop, in which IL-10Ra signaling reinforces IL-10 secretion by Tregs which potently controls Th17 development via direct and indirect mechanisms. IL-10R thus may be a promising therapeutic target for the treatment of GN.

scholarly journals MicroRNA-21 Regulates Diametrically Opposed Biological Functions of Regulatory T Cells

2021 ◽  
Vol 12 ◽  
Author(s):  
Jijun Sun ◽  
Ruiling Liu ◽  
Xiaozhen He ◽  
Jiang Bian ◽  
Wenbo Zhao ◽  
...  
Keyword(s):  
T Cells ◽  
Autoimmune Disease ◽  
Regulatory T Cells ◽  
Cell Activation ◽  
Treg Cells ◽  
Mechanistic Study ◽  
Dendritic Cell Activation ◽  
Adenoviral Delivery

Regulatory T cells (Tregs) are considered important for controlling the onset and development of autoimmune disease. Although studies have shown that miR-21 is expressed at higher levels in Treg cells, it remains largely elusive whether miR-21 regulates the immune-suppressive function of Tregs. In the current study, we generated mice lacking miR-21 specifically in their Tregs and investigated the role of miR-21 in regulating Treg function both in vitro and in vivo. Our study revealed that Tregs lacking miR-21 exhibit normal phenotype and unaltered function in suppressing T cell proliferation and dendritic cell activation in vitro. However, compared with miR-21-sufficient Tregs, they produce significant more IL-17 and IL-10 when under pathogenic Th17-priming condition. Adenoviral delivery of miR-21 into Treg cells is able to reduce the expression of both IL-17 and IL-10. Mechanistic study revealed that miR-21 down-regulates IL-10 expression through direct targeting of IL-10, and suppresses reprogramming of Tregs into IL-17-secreting cells through down-regulating Stat3 activity. However, we detected no significant or marginal difference in the development of various autoimmune diseases between wild type mice and mice with Treg-specific deletion of miR-21. In conclusion, our study demonstrated that miR-21 in Tregs regulates diametrically opposed biological Treg functions and is largely dispensable for the development of autoimmune disease.

Functional effects of chimeric antigen receptor co-receptor signaling domains in human regulatory T cells

2020 ◽  
Vol 12 (557) ◽  
pp. eaaz3866 ◽  
Author(s):  
Nicholas A. J. Dawson ◽  
Isaac Rosado-Sánchez ◽  
German E. Novakovsky ◽  
Vivian C. W. Fung ◽  
Qing Huang ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Cellular Therapy ◽  
In Vitro Assays ◽  
In Vivo Model ◽  
Receptor Signaling ◽  
Signaling Domain ◽  
Signaling Domains

Antigen-specific regulatory T cells (Tregs) engineered with chimeric antigen receptors (CARs) are a potent immunosuppressive cellular therapy in multiple disease models and could overcome shortcomings of polyclonal Treg therapy. CAR therapy was initially developed with conventional T cells, which have different signaling requirements than do Tregs. To date, most of the CAR Treg studies used second-generation CARs, encoding a CD28 or 4-1BB co-receptor signaling domain and CD3ζ, but it was not known if this CAR design was optimal for Tregs. Using a human leukocyte antigen–A2–specific CAR platform and human Tregs, we compared 10 CARs with different co-receptor signaling domains and systematically tested their function and CAR-stimulated gene expression profile. Tregs expressing a CAR encoding CD28wt were markedly superior to all other CARs tested in an in vivo model of graft-versus-host disease. In vitro assays revealed stable expression of Helios and an ability to suppress CD80 expression on dendritic cells as key in vitro predictors of in vivo function. This comprehensive study of CAR signaling domain variants in Tregs can be leveraged to optimize CAR design for use in antigen-specific Treg therapy.

Mechanisms of self–nonself discrimination and possible clinical relevance

Immunotherapy ◽  
2009 ◽  
Vol 1 (4) ◽  
pp. 631-644
Author(s):  
Carolin Daniel ◽  
Jens Nolting ◽  
Harald von Boehmer
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Immune Responses ◽  
Clinical Relevance ◽  
Molecular Mechanisms ◽  
Immunological Tolerance ◽  
Molecular Pathways ◽  
Clinical Protocols

This review discusses different mechanisms that result in immunological tolerance, such as intrathymic deletion of immature T cells, intrathymic and extrathymic generation of regulatory T cells, effector mechanisms of regulatory T cells as well as molecular pathways involved in extrathymic generation of regulatory T cells in vivo and in vitro. These molecular mechanisms should enable investigators to develop clinical protocols aiming at the specific prevention of unwanted immune responses, thereby replacing indiscriminate immunosuppression that often has fatal consequences.

5-Azacytidine Specifically Depletes Regulatory T Cells (Tregs) in Myelodysplastic Syndrome (MDS) Patients

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 787-787 ◽  
Author(s):  
Benedetta Costantini ◽  
Shahram Y Kordasti ◽  
Austin G Kulasekararaj ◽  
Jie Jiang ◽  
Thomas Seidl ◽  
...  
Keyword(s):  
T Cells ◽  
High Risk ◽  
Regulatory T Cells ◽  
Peripheral Blood ◽  
Th17 Cells ◽  
In Vitro Study ◽  
Tnf Α ◽  
T Helpers

Abstract Abstract 787 Introduction: The hypomethylating agent 5-azacytidine (5-azaC) leads to improved survival compared to conventional care regimens in patients with intermediate-2 and high-risk MDS and Acute Myeloid Leukaemia (AML) with less than 30% blasts. The precise mode of action of 5-azaC is uncertain, however a combination of cytotoxicity and demethylation is partly responsible for its anti-leukemic activity. In addition, 5-azaC has a profound effect on immune function and inhibits T cell proliferation and activation, blocking cell cycle in the G0 to G1 phase and decreases the production of pro-inflammatory cytokines, suggesting a possible in vivo and in vitro immunomodulating role that may contribute to its anti-leukemic activity. The aim of this study was to investigate the effects of 5-azaC on different subsets of CD4+ T cells, including regulatory T cells (Tregs) and T helpers (Th1, Th2, and Th17). Patients and methods: Seventy intermediate-2/high risk MDS patients and 10 healthy age matched donors (HDs) were studied. CD4+ and CD8+ T cells subsets (percentages and absolute numbers) were investigated by flow cytometry. All patients have received 5-azaC and peripheral blood samples were collected at diagnosis and after 1, 3, 6, 9 and 12 month from initial treatment. On average 3 samples were collected per patient. In vitro study: 5-azaC was added to pre-stimulated PBMCs from 4 HDs and 3 high-risk MDS patients to facilitate the drug incorporation. After 48 hours of initial stimulation, 5-azaC was added every 24 h up to 96 h on two different concentrations (1 μM and 2 μM). For each timepoint (t0, t+24, t+48, t+72, t+96) cells were stained with CD3, CD4, CD25, CD127 and Foxp3 for Tregs and with CD3, CD4, IFN-γ, TNF-α, IL-4, IL-17 for T helpers after an additional 4 hours stimulation with PMA/Ionomycin. Results: In vivo results: Numbers and percentages of Tregs were significantly higher in patients' peripheral blood prior to treatment compared to HDs (0.7% v 0.08%, p<0.001 and 1.1 × 107/L v 4.6 × 106/L, p=0.01). However, after 12 months of treatment with 5-azaC the number of Tregs decreased to the normal level. Absolute numbers and percentages of Tregs were also higher in non-responder patients compared to responders after treatment (1.2 × 107/L v 7.3 × 106/L, p=0.01). Although the number of Th1 and Th17 cells did not change significantly following treatment with 5-azaC, the Th1/Tregs and Th17/Tregs ratios were significantly decreased in non-responders (p=0.02), whereas these ratios remained stable in responder patients. In vitro results: There were no changes in the number or frequency of Th1, Th2 or Th17 when 5-azaC (1 μM and 2 μM) was added to patients' T cells. However, numbers and frequencies of Tregs dropped significantly compared to HDs' T cells (p=0.034). The ratio of Th1/Tregs and Th17/Tregs were also higher in patients' treated PBMCs after in vitro 5-azaC. There was a significant decrease in the percentages and numbers of Th1 cells (15.4% v 2.7%, p=0.043 and 1.42 × 103 v 6.44 × 104, p=0.021), Th17 cells (1.01% v 0.07%, p = 0.021 and 4.22 × 103 v 7.2 × 102, p=0.021) Th1/Tregs ratio (79.8 v 1.5, p=0.043), and Th17/Tregs ratio (5.2 v 0.1, p=0.021) in HDs' PBMCs treated with 2 μM compared to untreated cells. 5-azaC also reduced the absolute numbers of CD4+TNF-α+ T cells (1.07 × 105 v 5.37 × 103, p=0.021) and Th2 (9.24 × 103 v 1.25 × 102, p=0.021) in HDs' T cells. There was no preferential apoptosis in any subsets of T cells confirmed by Annexin V staining. However, it is interesting to note that the telomere length of Tregs treated with 5-azaC was longer than untreated Tregs, suggesting a decrease of their proliferation. Conclusion: Our study suggests that 5-azaC can induce a significant decrease in the number of Tregs in patients (in vivo and in vitro) and HDs, and therefore creates a pro-inflammatory state, despite a small decrease in the number of Th1 and Th17 cells. These changes are more significant in patients who responded to 5-azaC rather than in non-responders. Surprisingly, our in vitro study suggests that 5-azaC leads to a marked reduction in Tregs. As there is not a Tregs' specific apoptosis following 5-azaC treatment, we speculate that the reduction in Tregs' number is mainly due to de-methylation of transcription factors which leads to conversion of Tregs into other T cell subsets (ie Th1 or Th17). Disclosures: Mufti: Celgene: Research Funding.

scholarly journals In vitro generated Th17 cells support the expansion and phenotypic stability of CD4+Foxp3+ regulatory T cells in vivo

Cytokine ◽  
2014 ◽  
Vol 65 (1) ◽  
pp. 56-64 ◽  
Author(s):  
Qiong Zhou ◽  
Ya Hu ◽  
O.M. Zack Howard ◽  
Joost J. Oppenheim ◽  
Xin Chen
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Th17 Cells ◽  
Phenotypic Stability

Rap1-GTP Promotes the Generation of Regulatory T Cells in Vivo.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 110-110
Author(s):  
Lequn Li ◽  
Rebecca Greenwald ◽  
Esther M. Lafuente ◽  
Dimitrios Tzachanis ◽  
Alla Berezovskaya ◽  
...  
Keyword(s):  
T Cells ◽  
Lymph Node ◽  
T Cell ◽  
Regulatory T Cells ◽  
Immune Responses ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cd4 Cells

Abstract Elucidating the mechanisms that regulate T cell activation and tolerance in vivo will provide insights into the maintenance of physiologic homeostasis and will facilitate development novel strategies for induction of transplantation tolerance. Transient activation of the small GTPase Rap1 is one of the physiologic consequences of TCR ligation and is mandatory for β1 and β2 integrin-mediated adhesion. In contrast, sustained increase of active Rap1 inhibits T cell activation and IL-2 transcription in vitro. In order to understand the role of Rap1 in the immune responses of the intact host we generated transgenic (Tg) mice, which express the active Rap1 mutant Rap1E63 in T cells. Rap1E63-Tg mice had no defects in thymocyte development or maturation. Rap1E63-Tg thymocytes were capable of activating Ras and Erk1/2 and, compared to wild type (WT) thymocytes, displayed enhanced LFA-1:ICAM-1-mediated adhesion and increased proliferation in response to anti-CD3. Surprisingly, although lymph node and splenic CD4+ cells from the Rap1E63-Tg mice also displayed increased LFA-1:ICAM-1-mediated adhesion, they had significantly impaired activation of Erk1/2 and dramatically reduced proliferation and IL-2 production in response to anti-CD3 and WT antigen presenting cells (APC). The defective responses of CD4+ T cells suggest that Rap1E63-Tg mice may have impaired helper function in vivo. To address this issue we immunized Rap1E63-Tg and WT mice with TNP-OVA, a T-cell dependent antigen. Total IgG, IgG1 and IgG2a were dramatically reduced, indicating that Rap1E63-Tg mice had a defect in immunoglobulin class switching, consistent with defective helper T cell-dependent B cell activation. Because these results suggest that Rap1E63-Tg CD4+ cells may have an anergic phenotype, we tested rechallenge responses. We immunized Rap1E63-Tg and WT mice with TNP-OVA in vivo and subsequently we rechallenged T cells in vitro with WT APC pulsed with OVA. Compared with WT, Rap1E63-Tg T cells had dramatically reduced proliferation, IFN- γ and IL-2 production on rechallenge, findings consistent with T cell anergy. Using suppression subtraction hybridization we determined that Rap1E63 induced mRNA expression of CD103, a marker that defines a potent subset of regulatory T cells (Treg). Strikingly, Rap1E63-Tg mice had a 5-fold increase of CD103+CD25+CD4+ Treg compared to WT mice. Rap1E63-Tg CD103+CD25+CD4+ Treg expressed the highest level of Foxp3 among all T cell subsets and had the most potent inhibitory effect on proliferation and IL-2 production when added into cultures of WT CD4+CD25− cells. Importantly, removal of the CD103+ cells significantly restored Erk1/2 activation, proliferation and IL-2 production of Rap1E63-Tg CD4+ T cells. Generation of CD103+ Treg occurs after thymic development and requires encounter of peripheral autoantigen. Consistent with this, differences in CD103+ Treg were detected only between lymph node and splenic cells and not between thymocytes from Rap1E63-Tg and WT mice. Since generation of CD103+ Treg depends on the strength of TCR signal, these results suggest that by enhancing adhesion, active Rap1 regulates the generation of Treg. Moreover, these results provide evidence that active Rap1 is a potent negative regulator of immune responses in vivo and have significant implications for the development of immune-based therapies geared towards tolerance induction.

scholarly journals MARCH1-mediated MHCII ubiquitination promotes dendritic cell selection of natural regulatory T cells

2013 ◽  
Vol 210 (6) ◽  
pp. 1069-1077 ◽  
Author(s):  
Jaehak Oh ◽  
Nan Wu ◽  
Günther Baravalle ◽  
Benjamin Cohn ◽  
Jessica Ma ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
T Cell Activation ◽  
Cell Activation ◽  
Substantial Reduction ◽  
Surface Expression ◽  
Selection Of

Membrane-associated RING-CH1 (MARCH1) is an E3 ubiquitin ligase that mediates ubiquitination of MHCII in dendritic cells (DCs). MARCH1-mediated MHCII ubiquitination in DCs is known to regulate MHCII surface expression, thereby controlling DC-mediated T cell activation in vitro. However, its role at steady state or in vivo is not clearly understood. Here, we show that MARCH1 deficiency resulted in a substantial reduction in the number of thymus-derived regulatory T cells (T reg cells) in mice. A specific ablation of MHCII ubiquitination also significantly reduced the number of thymic T reg cells. Indeed, DCs deficient in MARCH1 or MHCII ubiquitination both failed to generate antigen-specific T reg cells in vivo and in vitro, although both exhibited an increased capacity for antigen presentation in parallel with the increased surface MHCII. Thus, MARCH1-mediated MHCII ubiquitination in DCs is required for proper production of naturally occurring T reg cells, suggesting a role in balancing immunogenic and regulatory T cell development.

scholarly journals Involvement of GANP in B Cell Activation in T Cell-dependent Antigen Response

2002 ◽  
Vol 9 (3) ◽  
pp. 169-172 ◽  
Author(s):  
Nobuo Sakaguchi ◽  
Satoru Fujimura ◽  
Kazuhiko Kuwahara
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cell Activation ◽  
Molecular Mechanisms ◽  
B Cell Differentiation ◽  
Region Gene

Adaptive immunity is dependent on proliferation of antigen-driven B cells for clonal expansion in germinal centers (GCs) against T cell-dependent antigens (TD-Ag), accompanied with somatic hypermutation of variable-region gene and class switching of B cell antigen receptors. To study molecular mechanisms for B cell differentiation in GCs, we have identified and studied a 210 kDa GANP protein expressed in GC-B cells. GANP has domains for MCM3-binding and RNA-primase activities and is selectively up-regulated in centrocytes surrounded with follicular dendritic cells (FDCs) upon immunization with TD-Agin vivoand in B cells stimulated with anti-CD40 monoclonal antibodyin vitro, which suggested that GANP plays a certain important role in the maturation of immunoglobulin or selection of B cells in GC during the immune response to TD-Ag. Since this up-regulation has not been detected in T cells in GCs and in Concanavalin A-stimulated T cellsin vitro, selective function of GANP molecule on B cell proliferation and differentiation might exist.

scholarly journals CTLA4Ig Improves Murine iTreg Induction via TGFβ and Suppressor Function In Vitro

2018 ◽  
Vol 2018 ◽  
pp. 1-10 ◽  
Author(s):  
Nina Pilat ◽  
Benedikt Mahr ◽  
Martina Gattringer ◽  
Ulrike Baranyi ◽  
Thomas Wekerle
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
T Cell Activation ◽  
Cell Activation ◽  
Functional Characterization ◽  
Immunological Tolerance ◽  
Suppressor Function ◽  
And Function

Blockade of the CD28:CD80/86 costimulatory pathway has been shown to be potent in blocking T cell activation in vitro and in vivo. The costimulation blocker CTLA4Ig has been approved for the treatment of autoimmune diseases and transplant rejection. The therapeutic application of regulatory T cells (Tregs) has recently gained much attention for its potential of improving allograft survival. However, neither costimulation blockade with CTLA4Ig nor Treg therapy induces robust tolerance on its own. Combining CTLA4Ig with Treg therapy would be an attractive approach for minimizing immunosuppression or for possibly achieving tolerance. However, since the CD28 pathway is more complex than initially thought, the question arose whether blocking CD80/86 would inadvertently impact immunological tolerance by interfering with Treg generation and function. We therefore wanted to investigate the compatibility of CTLA4Ig with regulatory T cells by evaluating direct effects of CTLA4Ig on murine Treg generation and function in vitro. For generation of polyclonal-induced Tregs, we utilized an APC-free in vitro system and added titrated doses of CTLA4Ig at different time points. Phenotypical characterization by flow cytometry and functional characterization in suppressor assays did not reveal negative effects by CTLA4Ig. The costimulation blocker CTLA4Ig does not impair but rather improves murine iTreg generation and suppressor function in vitro.

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