Deficient SOCS3 expression in CD4+CD25+FoxP3+ regulatory T cells and SOCS3-mediated suppression of Treg function

AbstractRegulatory T cells (Tregs), which characteristically express forkhead box protein 3 (Foxp3), are essential for the induction of immune tolerance. Here, we investigated microRNA-146a (miR-146a), a miRNA that is widely expressed in Tregs and closely related to their homeostasis and function, with the aim of enhancing the function of Tregs by regulating miR-146a and then suppressing transplant rejection. The effect of the absence of miR-146a on Treg function in the presence or absence of rapamycin was detected in both a mouse heart transplantation model and cell co-cultures in vitro. The absence of miR-146a exerted a mild tissue-protective effect by transiently prolonging allograft survival and reducing the infiltration of CD4+ and CD8+ T cells into the allografts. Meanwhile, the absence of miR-146a increased Treg expansion but impaired the ability of Tregs to restrict T helper cell type 1 (Th1) responses. A miR-146a deficiency combined with interferon (IFN)-γ blockade repaired the impaired Treg function, further prolonged allograft survival, and alleviated rejection. Importantly, miR-146a regulated Tregs mainly through the IFN-γ/signal transducer and activator of transcription (STAT) 1 pathway, which is implicated in Treg function to inhibit Th1 responses. Our data suggest miR-146a controls a specific aspect of Treg function, and modulation of miR-146a may enhance Treg efficacy in alleviating heart transplant rejection in mice.

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Selective expression of latency-associated peptide (LAP) and IL-1 receptor type I/II (CD121a/CD121b) on activated human FOXP3+ regulatory T cells allows for their purification from expansion cultures

Blood ◽

10.1182/blood-2009-01-199950 ◽

2009 ◽

Vol 113 (21) ◽

pp. 5125-5133 ◽

Cited By ~ 127

Author(s):

Dat Q. Tran ◽

John Andersson ◽

Donna Hardwick ◽

Lolita Bebris ◽

Gabor G. Illei ◽

...

Keyword(s):

T Cells ◽

Regulatory T Cells ◽

Ex Vivo ◽

Magnetic Bead ◽

Receptor Type ◽

Type I ◽

Graft Versus Host ◽

Treg Function ◽

Health And Disease ◽

Unique Cell

Abstract Although adoptive transfer of regulatory T cells (Foxp3+ Tregs) has proven to be efficacious in the prevention and treatment of autoimmune diseases and graft-versus-host disease in rodents, a major obstacle for the use of Treg immunotherapy in humans is the difficulty of obtaining a highly purified preparation after ex vivo expansion. We have identified latency-associated peptide (LAP) and IL-1 receptor type I and II (CD121a/CD121b) as unique cell-surface markers that distinguish activated Tregs from activated FOXP3− and FOXP3+ non-Tregs. We show that it is feasible to sort expanded FOXP3+ Tregs from non-Tregs with the use of techniques for magnetic bead cell separation based on expression of these 3 markers. After separation, the final product contains greater than 90% fully functional FOXP3+ Tregs. This novel protocol should facilitate the purification of Tregs for both cell-based therapies as well as detailed studies of human Treg function in health and disease.

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Phenotypic characterisation of regulatory T cells in dogs reveals signature transcripts conserved in humans and mice

Scientific Reports ◽

10.1038/s41598-019-50065-8 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 5

Author(s):

Ying Wu ◽

Yu-Mei Chang ◽

Anneliese J. Stell ◽

Simon L. Priestnall ◽

Eshita Sharma ◽

...

Keyword(s):

T Cells ◽

Regulatory T Cells ◽

Ex Vivo ◽

Treg Function ◽

Phenotypic Characterisation ◽

Transcriptomic Signature ◽

Healthy Dogs ◽

Neoplastic Disorders ◽

Cross Species Comparison ◽

Regulatory Phenotype

Abstract Regulatory T cells (Tregs) are a double-edged regulator of the immune system. Aberrations of Tregs correlate with pathogenesis of inflammatory, autoimmune and neoplastic disorders. Phenotypically and functionally distinct subsets of Tregs have been identified in humans and mice on the basis of their extensive portfolios of monoclonal antibodies (mAb) against Treg surface antigens. As an important veterinary species, dogs are increasingly recognised as an excellent model for many human diseases. However, insightful study of canine Tregs has been restrained by the limited availability of mAb. We therefore set out to characterise CD4+CD25high T cells isolated ex vivo from healthy dogs and showed that they possess a regulatory phenotype, function, and transcriptomic signature that resembles those of human and murine Tregs. By launching a cross-species comparison, we unveiled a conserved transcriptomic signature of Tregs and identified that transcript hip1 may have implications in Treg function.

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208 Epigenetic Regulation of FOXP3+ Regulatory T Cells (Treg) By Small Molecule Targeting of a Single HDAC, Hdac6, Enhances Treg Function and Ameliorates Development of IBD in Murine Models

Gastroenterology ◽

10.1016/s0016-5085(08)60176-6 ◽

2008 ◽

Vol 134 (4) ◽

pp. A-36

Author(s):

Edwin F. deZoeten ◽

James E. Bradner ◽

Ralph Mazitschek ◽

Wayne W. Hancock

Keyword(s):

T Cells ◽

Regulatory T Cells ◽

Small Molecule ◽

Epigenetic Regulation ◽

Murine Models ◽

Treg Function

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A genome-wide CRISPR screen reveals a role for the BRD9-containing non-canonical BAF complex in regulatory T cells

10.1101/2020.02.26.964981 ◽

2020 ◽

Author(s):

Chin-San Loo ◽

Jovylyn Gatchalian ◽

Yuqiong Liang ◽

Mathias Leblanc ◽

Mingjun Xie ◽

...

Keyword(s):

T Cells ◽

Regulatory T Cells ◽

Target Genes ◽

Saga Complex ◽

Baf Complex ◽

Treg Function ◽

Gene Ablation ◽

Genome Wide ◽

A Genome ◽

Novel Target

SummaryRegulatory T cells (Tregs) play a pivotal role in suppressing auto-reactive T cells and maintaining immune homeostasis. Treg development and function are dependent on the transcription factor Foxp3. Here we performed a genome-wide CRISPR/Cas9 knockout screen to identify the regulators of Foxp3 in mouse primary Tregs. The results showed that Foxp3 regulators are highly enriched in genes encoding SWI/SNF and SAGA complex subunits. Among the three SWI/SNF-related complexes, the non-canonical or ncBAF (also called GBAF or BRD9-containing BAF) complex promoted the expression of Foxp3, whereas the PBAF complex repressed its expression. Gene ablation of BRD9 led to compromised Treg function in inflammatory disease and tumor immunity. Functional genomics revealed that BRD9 is required for Foxp3 binding and expression of a subset of Foxp3 target genes. Thus, we provide an unbiased analysis of genes and networks regulating Foxp3, and reveal ncBAF complex as a novel target that could be exploited to manipulate Treg function.

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Reduced Numbers and Impaired Function of Regulatory T Cells in Peripheral Blood of Ischemic Stroke Patients

Mediators of Inflammation ◽

10.1155/2016/2974605 ◽

2016 ◽

Vol 2016 ◽

pp. 1-9 ◽

Cited By ~ 11

Author(s):

Johanna Ruhnau ◽

Juliane Schulze ◽

Bettina von Sarnowski ◽

Marie Heinrich ◽

Sönke Langner ◽

...

Keyword(s):

T Cells ◽

Regulatory T Cells ◽

Peripheral Blood ◽

Experimental Stroke ◽

Stroke Patients ◽

Effector Cells ◽

T Effector Cells ◽

Treg Function ◽

Age Dependent ◽

And Function

Background and Purpose. Regulatory T cells (Tregs) have been suggested to modulate stroke-induced immune responses. However, analyses of Tregs in patients and in experimental stroke have yielded contradictory findings. We performed the current study to assess the regulation and function of Tregs in peripheral blood of stroke patients. Age dependent expression of CD39 on Tregs was quantified in mice and men. Methods. Total FoxP3+ Tregs and CD39+FoxP3+ Tregs were quantified by flow cytometry in controls and stroke patients on admission and on days 1, 3, 5, and 7 thereafter. Treg function was assessed by quantifying the inhibition of activation-induced expression of CD69 and CD154 on T effector cells (Teffs). Results. Total Tregs accounted for 5.0% of CD4+ T cells in controls and <2.8% in stroke patients on admission. They remained below control values until day 7. CD39+ Tregs were most strongly reduced in stroke patients. On day 3 the Treg-mediated inhibition of CD154 upregulation on CD4+ Teff was impaired in stroke patients. CD39 expression on Treg increased with age in peripheral blood of mice and men. Conclusion. We demonstrate a loss of active FoxP3+CD39+ Tregs from stroke patient’s peripheral blood. The suppressive Treg function of remaining Tregs is impaired after stroke.

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Physiologic self antigens rapidly capacitate autoimmune disease-specific polyclonal CD4+CD25+ regulatory T cells

Blood ◽

10.1182/blood-2005-08-3088 ◽

2006 ◽

Vol 107 (3) ◽

pp. 1056-1062 ◽

Cited By ~ 38

Author(s):

Yulius Y. Setiady ◽

Katsuhiro Ohno ◽

Eileen T. Samy ◽

Harini Bagavant ◽

Hui Qiao ◽

...

Keyword(s):

T Cells ◽

Regulatory T Cells ◽

T Cell Receptors ◽

Neonatal Period ◽

Male And Female ◽

Treg Function ◽

Self Tolerance ◽

Disease Specific ◽

Better Than ◽

Striking Contrast

AbstractStudies on CD4+CD25+ regulatory T cells (Tregs) with transgenic T-cell receptors indicate that Tregs may receive continuous antigen (Ag) stimulation in the periphery. However, the consequence of this Ag encounter and its relevance to physiologic polyclonal Treg function are not established. In autoimmune prostatitis (EAP) of the day-3 thymectomized (d3tx) mice, male Tregs suppressed EAP 3 times better than Tregs from female mice or male mice without prostates. Importantly, the superior EAP-suppressing function was acquired after a 6-day exposure to prostate Ag in the periphery, unaffected by sex hormones. Thus, a brief exposure of physiologic prostate Ag capacitates peripheral polyclonal Tregs to suppress EAP. In striking contrast, autoimmune ovarian disease (AOD) was suppressed equally by male and female Tregs. We now provide evidence that the ovarian Ag develops at birth, 14 days earlier than prostate Ag, and that male Tregs respond to neonatal ovarian Ag in the Treg recipients to gain AOD-suppressing capacity. When d3tx female recipients were deprived of ovarian Ag in the neonatal period, AOD was suppressed by female but not by male Tregs, whereas dacryoadenitis was suppressed by both. We conclude that the physiologic autoAg quickly and continuously enhances disease-specific polyclonal Treg function to maintain self-tolerance.

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Loss of CD62L Expression in CD4+FoxP3+ Regulatory T Cells Following Freeze-and-Thaw Prevents Protection Against GVHD in Murine Models

Blood ◽

10.1182/blood.v124.21.2427.2427 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2427-2427

Author(s):

Mareike Florek ◽

Dominik Schneidawind ◽

Jeanette Baker ◽

Antonio Pierini ◽

Dennis B Leveson-Gower ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

Regulatory T Cells ◽

Lethal Dose ◽

Calf Serum ◽

Freezing Process ◽

Murine Models ◽

Freeze And Thaw ◽

Treg Function

Abstract Backgrounds: CD4+FoxP3+ regulatory T cells (Treg) have been shown in pre-clinical murine models and human clinical trials to protect against acute graft-versus-host (GVHD) disease following hematopoietic cell transplantation (HCT). In murine models, Treg expression of CD62L is required for in vivo protection of GVHD (Strober 2005/Blazer 2004). Certain aspects of Treg isolation and administration could affect CD62L expression, such as the use of G-CSF mobilization or freeze-and-thaw. We hypothesized that freeze-thaw of Tregs could reduce CD62L expression and prevent Tregs from protecting against GVHD. Furthermore, we hypothesized that the loss of CD62L expression is mediated at least in part by metalloproteinases (MP). Methods and Results: We used established protocols of GVHD, in which lethal dose of fractionated total body irradiation was followed by murine bone marrow transplantation across major histocompatibility barriers. We sort-purified CD4+Foxp3+ Tregs based on CD25+(high) expression and measured baseline expression of CD62L. Cells were then gradually frozen in fetal calf serum/10%DMSO, and finally stored in liquid nitrogen for at least 7 days. After thawing Tregs showed an average of 58% of the baseline CD62L expression (CD62L expression fresh vs. frozen p=0.0009, n=5). Binding of frozen Tregs to plate bound L-Selectin ligand Mucosal addressin cell adhesion molecule-1 (MAdCAM-1) in vitrowas significantly reduced as compared to fresh Tregs (p=0.0003). To test the potential effect of freeze and thaw in vivo, we used an established murine model in which Tregs are given prior to conventional T cells (Tcon) to protect against GHVD. At the time of transplant conditioned BALB/c donors received 5x10^6 whole bone marrow and 5x10^5 CD4+CD25(high) Tregs (day 0), followed by 1x10^6 Tcon (day +2). Frozen/thawed Tregs and fresh Tregs respectively were given at the same time point at the same quantity of viable cells. Using luciferase-expressing cells (luc+) and BLI imaging we could show that homing of frozen luc+Tregs into pLN at day+5 was impaired as compared to fresh Tregs (p=0.0005). Relative weight loss as an indicator for GVHD was significantly higher and survival was significantly reduced in mice receiving frozen Treg in comparison to mice receiving fresh Treg. To confirm the importance of CD62L expression on Treg function we blocked CD62L in vivo by incubating fresh Tregs with monoclonal blocking antibody Mel14 (120ug/ml) for 1 hour prior to injection. A significantly higher proliferation of luc+ Tcon was observed in mice injected with Mel14-treated Tregs as compared to isotype control-treated Tregs. To test the potential role of metalloproteinases in cleaving CD62L following freeze and thaw, we added various metalloproteinase inhibitors (MPI) during the freezing process. We found that the addition of at least one MPI GM6001 (50nM) can diminish the loss of CD62L. Conclusion: Augmenting or manipulating Tregs of the donor graft is a promising area of cellular therapy that could be used to prevent GVHD in HCT and in potentially many other clinical contexts. We have found that the process of freeze and thaw reduces CD62L expression on Tregs and reduces protection against GVHD. Further, MPs may play role in the loss of CD62L expression and the use of MPIs for frozen products might preserve Treg function. Disclosures No relevant conflicts of interest to declare.

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