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

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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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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Phenotypical and Functional Characteristics of Human Regulatory T Cells during Ex Vivo Maturation from CD4+ T Lymphocytes

Applied Sciences ◽

10.3390/app11135776 ◽

2021 ◽

Vol 11 (13) ◽

pp. 5776

Author(s):

Varvara G. Blinova ◽

Natalia S. Novachly ◽

Sofya N. Gippius ◽

Abdullah Hilal ◽

Yulia A. Gladilina ◽

...

Keyword(s):

T Cells ◽

Regulatory T Cells ◽

Ex Vivo ◽

Pcr Analysis ◽

Mrna Levels ◽

Rt Pcr ◽

Inflammatory Reactions ◽

Effector Cells ◽

Cycle Regulation ◽

Further Development

Regulatory T cells (Tregs) participate in the negative regulation of inflammatory reactions by suppressing effector cells. In a number of autoimmune disorders, the suppressive function and/or the number of Tregs is compromised. The lack of active functioning Tregs can be restored with adoptive transfer of expanded ex vivo autologous Tregs. In our study, we traced the differentiation and maturation of Tregs CD4+CD25+FoxP3+CD127low over 7 days of cultivation from initial CD4+ T cells under ex vivo conditions. The resulting ex vivo expanded cell population (eTregs) demonstrated the immune profile of Tregs with an increased capacity to suppress the proliferation of target effector cells. The expression of the FoxP3 gene was upregulated within the time of expansion and was associated with gradual demethylation in the promotor region of the T cell-specific demethylation region. Real-time RT-PCR analysis revealed changes in the expression profile of genes involved in cell cycle regulation. In addition to FOXP3, the cells displayed elevated mRNA levels of Ikaros zinc finger transcription factors and the main telomerase catalytic subunit hTERT. Alternative splicing of FoxP3, hTERT and IKZF family members was demonstrated to be involved in eTreg maturation. Our data indicate that expanded ex vivo eTregs develop a Treg-specific phenotype and functional suppressive activity. We suggest that eTregs are not just expanded but transformed cells with enhanced capacities of immune suppression. Our findings may influence further development of cell immunosuppressive therapy based on regulatory T cells.

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MiR-146a regulates regulatory T cells to suppress heart transplant rejection in mice

Cell Death Discovery ◽

10.1038/s41420-021-00534-9 ◽

2021 ◽

Vol 7 (1) ◽

Author(s):

Jian Lu ◽

Weiwei Wang ◽

Peiyuan Li ◽

Xiaodong Wang ◽

Chao Gao ◽

...

Keyword(s):

T Cells ◽

Regulatory T Cells ◽

Heart Transplant ◽

Transplant Rejection ◽

Mouse Heart ◽

Allograft Survival ◽

Treg Function ◽

Ifn Γ ◽

Heart Transplant Rejection

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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Rapamycin enriches for CD4+ CD25+ CD27+ Foxp3+ regulatory T cells in ex vivo-expanded CD25-enriched products from healthy donors and patients with multiple sclerosis

Cytotherapy ◽

10.1080/14653240601145223 ◽

2007 ◽

Vol 9 (2) ◽

pp. 144-157 ◽

Cited By ~ 30

Author(s):

Ca Keever-Taylor ◽

Mb Browning ◽

Bd Johnson ◽

Rl Truitt ◽

Cn Bredeson ◽

...

Keyword(s):

Multiple Sclerosis ◽

T Cells ◽

Regulatory T Cells ◽

Ex Vivo ◽

Healthy Donors

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Changes in Chemokine Receptor Expression of Regulatory T Cells After Ex Vivo Culture

Journal of Immunotherapy ◽

10.1097/cji.0b013e318255adcc ◽

2012 ◽

Vol 35 (4) ◽

pp. 329-336 ◽

Cited By ~ 5

Author(s):

Rikhia Chakraborty ◽

Cliona Rooney ◽

Gianpietro Dotti ◽

Barbara Savoldo

Keyword(s):

T Cells ◽

Regulatory T Cells ◽

Chemokine Receptor ◽

Ex Vivo ◽

Receptor Expression ◽

Chemokine Receptor Expression ◽

Ex Vivo Culture

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Transcriptional heterogeneity in cancer-associated regulatory T cells is predictive of survival

10.1101/478628 ◽

2018 ◽

Cited By ~ 2

Author(s):

Nicholas Borcherding ◽

Kawther K. Ahmed ◽

Andrew P. Voigt ◽

Ajaykumar Vishwakarma ◽

Ryan Kolb ◽

...

Keyword(s):

T Cells ◽

Regulatory T Cells ◽

Immune Cells ◽

Ex Vivo ◽

Expression Patterns ◽

Suppressive Effect ◽

Renal Clear Cell Carcinoma ◽

Cell Fates ◽

Treg Subset

Regulatory T cells (Tregs) are a population of T cells that exert a suppressive effect on a variety of immune cells and non-immune cells. The suppressive effects of Tregs are detrimental to anti-tumor immunity. Recent investigations into cancer-associated Tregs have identified common expression patterns for tumor-infiltration, however the functional heterogeneity in tumor-infiltrating (TI) Treg is largely unknown. We performed single-cell sequencing on immune cells derived from renal clear cell carcinoma (ccRCC) patients, isolating 160 peripheral-blood (PB) Tregs and 574 TI Tregs. We identified distinct transcriptional TI Treg cell fates, with a suppressive subset expressing CD177. We demonstrate CD177+ TI-Tregs have preferential suppressive effects in vivo and ex vivo. Gene signatures derived the CD177+ Treg subset had superior ability to predict survival in ccRCC and seven other cancer types. Further investigation into the development and regulation of TI-Treg heterogeneity will be vital to the application of tumor immunotherapies that possess minimal side effects.

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Comparing the Effects of the mTOR Inhibitors Azithromycin and Rapamycin on In Vitro Expanded Regulatory T Cells

Cell Transplantation ◽

10.1177/0963689719872488 ◽

2019 ◽

Vol 28 (12) ◽

pp. 1603-1613 ◽

Cited By ~ 1

Author(s):

Marcus Bergström ◽

Malin Müller ◽

Marie Karlsson ◽

Hanne Scholz ◽

Nils Tore Vethe ◽

...

Keyword(s):

T Cells ◽

Regulatory T Cells ◽

Ex Vivo ◽

Mtor Inhibitors ◽

Allogeneic Transplantation ◽

Suppressive Effect ◽

And Function ◽

Treg Phenotype ◽

Suppression Assay

Adoptive transfer of autologous polyclonal regulatory T cells (Tregs) is a promising option for reducing graft rejection in allogeneic transplantation. To gain therapeutic levels of Tregs there is a need to expand obtained cells ex vivo, usually in the presence of the mTOR inhibitor Rapamycin due to its ability to suppress proliferation of non-Treg T cells, thus promoting a purer Treg yield. Azithromycin is a bacteriostatic macrolide with mTOR inhibitory activity that has been shown to exert immunomodulatory effects on several types of immune cells. In this study we investigated the effects of Azithromycin, compared with Rapamycin, on Treg phenotype, growth, and function when expanding bulk, naïve, and memory Tregs. Furthermore, the intracellular concentration of Rapamycin in CD4+ T cells as well as in the culture medium was measured for up to 48 h after supplemented. Treg phenotype was assessed by flow cytometry and Treg function was measured as inhibition of responder T-cell expansion in a suppression assay. The concentration of Rapamycin was quantified with liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Azithromycin and Rapamycin both promoted a FoxP3-positive Treg phenotype in bulk Tregs, while Rapamycin also increased FoxP3 and FoxP3+Helios positivity in naïve and memory Tregs. Furthermore, Rapamycin inhibited the expansion of naïve Tregs, but also increased their suppressive effect. Rapamycin was quickly degraded in 37°C medium, yet was retained intracellularly. While both compounds may benefit expansion of FoxP3+ Tregs in vitro, further studies elucidating the effects of Azithromycin treatment on Tregs are needed to determine its potential use.

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