Regulatory T cells

Regulatory T cellsRegulatory T cells (Tregs) modulate the immune system, maintaining its homeostasis, tolerance to autoantigens, and limiting exaggerated responses. Deficiencies in their development or function are associated with inflammatory disorders and autoimmune diseases, while overactive cell function contributes to the suppression of tumor immunity and may impact host defense in infectious diseases. They are therefore attractive therapeutic targets in these areas.

Formation

Naturally-derived Tregs can be divided into thymus-derived (tTregs) and peripheral-derived Tregs (pTregs), which form through different pathways.1 iTregs are a further subgroup generated in vitro when FoxP3- T cells are induced to express FoxP3 in the presence of TGFß.2

tTregs

tTregs develop from thymocytes in the thymus. During this process, a pool of CD4+CD8- cells is produced, which further differentiate into Treg and helper T cells. The selection of Tregs in the thymus occurs based on autoreactivity with hematopoietically-derived MHC class II-expressing cells, beginning the transcription of FoxP3 – a key marker for this cell type.1

pTregs

pTregs form in the periphery from conventional T cells, as a result of antigen exposure under both non-inflammatory and inflammatory conditions, leading to the transcription of FoxP3. It is now becoming clear that pTregs arise in various conditions and could make up a large proportion of Tregs in the periphery.3

Cell function

Regulatory T cells suppress activation, proliferation and cytokine production of helper T cells and cytotoxic T cells, as well as modulating the activity of other immune cells, such as dendritic cells and B cells. It has been shown that mutations in the Treg marker gene, FoxP3, impair Treg function and cause severe autoimmunity, including immune dysregulation, polyendocrinopathy, enteropathy, and X-linked (IPEX) syndrome in mice and humans.4,5

Tregs have several mechanisms which they use to mediate their suppressor activity. These include5:

  • production of inhibitory cytokines, including TGF-ß, IL-35 and IL-10;6
  • generation of extracellular adenosine, a known suppressor of pro-inflammatory activities, through expression of CD39 and CD73;7
  • induction of cytolysis of effector cells through the release of granzyme and perforin;8
  • sequestering IL-2 from the microenvironment by the high expression of CD25, reducing effector T cells proliferation;9
  • regulation of innate immune cells, including dendritic cells, macrophages and neutrophils.10

Emerging research

The therapeutic potential of Tregs is an area of significant interest, and multiple polyclonal Treg­based cell therapies have been used in clinical trials, showing promise for treating both autoimmune diseases and transplant rejection.11 However, the number of cells needed for infusions is often large and there is a risk of non-specific immunosuppression. Recently, chimeric antigen receptor (CAR) technology – which was initially used to generate antigen-specific CAR-T cells for treatment of hematologic malignancies – has been applied to Treg cells, to produce CAR-Treg cells for the target antigen-guided treatment of allograft rejection and various autoimmune diseases.12,13

Tools to study regulatory T cells

Identification and isolation of Tregs

Tregs make up a small proportion of CD4+ T cells in human peripheral blood, and therefore must often be enriched or isolated to be studied. This can be achieved by sorting based on specific cell surface markers, outlined in the table below.

When identifying Treg cells it can be complicated by the fact that other T cell types can transiently express Treg markers at low levels, following activation of the TCR. The methylation status of the Treg-specific demethylated region (TSDR), a conserved non-coding element within the FoxP3 gene locus, can be used for the identification of ’real‘ stable human Tregs.14

Treg suppression assays

Treg suppression assays can be carried out by flow cytometry. They are used to study the effects of Tregs on effector cells, and to assess whether potential therapies can inhibit or promote Treg function. In the assay, Tregs (either iTregs or those isolated from tissue) are co-cultured with effector cells, and functional readouts of the responder cells – such as inhibition of proliferation and inhibition of cytokine production – are performed at the end of the culture period.

Cell markers

tTreg cells are characterized by expression of CD4, CD25 and FoxP3. Lack of surface CD127 (α-chain of IL-7R) can also be used to further improve characterization of Treg cells and, if applied without intracellular FoxP3 staining, to allow for sorting of living Tregs.15 CD39 can be used for isolation of functionally-active Treg cells.16 The transcription factor Helios is expressed in a large subset of FoxP3+ Tregs, and is associated with a more activated phenotype and a slightly higher suppressive capacity.17

Surface marker Alternative names Location
CD3 T3 Surface
CD4 T4 Surface
CD25 Interleukin-2 receptor subunit alpha, IL2RA, TAC antigen, TCGFR Surface
CD39 NTPDase 1, TPDase Surface
CD45RA GP180 Surface
CD49d VLA-4 α chain Surface
CD73 5'-nucleotidase, NT5E, E5NT Surface
CD152 CTLA-4 Surface
CD161 NKR-P1A Surface
CD185 CXCR5 Surface
CD223 LAG-3 Surface
CD357 TNFRSF18, AITR, GITR Surface
CD366 TIM-3 Surface
FoxP3 Forkhead box P3 Nucleus
Helios IKZF2 Nucleus

References

1. Abbas A, Benoist C, et al (2013) Regulatory T cells: recommendations to simplify the nomenclature. Nat Immunol. 14:307–8. doi:10.1038/ni.2554

2. Shevach E and Thornton A (2014), tTregs, pTregs, and iTregs: similarities and differences. Immunol Rev. 259:88-102. doi:10.1111/imr.12160

3. Yadav M, Bluestone J, et al (2013) Peripherally Induced Tregs – Role in Immune Homeostasis and Autoimmunity. Front. Immunol. 4:232. doi:10.3389/fimmu.2013.00232

4. Bennett C, Christie J, et al (2001) The immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is caused by mutations of FOXP3. Nat Genet. 27:20–1. doi:10.1038/83713

5. Brunkow ME, Jeffery EW, et al (2001) Disruption of a new forkhead/winged-helix protein, scurfin, results in the fatal lymphoproliferative disorder of the scurfy mouse. Nat Genet. 27:68–73. doi:10.1038/83784

6. Roamno M, Fanelli G, et al (2019) Past, Present, and Future of Regulatory T Cell Therapy in Transplantation and Autoimmunity. Front. Immunol. 10:43. doi:10.3389/fimmu.2019.00043

7. Ohato A, Sitkovsky M (2014) Extracellular Adenosine-Mediated Modulation of Regulatory T Cells. Front. Immunol. 5:304. doi:10.3389/fimmu.2014.00304

8. Vignali A, Collison L, et al (2008) How regulatory T cells work. Nat Rev Immunol 8:523–532. doi:10.1038/nri2343

9. Sojka D, Huang Y-H, et al (2008) Mechanisms of regulatory T-cell suppression – a diverse arsenal for a moving target. Immunology 124:13–22. doi:10.1111/j.1365-2567.2008.02813.x

10. Okeke E, Uzonna J (2019) The Pivotal Role of Regulatory T Cells in the Regulation of Innate Immune Cells. Front. Immunol. 10:680. doi:10.3389/fimmu.2019.00680

11. Biswas M, Kumar SRP, et al (2018) Gene Therapy With Regulatory T Cells: a Beneficial Alliance. Front Immunol. 9:554. doi:10.3389/fimmu.2018.00554

12. Zhang Q, Lu W, et al (2018) Chimeric Antigen Receptor (CAR) Treg: A Promising Approach to Inducing Immunological Tolerance Front Immunol. 9:2359. doi:10.3389/fimmu.2018.00554

13. Mohseni R, Tung S, et al (2020) The Future of Regulatory T Cell Therapy: Promises and Challenges of Implementing CAR Technology. Front Immunol. 11:1608. doi:10.3389/fimmu.2020.01608

14. Polansky JK, Kretschmer K, et al (2008) DNA methylation controls Foxp3 gene expression. Eur J Immunol. 38:1654–63. doi:10.1002/eji.200838105

15. Liu W, Putnam AL, et al (2006) CD127 expression inversely correlates with FoxP3 and suppressive function of human CD4+ T reg cells. J Exp Med. 203:1701–11. doi:10.1084/jem.20060772

16. Mandapathil, M, Lang, S, et al (2009). Isolation of functional human regulatory T cells (Treg) from the peripheral blood based on the CD39 expression. Journal of immunological methods, 346(1-2): 55–63. doi:10.1016/j.jim.2009.05.004

17. Thornton A, Lu J, et al (2019) Helios + and Helios - Treg subpopulations are phenotypically and functionally distinct and express dissimilar TCR repertoires. Eur J Immunol, 49(3):398-412. doi:10.1002/eji.201847935

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