ABSTRACT
Title
CD4+CD25lowGITR+ cells: a novel human CD4+ regulatory T cell subset disregulated in autoimmune diseases.
Authors
M.G. Petrillo1, A. Alunno2, R. Bianchini1, O. Bistoni2, S. Ronchetti1, G. Nocentini1, R. Gerli2 and C. Riccardi1
1Dept. Of Clinical and Experimental Medicine, Section of Pharmacology, Toxicology and Chemotherapy, University of Perugia, Italy
2Dept. Of Clinical and Experimental Medicine, Rheumatology Unit, University of Perugia, Italy
1Dept. Of Clinical and Experimental Medicine, Section of Pharmacology, Toxicology and Chemotherapy, University of Perugia, Italy
2Dept. Of Clinical and Experimental Medicine, Rheumatology Unit, University of Perugia, Italy
Abstract
CD4+ T regulatory cells (Tregs) are specialized T cell subsets able to suppress immune responses. Their impairment is involved in the pathogenesis of many autoimmune disorders and the modulation of their activity may result in amelioration of these diseases.
A number of different markers are employed to identify Tregs, including the T cell surface markers CD25 (IL-2R) and CD127 (IL-7R), expressed at high levels and at low levels, respectively. A recently characterised intracellular marker of Tregs is the forkhead wing helix box P3 (FoxP3) transcription factor, which is considered a regulator of Treg phenotype. Other additional markers are Cytotoxic T-Lymphocyte Antigen (CTLA)-4, Transforming growth factor(TGF)-beta and Interleukin (IL)-10. Some molecules belonging to the Tumor Necrosis Factor Receptor superfamily (TNFRSF), deeply involved in activation, differentiation and survival of cells of the immune system, are over-expressed on Tregs. Among them, glucocorticoid-induced TNF receptor family-related (GITR) protein is expressed at high levels in Tregs and also in several other cell types of the immune system in which it regulates their activity when triggered by its ligand (GITRL) (Nocentini et al. 1997, Ronchetti et al. 2004).
GITR is expressed on naïve murine T cells, up-regulated for several days following T cell activation, plays a co-stimulatory role and is considered a marker of murine Tregs (Shimizu et al. 2002). Its expression correlates with CD25, even if murine CD25-GITR+ cells with Treg activity have been described.
Despite human GITR has not been deeply investigated, preliminary studies suggest it plays a role similar to murine GITR in effector T cells activation, but its role has still not been defined in Treg subsets.
In our studies, we found that a small subset of peripheral blood CD4+ T lymphocytes (0.32-1.74% of CD4+ T lymphocytes) is characterized by expression of GITR together with low expression of CD25 (CD4+CD25lowGITR+).
To characterize these cells, peripheral blood samples were obtained from a total of 40 healthy donors. CD4+ T lymphocytes were isolated from Ficoll/Hypaque-purified PBMC using a CD4+ T Cell Isolation Kit (Miltenyi). For isolation of CD4+ subpopulations, positive and negative selection strategies were used. Cell phenotype was analysed by real-time PCR, ELISPOT and flow-cytometry. Results suggested that CD4+CD25lowGITR+ cells represent a new Treg subset since they express FoxP3, IL-10, TGF-beta, are anergic and have a prevalent memory phenotype. However, unlike classical Tregs, they do not express CTLA-4 and express high levels of CD127.
Functional data were obtained by flow-cytometric evaluation of CFSE-labeled anti-CD3 triggered cells. Our findings demonstrate that, although peripheral CD4+CD25lowGITR+ T cell subset represents a small percentage of the entire circulating CD4+ T cell population, it shows a regulatory activity on TCR/CD3-driven proliferation of effector T cells which is inhibited by the presence of anti-TGF-beta and anti-IL-10 neutralizing antibodies. However, when an antagonistic anti-GITR antibody is added, regulatory activity of CD4+CD25lowGITR+ T cell is lost, suggesting that this antibody might be used in treating disease where Treg activity overcome effector activity, such as tumors or autoimmunity diseases.
To evaluate whether CD4+CD25lowGITR+ T cell subset participate in the pathogenesis of autoimmune disorders, we studied 25 SLE patients and demonstrated that half of them have an increased number of peripheral blood CD4+CD25lowGITR+ cells.
Additional studies are required to further examine the functional role of CD4+CD25lowGITR+ cells in vivo and to clarify their specific role in protection and/or progression of chronic autoimmune/inflammatorydisorders (Gerli et al. 2009).
Nocentini G et al. (1997) P Natl Acad Sci USA 94:6216
Ronchetti S et al. (2004) Eur. J Immunol 34(3):613
Shimizu J et al. (2002) Nat Immunol 3:135
Gerli R et al. (2009) Autoimmun Rev 8(5):426
A number of different markers are employed to identify Tregs, including the T cell surface markers CD25 (IL-2R) and CD127 (IL-7R), expressed at high levels and at low levels, respectively. A recently characterised intracellular marker of Tregs is the forkhead wing helix box P3 (FoxP3) transcription factor, which is considered a regulator of Treg phenotype. Other additional markers are Cytotoxic T-Lymphocyte Antigen (CTLA)-4, Transforming growth factor(TGF)-beta and Interleukin (IL)-10. Some molecules belonging to the Tumor Necrosis Factor Receptor superfamily (TNFRSF), deeply involved in activation, differentiation and survival of cells of the immune system, are over-expressed on Tregs. Among them, glucocorticoid-induced TNF receptor family-related (GITR) protein is expressed at high levels in Tregs and also in several other cell types of the immune system in which it regulates their activity when triggered by its ligand (GITRL) (Nocentini et al. 1997, Ronchetti et al. 2004).
GITR is expressed on naïve murine T cells, up-regulated for several days following T cell activation, plays a co-stimulatory role and is considered a marker of murine Tregs (Shimizu et al. 2002). Its expression correlates with CD25, even if murine CD25-GITR+ cells with Treg activity have been described.
Despite human GITR has not been deeply investigated, preliminary studies suggest it plays a role similar to murine GITR in effector T cells activation, but its role has still not been defined in Treg subsets.
In our studies, we found that a small subset of peripheral blood CD4+ T lymphocytes (0.32-1.74% of CD4+ T lymphocytes) is characterized by expression of GITR together with low expression of CD25 (CD4+CD25lowGITR+).
To characterize these cells, peripheral blood samples were obtained from a total of 40 healthy donors. CD4+ T lymphocytes were isolated from Ficoll/Hypaque-purified PBMC using a CD4+ T Cell Isolation Kit (Miltenyi). For isolation of CD4+ subpopulations, positive and negative selection strategies were used. Cell phenotype was analysed by real-time PCR, ELISPOT and flow-cytometry. Results suggested that CD4+CD25lowGITR+ cells represent a new Treg subset since they express FoxP3, IL-10, TGF-beta, are anergic and have a prevalent memory phenotype. However, unlike classical Tregs, they do not express CTLA-4 and express high levels of CD127.
Functional data were obtained by flow-cytometric evaluation of CFSE-labeled anti-CD3 triggered cells. Our findings demonstrate that, although peripheral CD4+CD25lowGITR+ T cell subset represents a small percentage of the entire circulating CD4+ T cell population, it shows a regulatory activity on TCR/CD3-driven proliferation of effector T cells which is inhibited by the presence of anti-TGF-beta and anti-IL-10 neutralizing antibodies. However, when an antagonistic anti-GITR antibody is added, regulatory activity of CD4+CD25lowGITR+ T cell is lost, suggesting that this antibody might be used in treating disease where Treg activity overcome effector activity, such as tumors or autoimmunity diseases.
To evaluate whether CD4+CD25lowGITR+ T cell subset participate in the pathogenesis of autoimmune disorders, we studied 25 SLE patients and demonstrated that half of them have an increased number of peripheral blood CD4+CD25lowGITR+ cells.
Additional studies are required to further examine the functional role of CD4+CD25lowGITR+ cells in vivo and to clarify their specific role in protection and/or progression of chronic autoimmune/inflammatorydisorders (Gerli et al. 2009).
Nocentini G et al. (1997) P Natl Acad Sci USA 94:6216
Ronchetti S et al. (2004) Eur. J Immunol 34(3):613
Shimizu J et al. (2002) Nat Immunol 3:135
Gerli R et al. (2009) Autoimmun Rev 8(5):426