Soluble glucocorticoid-induced tumor necrosis factor receptor regulates Helios expression in myasthenia gravis.

BACKGROUND: Helios is important for functional and phenotype stability of regulatory T cells (Tregs). However, the role of Helios in autoimmune diseases and its regulation remains unclear. This study aimed to investigate the role of Helios+ Tregs in myasthenia gravis (MG) and glucocorticoid-induced tumor necrosis factor receptor (GITR) and its ligand (GITRL) in the modulation of Helios. METHOD: Multicolor flow cytometry was performed to analyze Helios+ Tregs in peripheral blood from MG patients and healthy donors (HDs). Enzyme-linked immunosorbent assay (ELISA) was used to determine the levels of soluble GITRL/GITR in plasma. Tregs were isolated via magnetic separation and treated with recombinant GITRL and GITR-Fc. Membrane GITRL on Tregs and expression of Helios and other markers (FOXP3, CD25, CD39, CTLA-4, PD-L1 and IL-10) involved in immunosuppressive activity were determined by flow cytometry. RESULT: Both Helios+ Tregs and soluble GITR were decreased in generalized MG (GMG) patients (n = 14), compared with HDs (n = 14) and ocular MG (OMG) patients (n = 16). Helios+ Tregs possessed greater immunosuppressive capacity compared to Helios- Tregs. Further analysis indicates soluble GITR was negatively correlated with quantitative MG score and promoted Helios expression and enhanced function of Tregs independently of membrane GITRL. CONCLUSION: This work demonstrates abnormal changes in Helios+ Tregs and soluble GITR in MG, as well as direct regulation of Helios by GITR in the context of Tregs. This work provides new insight into the role of GITR in the regulatory pathway of Helios and pathogenesis of MG.

Correlation of increased blood levels of GITR and GITRL with disease severity in patients with primary Sjögren's syndrome.

Glucocorticoid-induced tumor necrosis factor receptor family-related protein (GITR) is a type I transmembrane protein belonging to the TNFR superfamily. After activated by its ligand GITRL, GITR could influence the activity of effector and regulatory T cells, participating in the development of several autoimmune and inflammatory diseases included rheumatoid arthritis and autoimmune thyroid disease. We previously reported that serum GITRL levels are increased in systemic lupus erythematosus (SLE) patients compared with healthy controls (HC). Here, we tested serum soluble GITR (sGITR) and GITRL levels in 41 primary Sjögren's syndrome (pSS) patients and 29 HC by ELISA and correlated sGITR and GITRL levels with clinical and laboratory variables. GITR and GITRL expression in labial salivary glands was detected by immunohistochemistry. pSS patients had significantly increased serum levels of sGITR and GITRL compared with controls (GITR: 5.66 ± 3.56 ng/mL versus 0.50 ± 0.31 ng/mL; P < 0.0001; GITRL: 6.17 ± 7.10 ng/mL versus 0.36 ± 0.28 ng/mL; P < 0.0001). Serum sGITR and GITRL levels were positively correlated with IgG (GITRL: r = 0.6084, P < 0.0001; sGITR: r = 0.6820, P < 0.0001) and ESR (GITRL: r = 0.8315, P < 0.0001; sGITR: r = 0.7448, P < 0.0001). Moreover, GITR and GITRL are readily detected in the lymphocytic foci and periductal areas of the LSGs. In contrast, the LSGs of HC subjects did not express GITR or GITRL. Our findings indicate the possible involvement of GITR-GITRL pathway in the pathogenesis of pSS. Further studies may facilitate the development of targeting this molecule pathway for the treatment of pSS.

Correlation of circulating glucocorticoid-induced TNFR-related protein ligand levels with disease activity in patients with systemic lupus erythematosus.

The aim of this paper is to investigate the correlation of glucocorticoid-induced tumor necrosis factor receptor- (TNFR-) related protein ligand (GITRL) with disease activity and organ involvement in patients with systemic lupus erythematosus (SLE). Serum GITRL levels were measured in 58 patients with SLE and 30 healthy controls matched for age and sex. Patients were assessed for clinical and laboratory variables. Correlations of serum GITRL levels with SLEDAI, laboratory values, and clinical manifestations were assessed. Serum GITRL levels were determined by ELISA. Serum GITRL levels were markedly increased in patients with SLE compared with healthy controls (mean 401.3 ng/mL and 36.59 ng/mL, resp.; P < 0.0001). SLE patients with active disease showed higher serum GITRL levels compared to those with inactive disease (mean 403.3 ng/mL and 136.3 ng/mL, resp; P = 0.0043) as well as normal controls (36.59 ng/mL; P < 0.0001). Serum GITRL levels were positively correlated with SLEDAI, titers of anti-dsDNA antibody, erythrocyte sedimentation rate (ESR), and IgM and negatively correlated with complement3 (C3). Serum GITRL levels were higher in SLE patients with renal involvement and vasculitis compared with patients without the above-mentioned manifestations.

Phenotypic characterization of ex vivo CD4+CD25highCD127low immune regulatory T cells in allergic asthma: pathogenesis relevance of their FoxP3, GITR, CTLA-4 and FAS expressions.

BACKGROUND: CD4+CD25high regulatory T (Treg) cells are crucial for immune homeostasis and peripheral tolerance, but their relevance to allergic asthma has not been fully elucidated. OBJECTIVE: To assess peripheral blood CD4+ T cells, and CD4+CD25highCD127low Treg cells expressing phenotypic markers (FoxP3, GITR, CTLA-4, and FAS) in allergic asthma subjects. MATERIALS AND METHODS: Peripheral blood mononuclear cells were isolated from 60 allergic asthma (AA) subjects and 30 healthy controls (HC). We examined by flow cytometry, the proportion of CD4+ T cells and CD4+CD25highCD127low Treg cells as well as the expression of FoxP3, GITR, CTLA-4, and FAS by CD4+CD25highCD127low Treg cells. Moreover, FOXP3 mRNA expression was measured by quantitative real time polymerase chain reaction (real-time RT-PCR). RESULTS: The absolute number of CD4+CD25highCD127low Treg cells and the percentages of CD4+CD25highCD127low Treg cells expressing one of the four selected markers were significantly lower in allergic asthma subjects compared with controls. We observed no significant decreased absolute CD4+ T cell count in the examined groups compared to the control group. Except for GITR, circulatory CD4+CD25highCD127low Treg cells of severe allergic asthma (SA) subjects showed significantly lower expressions of FoxP3, CTLA-4, and CD95 than did those isolated from mild to moderate asthma (MA) patients. There was no statistically significant difference in the level of mRNA FoxP3 expression in CD4+CD25+ Treg cells between allergic asthma subjects and healthy controls groups, and within the examined groups (p>0.05). CONCLUSIONS: These findings suggest that allergic asthma and the use of glucocorticosteroids are associated with decreased absolute number of circulatory CD4+CD25highCD127low Treg cells and the decreased frequencies of CD4+CD25highCD127low Treg cells expressing one of the four selected markers.

Abrogation of Treg function deteriorates rheumatoid arthritis.

An early prognostic indicator which warns of progressive joint destruction of rheumatoid arthritis (RA) was explored using a novel suspension-array technique in moderate (Steinbrocker stage I and II) and severe (Steinbrocker stage IV) RA patients. DNA microarray analysis of peripheral blood lymphocytes showed significant increase of interleukin (IL)-2 receptor α-chain (CD25) gene expression, a regulatory T cell (Treg) surface marker in severe RA patients. In contrast, suspension array, a comprehensive bead-based enzyme-linked immunosorbent assay (ELISA), revealed decreased production of IL-10 and increased production of interferon (IFN)-γ in sera in the incipient stage of the aggressive disease process. Both in moderate and in severe RA patients, the IFN-γ/IL-10 ratio indicated deterioration of the disease with universal validity. Fluorescence-activated cell sorting (FACS) and reverse-transcription polymerase chain reaction (RT-PCR) analysis showed extant CD4+CD25+ regulatory T cells in severe RA patients, however Foxp3, a regulatory T cell-specific transcription factor, gene expression was absent, while glucocorticoid-induced tumor necrosis factor (TNF) receptor family-related protein (GITR), which transmits a signal that abrogates regulatory T cell functions, was elevated. In the current study, we showed the validity of suspension-array analysis for enabling more complete understanding of RA, and showed that IFN-γ/IL-10 ratio can be a prognostic tool for early lesion and more aggressive RA.

Identification of Chicken GITR and GITR Ligand, Proof of Their Mutual Interaction, and Analysis of Chicken GITR Tissue Distribution by a Novel Antibody That Reveals Expression on Activated T Cells and Erythrocytes.

Glucocorticoid-induced TNFR (GITR) and its ligand, GITRL, belong to the costimulatory members of the TNF superfamily and are crucially involved in the formation and modulation of an effective immune response, comprising innate as well as adaptive mechanisms. In this study, we identify and describe chicken GITR and GITRL, and provide an initial characterization of the newly developed chGITR-specific mAb 9C5. Structural analyses of the putative chicken molecules GITR and GITRL confirmed the conservation of classic topological features compared with their mammalian homologs and suggested the ability of mutual interaction, which was verified via flow cytometry. Whereas only minute populations of native lymphocytes isolated from spleen, bursa, and thymus expressed GITR, it was strongly upregulated upon activation on αß and γδ T cells, comprising CD4+ as well as CD8+ subsets. In blood, a fraction of CD4+CD25+ T cells constitutively expressed GITR. In addition, virtually all chicken erythrocytes displayed high levels of GITR. Our results verify the existence of both GITR and its ligand, GITRL, in chickens; they provide the basis and novel tools to further characterize their impact within the immune response and reveal the so-far unrecognized expression of GITR on erythrocytes.

T regulatory markers expression in unexplained recurrent spontaneous abortion.

OBJECTIVE: To evaluate expression of glucocorticoid-induced tumor necrosis factor receptor (GITR), cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and IL-10 in peripheral blood mononuclear cells (PBMCs) of 20 women with unexplained recurrent spontaneous abortion (URSA) compared to 20 normal non-pregnant women (NNP) during luteal phase in the window of implantation. METHODS: Quantitative real-time PCR (qRT-PCR) was performed using the Taqman method for expression of GITR and SYBR Green method for expression of CTLA-4 and IL-10. RESULTS: Expression of CTLA-4 in the NNPs (median; interquartile range; 3; 1.8-10) was significantly higher than the URSAs (0.72; 0.26-3.81, p = 0.015). Expression of GITR in the NNPs (53; 10-139) was significantly higher than the URSAs (6; 3-27, p = 0.005). However, IL-10 expression in the URSAs was significantly higher than the NNPs, did not meet a significant value. A significant correlation was found between CTLA-4 and GITR expression in the study population (p = 0.0001). CONCLUSIONS: Expression of CTLA-4 and GITR were significantly down-regulated in the URSAs compared to NNPs at the window of implantation, which shows the essential role of Treg cells in creating an immunological privileged site for fetus as an allograft at the maternal-fetal interface by high expression levels of CTLA-4 and GITR during a normal pregnancy.

In vivo fluctuation of Tax, Foxp3, CTLA-4, and GITR mRNA expression in CD4(+)CD25(+) T cells of patients with human T-lymphotropic virus type 1-associated myelopathy.

HTLV-1 Tax expression exerts an inhibitory effect on the Foxp3 transcription factor in CD4(+)CD25(+) T-regulatory cells (Treg). For a better understanding of the role of Tax mRNA in the gene expression of cellular markers we measured Tax, Foxp3, CTLA-4, GITR, TGF-ß, and IL-10 mRNA in Treg cells of 50 patients with human T-lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP; 27 women and 23 men; mean age: 56.7 years). The control group consisted of 23 non-infected subjects (12 women and 11 men) with a mean age of 51.3 years. Real-time PCR was used to measure mRNA of Tax proteins and several cellular markers of Treg function. Determinations revealed a high level of Tax mRNA in HAM/TSP (124.35 copies/100 CD4(+)CD25(+) T cells). Foxp3, GITR, and CTLA-4 mRNA levels were lower in HAM/TSP patients (mean ± SD, 22.07 ± 0.78, 9.63 ± 0.36, and 4.54 ± 0.39, respectively) than in non-infected controls (47.15 ± 12.94, 22.14 ± 1.91, and 21.07 ± 2.31). Both groups had similar levels of TGF-ß and IL-10. An inverse relationship was found between Tax levels and Foxp3, CTLA-4, and GITR levels. Conversely, there was a direct correlation between levels of Foxp3, GITR, and CTLA-4. Disease severity and evolution time did not correlate with Tax or Foxp3 levels. The present results suggest that Tax and Foxp3 mRNA vary with the same degree of disease severity in HAM/TSP patients. Tax fluctuations may affect CTLA-4 and GITR expression via the Foxp3 pathway, causing virus-induced dysfunction of CD4(+)CD25(+) T cells in HAM/TSP patients.

In vivo fluctuation of Tax, Foxp3, CTLA-4, and GITR mRNA expression in CD4+CD25+ T cells of patients with human T-lymphotropic virus type 1-associated myelopathy

HTLV-1 Tax expression exerts an inhibitory effect on the Foxp3 transcription factor in CD4+CD25+ T-regulatory cells (Treg). For a better understanding of the role of Tax mRNA in the gene expression of cellular markers we measured Tax, Foxp3, CTLA-4, GITR, TGF-β, and IL-10 mRNA in Treg cells of 50 patients with human T-lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP; 27 women and 23 men; mean age: 56.7 years). The control group consisted of 23 non-infected subjects (12 women and 11 men) with a mean age of 51.3 years. Real-time PCR was used to measure mRNA of Tax proteins and several cellular markers of Treg function. Determinations revealed a high level of Tax mRNA in HAM/TSP (124.35 copies/100 CD4+CD25+ T cells). Foxp3, GITR, and CTLA-4 mRNA levels were lower in HAM/TSP patients (mean ± SD, 22.07 ± 0.78, 9.63 ± 0.36, and 4.54 ± 0.39, respectively) than in non-infected controls (47.15 ± 12.94, 22.14 ± 1.91, and 21.07 ± 2.31). Both groups had similar levels of TGF-β and IL-10. An inverse relationship was found between Tax levels and Foxp3, CTLA-4, and GITR levels. Conversely, there was a direct correlation between levels of Foxp3, GITR, and CTLA-4. Disease severity and evolution time did not correlate with Tax or Foxp3 levels. The present results suggest that Tax and Foxp3 mRNA vary with the same degree of disease severity in HAM/TSP patients. Tax fluctuations may affect CTLA-4 and GITR expression via the Foxp3 pathway, causing virus-induced dysfunction of CD4+CD25+ T cells in HAM/TSP patients.