| Background:T follicular helper (Tfh) cell is a T cell subset, which provides help for B cell activation and antibody secretion. There are high levels of autoantibodies in the serum of autoimmune disease patients, suggesting that autoreactive B cell might obtain excess help from uncontrolled Tfh cell. Mesenchymal stem cells (MSCs) are multipotential nonhematopoietic progenitor cells, which are capable of differentiating into a wide range of cell types, including osteocytes and chondrocytes. Besides, MSCs have immunoregulatory capacity.Objective:The objective of this study is to observe whether the quantity of Tfh cell is abnormal in rheumatoid arthritis (RA) and primary Sjogren’s syndrome (pSS) patients, whether it is correlated with disease. activity and whether umbilical cord-mesenchymal stem cells (UC-MSCs) are able to modulate Tfh cell and underlying mechanism.Methods:Peripheral blood mononuclear cells (PBMCs) were isolated from RA, pSS patients and healthy controls (HC), respectively. The frequency of CD4+CXCR5+PD-1+T cell was examined by flow cytometry. The serum level of IL-21was tested by enzyme linked immunosorbent assay (ELISA). PBMCs from RA patients and HC were stimulated with recombinant human IL-21and the frequency of Tfh cell was examined by flow cytometry. UC-MSCs were isolated from human umbilical cord and were cocultured with PBMCs through cell-to-cell contact or in a transwell system. Purified Naive T cells from peripheral blood of RA, pSS patients and HC were cocultured with UC-MSCs under Tfh cell-polarizing condition. Carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled purified CD4+T cells from peripheral blood were cocultured with UC-MSCs. Tfh cell proliferation and apoptosis were deterrmined-by flow cytometry. The level of IL-21in the supernatant of coculture system was detected by Luminex. Purified naive T cells from RA and pSS patients were cocultured with UC-MSCs under Tfh cell-polarizing condition. Then, UC-MSCs were collected and fixed by Trizol. The mRNA levels of indoleamine2,3-dioxygenase (IDO), IL-10, cyclooxygenase2(COX2)/prostaglandin E2(PGE2), hepatocyte growth factor (HGF), transforming growth factor-β (TGF-β) and human leukocyte antigen-G (HLA-G) in UC-MSCs were tested by RT-PCR. The activity of IDO was measured by high performance liquid chromatography (HPLC). The IL-10level was detected by Luminex. IDO inhibitor1-MT or anti-IL-10antibody was added into UC-MSCs and Tfh cell differentiation coculture system and then the frequency of Tfh cell was examined by flow cytometry. The level of IFN-y in the supernatant was detected by Luminex. Anti-IFN-γR antibody was added into coculture system of UC-MSCs and Tfh cell differentiation.UC-MSCs were fixed by Trziol. Erk, Akt, Stat-1/3/5and NF-κB pathways in UC-MSCs that cocultured with Naive T cells under Tfh cell-polarizing condition were tested by Western blot. DBA/1J mice were injected at the base of the tail with emulsified bovine type Ⅱ collagen. On day21, the mice received a booster injection. Treatment was begun when arthritis score>1(Day28). Mice were injected intravenously with1×106human UC-MSCs and killed on Day62. Serum anti-CII antibody level was examined by ELISA. Inflammatory cell infiltration and joint space in the ankle was observed by HE staining. The frequencies of Th1,Th2, Th17, Tfh and Treg cells in the spleen were tested by flow cytometry. Purified CD4+CXCR5+T cells, which were isolated from collagen-induced arthritis (CIA) mice with or without treatment, were cocultured with B220+B cell from normal mice. Then, the frequency of plasma cells was determined by flow cytometry and the levels of IgG and IgM were determined by ELISA. Purified CD4+T cells from CIA mice were cocultured with UC-MSCs and then UC-MSCs were collected and fixed by Trizol. IDO, IL-10, COX2/PGE2, HGF, TGF-β, HLA-G and inducible NO synthase (iNOS) mRNA levels in UC-MSCs were tested by RT-PCR.Results:The frequency of peripheral Tfh cell was markedly increased in RA and pSS patients. The percentages of Tfh cells were positively correlated with DAS28and anti-CCP antibody in high Tfh cell group in RA. Furthermore, the serum IL-21level in RA patients was significantly increased than that of HC. Consistantly, IL-21concentration was positively correlated with DAS28and anti-CCP antibody in RA patients with high IL-21level. Moreover, elevated IL-21level was also positively correlated with the frequency of Tfh cells. In vitro, IL-21was able to upregulate the frequency of Tfh cells in RA patients. The frequency of Tfh cells in pSS patients was positively correlated with serum anti-La/SSB level and the SS disease activity index (SSDAI) score. UC-MSCs were able to suppress the generation of Tfh cells in RA, pSS patients and HC. This suppression was dose-dependent and independent of cell-to-cell contact. UC-MSCs inhibited the differentiation and proliferation of Tfh cells in RA, pSS patients and HC but had no effect on Tfh cell apoptosis. The level of IL-21in the supernatant of UC-MSCs and Tfh cell differentiation coculture system was also decreased. RT-PCR analysis showed significantly higher IDO and IL-10mRNA expression in UC-MSCs when cocultured with Naive T cells under Tfh cell-polarizing condition both in RA and in pSS. In addition, HPLC result showed increased IDO enzymatic activity in the supernatant of UC-MSCs cocultured with RA or pSS Naive T cells under Tfh cell-polarizing condition. The level of IL-10in the supernatant was also upregulated. However, only the addition of the IDO inhibitor1-MT, but not anti-IL-10antibody, partly reversed the suppressive effect of UC-MSCs on Tfh cell differentiation both in RA and in pSS. The level of IFN-y in the coculture system was significantly increased in UC-MSCs coculture group compared with UC-MSCs cultured with Tfh cell inducer in RA. IDO mRNA expression was markedly downregulated in UC-MSCs, when anti-IFN-yR antibody was added to UC-MSCs and Tfh cell differentiaion coculture system. Finally, Western blot results showed that Erk and Stat-1pathways in UC-MSCs that cocultured with Naive T cells under Tfh cell-polarizing condition were activated. However, only neutralizing Stat pathway, the IDO mRNA level was significantly inhibited in UC-MSCs after stimulation with IFN-γ. Decreased levels in arthritis score, serum anti-CII antibody, inflammatory cell infiltration and joint space in the ankle were observed in UC-MSCs-treated CIA mice compared with control mice. The frequencies of Thl, Th17and Tfh cells were significantly downregulated and Treg cells were upregulated in UC-MSCs-treated CIA mice. However, no marked differences were observed in Th2cell subset. Moreover, UC-MSCs-treated group also showed lower levels of plasma cells and IgG after coculturing splenic CD4+CXCR5+T cells from each group with B220+B cells from normal mice. Inconsistent with human results, RT-PCR analysis demonstrated that there were no differences in mRNA levels of IDO, IL-10, COX2/PGE2, HGF, TGF-β, HLA-G or iNOS expressed by UC-MSCs after cultured with CD4+T cells from CIA mice.Conclusion:Tfh cell might involve in the pathogenesis of RA and pSS. UC-MSCs suppress the differentiation of Tfh cells in RA via IFN-γ-IDO-Stat-1axis in vitro. UC-MSCs transplantation (MSCT) into CIA mice is effective through inhibiting both quantity and function of Tfh cells.
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