Objectives The aim of our study was to elucidate whether umbilical cord derived mesenchymal stem cell (UC-MSCs) had immunomodulatory effect on T follicular helper (Tfh) cell under rheumatoid arthritis (RA) background.
Methods PBMCs that isolated from RA patients and healthy contorl (HC) were cocultured with UC-MSCs at a ratio of 100:1, 10:1 and 1:1 respectively through cell-to-cell contact or in a transwell system at a ratio of 1:10. After 3 days' coculture, PBMCs were collected and the frequency of CD4+CXCR5+PD-1+T cell was examined by flow cytometry. Purified naïve T cells isolated from PBMC of RA patients were cocultured with human UC-MSCs for 4 days at a ratio of 1:10 under Tfh cell-polarizing condition. To detect the effect of MSC on Tfh proliferation, CFSE-labeled purified CD4+ T cells isolated from PBMC of RA patients or HC were stimulated with anti-CD3/CD28 and cocultured with UC-MSCs for 5 days. The frequency of CD4+CXCR5+PD-1+T or CD4+CXCR5+PD-1+AnnexinV+T was examined by flow cytometry and the level of IL-21 in the cultured supernatant was measured by ELISA. The mRNA levels of indoleamine 2,3-dioxygenase (IDO), IL-10, PGE2, HGF, TGF-β and HLA-G in UC-MSCs were tested by RT-PCR. IDO activity of was measured by high-performance liquid chromatography (HPLC), and P-STAT-1/3/5 and p-Akt in UC-MSCs were tested by Western blot. IDO inhibitor 1-MT or anti-IL-10 antibody was added into UC-MSCs and Tfh cell differentiation coculture system for 5 days and then the frequency of CD4+CXCR5+PD-1+T was examined by flow cytometry.
Results UC-MSCs were able to suppress the generation of Tfh cell both in RA patients and in HC in vitro, which was dose-dependent and not relied on cell-to cell contact. UC-MSCs suppressed the differentiation and proliferation of Tfh cell that was more prominent in samples from RA patients, but had no effect on Tfh cell apoptosis. The level of IL-21 in UC-MSCs and Tfh cell differentiation coculture system was significantly decreased. Dramatic increases of both IDO mRNA expression and IDO enzymatic activity were detected in UC-MSCs after coculturing with naïve T cells under Tfh cell-polarizing condition. Meanwhile, pSTAT-1/3/5 and pAkt in these UC-MSCs were increased. The addition of the IDO inhibitor 1-MT, but not anti-IL-10 antibody, could reverse the suppressive effect of UC-MSCs on the differentiation of Tfh cell.
Conclusions UC-MSCs suppress Tfh cell differentiation and proliferation in RA patients, which is partially mediated by the secretion of IDO.
Disclosure of Interest None declared
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