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 DOI 10.1136/annrheumdis-2014-eular.2202

Background Interleukin (IL)-21 is a member of type I cytokine family. Recent studies have indicated that IL-21 is an important regulator for human B cell activation, proliferation, plasma cell (PC) differentiation, immunoglobulin (Ig) production and isotype switching. Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by abnormal production of cytokines and autoantibodies including rheumatoid factor (RF) and anti-cyclic citrullinated peptide antibodies (anti-CCP), suggesting that B cells play a key role in pathogenesis of RA. Objectives The aim of this study was to investigate the effect of IL-21 on B cell proliferation and differentiation of RA patients. Methods Concentrations of IL-21 in serum were measured by ELISA. The correlation between serum IL-21 levels and clinical features of RA patients were assessed. The percentages of IL-21R+CD19+ cells were analyzed by flow cytometry (FACS) in peripheral blood mononuclear cells (PBMC) from RA patients and healthy controls. PBMC from RA patients were stimulated with rIL-21 (50 or 100ng/ml) after motivation of anti-CD40 and anti-IgM. The percentages of IL-21R, activation markers (CD40, CD69 and CD25) on B cells and the proliferation labeled with CFSE as well as differentiation of B cells were determined by using FACS analysis Results The results showed that serum IL-21 concentrations in RA patients (191.3±34.42pg/ml, n=104) were significantly higher than in healthy controls (10.33±11.43pg/ml, n=56, p<0.01). The levels of IL-21 in RA patients were positively related to RF-IgM (r=0.23, p<0.05), RF-IgA (r=0.34, p<0.05), RF-IgG (r=0.35, p<0.05) and anti-CCP (r=0.32, p<0.05). Moreover, the percentages of IL-21R+CD19+ cells were found to be markedly higher in PBMC of RA patients (48.55%±2.63%, n=50) compared to healthy controls (34.12%±2.37%, n=40, p<0.01) and IL-21 could up-regulate IL-21R expression on B cells in vitro. Meanwhile, IL-21 stimulated the proliferation of B cells and activated markers expressions (CD40, CD69 and CD25). IL-21 induced more production of CD138+CD19+/low cells in RA patients, indicating that IL-21 can promote B cell differentiation. Conclusions These results suggest that increased IL-21 expression from RA patients might support B cells proliferation, activation and antibody secretion. Thus, antagonizing IL-21 may be a novel strategy for treating RA. Disclosure of Interest None Declared

Background Interleukin (IL)-27 is a new member of IL-6/IL-12 family involved in isotype switching and plasma cell differentiation of naïve B cells. Objectives The objective of this study is to investigate the association of IL-27 with B cell functions in patients with Sjogren's syndrome (SS), an autoimmune disease characterized by B-cell hyperactivity. Methods B cell activation was measured by the percentages of CD40 and CD69 positive cells in total CD19+ B cells under flow cytometry. Carboxyfluorescein diacetate succinimidyl ester (CFSE) and Annexin V were marked to detect B cell proliferation and apoptosis respectively. CD27 and CD138 were used to identify the trend of CD19+ B cell differentiation. To verify the role of IL-27 on B cell regulation, peripheral blood mononuclear cells (PBMCs) collected from SS patients and healthy controls were stimulated by B cell activators including anti-CD40, recombinant human IL-4, CpG-ODN and F(ab)2 goat anti-human IgM with or without the presence of IL-27 at the concentration of 50ng/ml. 72 or 96 hours later, cells were harvested and detected for B cell activation, proliferation, apoptosis and differentiation. Levels of several cytokines that have been reported to participate in B cell regulation, including IL-27, IFN-γ, IL-10, IL-4 and BAFF, were detected by ELISA. Results Compared with healthy controls, the percentages of active B cells (CD40 or CD69 positive in total CD19+ B cells) were elevated in SS patients (p both <0.05). B cells from SS patients displayed higher proliferation and apoptosis rate (p both <0.05), and an increased cell differentiation into plasma cells was observed (p<0.05). Serum levels of IL-27 were elevated in SS patients, especially in those with high titers of ANA (197.2±24.48 pg/ml vs. 179.8±9.37 pg/ml, p<0.01) or positive anti-SSA/SSB antibodies. In vitro cultures showed that IL-27 stimulation could increase the percentages of CD40/CD69 and CD138 cells, promote B cell proliferation and IgG secretion. Meanwhile, IL-27 could upregulate several B cell related factors in vitro (484.2±235.9 pg/ml vs. 4718±259.9 pg/ml for IFN-γ, 119.2±39.7 pg/ml vs. 98.58±29.17 for IL-4 and 1711±556.6 pg/ml vs. 1436±660.3 pg/ml for IL-10, p all <0.05). Conclusions IL-27 elevation contributes to B cell hyperactivity in SS patients. Treatment targeting IL-27 may be promising for the amelioration of this disease. Disclosure of Interest : None declared DOI 10.1136/annrheumdis-2014-eular.2416

Background It has been shown that human umbilical cord mesenchymal stem cells (MSCs) could provide some benefits for the treatment of rheumatoid arthritis (RA) patients with refractory disease. Objectives The aim of this study is to explore whether the MSCs from bone marrow in RA patients are defective and dysfunctional. Methods We evaluated the proliferative potential of MSCs in 7 days of culture. Cellular apoptosis, immigration and senescence were detected by using 7-AAD flow-cytometry, transwell assay and the senescence-associated β-galactosidase (SA-β-Gal) staining respectively. Cultured supernatants of MSCs from RA patients and healthy controls (HC) were collected, and analyzed for cytokine production using a protein array. The immunomodulatory capacity of MSCs on PBMCs proliferation and on the distribution of Th17, Treg and Tfh cells was also investigated. Results The growth rate was lower in RA MSCs than that in HC MSCs. RA MSCs had deficient migration ability compared to HC MSCs after 24 hours of culture. More SA-β-Gal stained cells were observed in RA MSCs, and the expression of cell cycle inhibitor p21 was higher in RA MSCs than HC MSCs. However, no significant differences were found between RA MSCs and HC MSCs in the percentage of early apoptotic and late apoptotic cells. Protein arrays showed no significant differences in the cytokine production between RA MSCs and HC MSCs. MSCs from both the RA patients and controls were able to suppress the proliferation of PBMCs, and no significant differences were found between RA MSCs and HC MSCs in regulating Treg and Tfh cells. However, the ability to inhibit Th17 cells was impaired in MSCs from RA patients. Conclusions MSCs in RA patients have abnormalities compared to those in healthy control, which may play an important role in the development of RA disease. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.2194

Background The immune suppressive properties of mesenchymal stem cells (MSCs) have garnered increasing attention over the past decades. MSCs that derived from human umbilical cord (UC) share similar immunosuppressive properties as MSCs obtained from bone marrow and cord blood in vitro. Rheumatoid arthritis (RA) is characterized by persistent synovitis and systemic inflammation, frequently leading to cartilage and bone destruction. Previous studies showed that MSCs transplantation had effect in an animal model of RA. However, the mechanism remains largely unknown. Objectives The purpose of this study was to confirm the hypothesis that UC-MSCs might suppress the generation of T follicular helper (Tfh) cells in RA patients. Methods The percentages of CXCR5+PD-1+CD4+ cells were analyzed by flow cytometry in peripheral blood mononuclear cells (PBMC) from RA patients and healthy controls. PBMC from RA patients stimulated with or without phytoagglutinin (PHA) were cultured with UC-MSCs supernatant or UC-MSCs at a ratio of 1 to 1, 1 to 10 or 1 to 100 in a cell-to-cell contact or in a transwell system for 3 days. Naïve T cells were isolated from PBMC of RA patients and then co-cultured with UC-MSC in the presence of anti-CD3, anti-CD28, anti-IFNg, anti-IL-4, anti-TGFb, IL-6, IL-21 and IL-12 for 4 days. The percentages of CXCR5+PD-1+CD4+ cells were tested by means of flow cytometry. Results The results showed that the circulating percentages of CXCR5+PD-1+CD4+ cells were significantly higher than that of healthy controls. UC-MSCs did not inhibit non-activated Tfh cells but dose-dependently inhibited the generation of Tfh cells when stimulated with PHA whether in a cell-to-cell contact or in a transwell system. The differentiation of Tfh cells were also blocked by UC-MSCs significantly. However, UC-MSCs supernatant was not able to suppress the generation of Tfh cells in RA. Conclusions These results suggest an inhibitory effect of UC-MSCs on the generation of Tfh cells via soluble factors secreted from UC-MSCs, which may be one the mechanism of UC-MSCs treatment in RA. Disclosure of Interest None Declared