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BackgroundCD19+CD24highCD38high B cells have been described to have a regulatory capacity and their frequency is altered in the peripheral blood of patients with various autoimmune diseases.ObjectivesTo determine the frequency of circulating CD19+CD24highCD38high B cells in patients with RA, and establish a correlation with clinical parameters.MethodsPeripheral blood was drawn from RA patients treated with non-biological DMARDs (n=48), RA patients treated with anti-TNFalpha drugs (n=15) and healthy controls (n=63) that were matched with patients for age and gender. After isolation by Ficoll-Hypaque gradient, PBMCs were stained with antibodies to CD3, CD4, CD19, CD24, and CD38, and examined by flow cytometry.ResultsA decreased frequency of CD19+CD24highCD38high B cells was apparent in in ACPA+ but not ACPA- RA patients, and this was observed not only in patients treated with non-biological DMARDs but also in patients receiving anti-TNFalpha drugs. In addition, the frequency of CD19+CD24highCD38high B cells showed a significant negative correlation with ACPA titers (r = -0.43, p=0.023). In contrast, no correlation was found between the frequency of circulating CD19+CD24highCD38high B cells and either rheumatoid factor titres or disease activity as determined by the DAS28 score.ConclusionsA decreased frequency of CD19+CD24highCD38high B cells is observed in ACPA+ but not ACPA- RA patients treated with either nonbiological DMARDs or anti-TNFalpha drugs, that is related with ACPA titres but not with rheumatoid factor titres or disease activity.Disclosure of InterestNone declared

Objectives To examine the frequency and phenotype of Th17 cells in the peripheral blood of early RA (eRA) patients. Methods CD4+ T cells were isolated from the peripheral blood of 33 eRA patients and 33 healthy controls (HC), and from the synovial fluid of 20 established RA patients (RASF), by ficoll-hypaque gradient and magnetical negative selection. After polyclonal stimulation, the frequency of Th17 and Th1 cells was determined by flow cytometry and concentrations of IL-17, IFN-g, TNF-a and IL-10 were measured by ELISA in cell-free supernatants. Results When all of our eRA patients were analyzed together, a significantly lower percentage of circulating Th17 cells and a lower CD4-derived IL-17 secretion were observed in comparison with HC. However, after stratifying by anti-CCP antibody status, circulating Th17 cells were decreased in anti-CCP(+) but not in anti-CCP(–)-eRA. All Th17 cells were CD45RO+CD45RA- and CCR6+. Dual Th17/Th1 cells were also exclusively decreased in anti-CCP(+)-eRA. Circulating Th17 and Th17/Th1 cells were negatively correlated with anti-CCP titres. When anti-CCP(+)-eRA patients were retested one year after initiating treatment with oral methotrexate, their circulating Th17 frequency was no longer different from HC. Of note, the percentage of circulating Th1 cells and the secretion of CD4-derived IFN-g, TNF-a and IL-10 were not different between eRA patients and HC. In RASF, both Th17 and Th1 cells were increased when compared with blood. Conclusions Decreased circulating Th17 levels in eRA seem to be a marker of anti-CCP seropositivity, and return to levels observed in healthy controls after treatment with methotrexate. Disclosure of Interest None Declared

BackgroundIL-17-expressing FoxP3 regulatory T cells have been described, and their suppressive capacity has been questioned. An inflammatory environment seems to favor IL-17 secretion by regulatory CD4+CD25+FoxP3+ T cells.ObjectivesTo assess the suppressive function and IL-17 producing capacity of CD4+CD25+CD127-FoxP3+ T cells in the synovial fluid of RA patients (RASFd).MethodsSynovial fluid was drawn from 35 patients with established RA who were receiving methotrexate and low-dose oral prednisone. The frequency of CD4+CD25+CD127-FoxP3+ T cells was assessed by flow cytometry. Total CD4+ T cells, CD4+CD25+CD127- T reg cells and CD4+CD25- Tresp cells were isolated by Ficoll-Hypaque gradient, followed by sorting. After isolation, cells were stimulated for 5 hours with PMA+ionomycin or cultured for 5 days in flat-bottom 96-well plates coated with an anti-CD3 monoclonal antibody. Treg cell function was assessed using two different approaches: A.The regulatory function of natural proportions of Tregs was inferred by comparing the proliferative and cytokine responses of total CD4+ T cells (TCD4T) versus CD25+ depleted CD4+ T cells (CD4+CD25-T cells or Tresp cells); B. The per cell suppressor potency of Tregs was assessed in cocultures of isolated Tregs with Tresp, established at different Treg/Tresp ratios. Proliferation was determined by 3Hthymidine incorporation and CFSE dilution; cytokine secretion was measured by ELISA of culture supernatants.ResultsA high proportion of CD4+CD25+CD127- T cells was present in RASFd (mean ± SD, 21.1%±6.2), which is significantly higher than reported frequencies of this cell population in the peripheral blood of both RA and healthy subjects. These RASFd CD4+CD25+CD127- T cells expressed FoxP3 but did not express CD69. The proliferation rate, TNFα and IFNγ secretion were significantly higher for isolated Tresp as compared with TCD4T cells, indicating that natural proportions of Treg cells present in the synovial fluid of RA are funcionally suppressive. Surprisingly, TCD4T cells secreted higher amounts of IL-17 as compared with Tresp cells, although the difference did not reach statistical significance. On a per cell basis, RAPB Tregs were potent suppressors of Tresp proliferation, TNFα and IFNγ but not of IL-17 secretion; in fact IL-17 secretion did not decrease but was enhanced in the presence of increasing proportions of Treg cells. Isolated Treg cells did not proliferate or produce cytokines when cultured alone in anti-CD3 coated plates. However, in the presence of plate-bound anti-CD3 plus anti-CD28 and recombinant human IL-2, isolated Treg cells secreted significant amounts of IL-17 whereas no TNFα or IFNγ could be detected in supernatants.ConclusionsCD4+CD27+CD127- FoxP3+ Treg cells present in the synovial fluid of RA patients are potent suppressors of Tresp proliferation, TNFα and IFNγ secretion, and at the same time produce significant amounts of IL-17.References Voo KS, et al. Proc Natl Acad Sci U S A. 2009;106:4793.Beriou G et al. Blood. 2009;113:4240.Wang T, et al. Ann Rheum Dis. 2015;74:1293.Komatsu N, et al. Nat Med. 2014;20:62.Yang BH, et al. Mucosal Immunol. 2016;9:444. Disclosure of InterestNone declared