Background IL-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.
Objectives To assess the suppressive function and IL-17 producing capacity of CD4+CD25+CD127-FoxP3+ T cells in the synovial fluid of RA patients (RASFd).
Methods Synovial 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.
Results A 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.
Conclusions CD4+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.
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Disclosure of Interest None declared