In this regard, it has been widely accepted that effector T cells have an important role in the pathogenesis of RA [2,3,5], and it is evident that these lymphocytes should be susceptible to be inhibited by regulatory T cells. UNAM, Mxico) for 72 h. 3H-TdR was added for the last 12 h of cell culture and at the end of incubation cells were harvested and proliferation was decided using a liquid scintillation counter. These experiments were run by triplicate and results expressed as the stimulation index. The reactivity against was determined by a standard PPD skin test (50 U, Connaught Laboratories, Willowdale, Ontario, Canada). Statistical analysis Data were compared with the Sigma STAT software (SPSS Inc., Chicago, IL, USA) using Wilcoxon, MannCWhitney U, and T paired tests with a level of significance of < 005. Results Before starting anti-TNF- therapy, we found a variable number of CD4+CD25bright cells Bentiromide in the eight patients studied (Fig. 1a). Although these percentages tended to be lower than those detected in five healthy volunteers (41 11%, = 5), no significant differences were seen (> 005). A significant increase of the percent of TREG lymphocytes was observed at day 15 of Adalimumab therapy (< 005 compared to day 0, Fig. Bentiromide 1a). Although this increase was also observed at day 180 (< 005 compared to day 0), in 6 out of 8 patients an important diminution in CD4+CD25bright cells was detected when compared with day 15 (Fig. 1a). No significant changes in the levels of CD4+CD25bright cells were observed in the five healthy individuals studied (data not shown). Open in a separate window Fig. 1 Rabbit Polyclonal to LRG1 Quantification of regulatory T cells in RA patients under Adalimumab therapy. PBMNC from eight RA patients were isolated, and then the levels of CD4+CD25bright, and CD4+CTLA-4+ T cells (a), and the synthesis of TGF-, and IL-10 by CD4+ lymphocytes (b) were assessed by flow cytometry before (day 0) and at days 15 and 180 of Adalimumab therapy, as described in Materials and Methods. Horizontal lines correspond to the arithmetic mean and vertical lines to standard deviation. Representative histograms of the quantification of CD4+ TGF-+ cells in a patient with RA are shown in (c). Numbers correspond to Bentiromide the percent of double positive cells. We also found a significant increase in the percent of CD4+CTLA-4+ and Tr1-like lymphocytes at day 15 of anti-TNF- therapy (< 005, Fig. 1a, b). However, at day 180 no significant differences were observed when compared to day 0. Similar results were observed in cells stimulated with an anti-CD3 mAb, but a significant enhancement of CD4+CTLA-4+ cells at day 180 was observed in these cells (data not shown). We then studied the function of TREG cells. We found that CD4+CD25+ lymphocytes from all patients were able to inhibit the proliferation of autologous CD4+CD25C cells stimulated with PHA. According with results obtained by us in five healthy volunteers, TREG cells from RA patients showed a diminished regulatory function (288 83 and 483 88 of stimulation index in controls and patients, respectively, < 005, Fig. 2 and data not shown). On the other hand, we observed in all patients studied a modest but significant increase in the function of TREG cells at day 15 of Adalimumab therapy (< 005 compared to day 0, Fig. 2). Interestingly, when these assays were performed at day 180, a diminution in TREG function was observed when compared with values of day 15 (Fig. 2). Accordingly, no significant differences were detected between values obtained at day 0 and 180 (> 005, Fig. 2). These results showed that although significant changes in the number and function.