Objectives To analyze the effect of methotrexate (MT) therapy on the percentage and absolute number of FoxP3+ regulatory T (Treg) cells in the peripheral blood of MT-naïve patients (pts) with early RA we used Treg immunostainingwith antibodies to surface markers and Foxp3 with subsequent flow cytometric analysis
Methods 45 MT-naïve pts with early RA (39 females, Me;IQR age 52.0 (32.5–57.5) years, RA duration 5 (4–6) months, DAS 28 5.01 (4.18–5.8), RF positive -71.1%, ACPA - positive-88.9%) were included into the study. All pts were administered subcutaneous MT as the first-line DMARD at 10 mg/week with rapid dose escalation up to 20–25 mg/week. Human blood mononuclear cells were isolated from whole venous blood by Ficoll–Hypaque centrifugation and subjected to multicolor flow cytometry analysis. Tregs were stained for different surface markers, and proportions of marker-positive subsets (FoxP3+CD25+; CD152+surface; CD152+intracellular; FoxP3+CD127-; CD25+CD127-; FoxP3+ICOS+; FoxP3+CD154+; FoxP3+CD274+) were determined; 20 healthy donors made the control group
Results After 24 weeks of MT therapy value DAS 28 was 3.1 (2.7–3.62); SDAI 7.4 (4.2–11.4); DAS 28 remission/low disease activity was achieved in 22 (56.4%) pts, based on SDAI – in 25 (64.1%) pts; while MT failure based on EULAR response was documented in 4 (10.3%) pts. Lower percentages of FoxP3+CD25+ cells ((5.53 (4.09–6.48 vs 6.92 (5.84–7.96)), percentages and absolute number of FoxP3+ICOS+ cells ((6.91 (2.14–11.47) vs 10.83 (9.27–13.7); 0.0035 (0.0013–0.0067) vs 0.0068 (0.0039–0.009)), and percentages and absolute number of FoxP3+CD154+ cells ((0.47 (0.19–0.83) vs 1.51 (1.12–2.08); 0.0002 (0.00009–0.0005) vs 0.00087 (0.00047–0.0014)), and FoxP3+CD274+ T-cells (0.63 (0.34–1.49) vs 1.94 (1.16–2.25); 0.0003 (0.0002–0.00065) vs 0.001 (0.0006–0.0016), p<0,05 in all cases) were documented in early RA pts versus healthy donors.
An increase in the percentage of CD4+cells (from 45.0 (38.0–49.2) to 46.8 (39.9–53.2)); an increase in the relative and absolute number of CD152+surface 0.65 (0.22–1.67) vs 2.07 (1.11–3.81); 0.0002 (0.0001–0.0008) vs 0.0007 (0.0004–0.002); and moderate decrease in the relative and absolute number of FoxP3+ICOS+ cells 5.3 (2.1–11.3) vs 4.1 (1.6–6.6); 0.002 (0.001–0.006) vs 0.0015 (0.0006–0.003), p<0.05 in all cases, was registered after 24 weeks of MT therapy. After 24 weeks of MT therapy the level of CD152+surface in the RA pts group was higher compared with healthy donors 2.1 (1.11–3.81) and 0.51 (0.34–1.2); 0.0007 (0.0004–0.002) and 0.0003 (0.00014–0.0008), respectively, p<0.05
Treg levels and phenotype were analyzed in pts groups based on MT efficacy by 24th week of treatment. Higher percentages and absolute number of FoxP3+CD274+ cells (1.25 (0.43–2.3) 0.0004 (0.0002–0.001) were detected in patients achieving SDAI remission (n=25), compared to pts with moderate disease activity (SDAI>11 n=14) (0.44 (0.2–0.69) 0.00016 (0.0001–0.0004), p<0.05)
Conclusions MT therapy in early RA pts results in increased Treg suppressor activity according to growing percentages and absolute number of CD152+surface and FoxP3+CD274+ cells, more pronounced among pts, achieving remission/low disease activity following treatment. Increased levels of these markers are indicative of improved Tregs functional activity after successful MT therapy
Disclosure of Interest None declared
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