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BackgroundAlterations of B-cell subpopulation have been described in the peripheral blood of rheumatoid arthritis (RA) patients (pts), but differences between B-cell subsets distributions in early and late onset RA (LORA) are still unclear [1,2].ObjectivesTo examine B-cell subsets in peripheral blood of healthy donors, early (disease duration <6 months) RA (ERA) and LORA pts by flow cytometry, and to analyze the association between B-cell subsets and RA activity.Methods24 active ERA pts (20F/4M); median age 54 years (range) (38–64); disease duration 5 months (4–6); DAS28 score 5.2 (4.7–5.9); RF+75%, ACCP+ 87.5% and 20 LORA pts (16F/4M); median age 58 years (range) (44–62); disease duration 12 years (4.5–17.5); DAS28 score 5.3 (4.6–6.2); both RF+ and ACCP+ 80%, were assessed for B-cell subpopulations and laboratory data: ESR, CRP. LORA pts were treated with DMARDs (anti-TNF-α, Methotrexate), glucocorticoids, and NSAIDS; ERA pts received NSAIDS only. CD19+B cells, memory B cells (CD19+CD27-), non-switched memory B cells (CD19+IgD+CD27+), switched memory B cells (CD19+IgD-CD27+), naïve (CD19+IgD+CD27-), double-negative (CD19+IgD-CD27-), transitional (CD19+IgD+CD10+CD38++CD27-) B cells, and plasmablasts (CD19+CD38+++IgD-CD27+CD20-) were analyzed using multicolor flow cytometry.ResultsThe median percentages (range) of non-switched memory B cells (CD19+IgD+CD27+) were lower in ERA and LORA RA cohorts compared to donors: 3.0% (1.6–5.5) and 6.2% (2.4–10.2) vs 7.4% (3.7–11.1), respectively, p<0.05 for both cases. In LORA pts, the median percentages (range) and absolute numbers of switched memory B cells (CD19+IgD+CD27-) were higher compared to both ERA pts and donors: 24,1% (14,0–42.3) vs 8,3% (4.4–12.9) and 12.8% (9.3–17.0); and 0.03 (0.02–0.05) vs 0.01 (0.006–0.02) and 0.02 (0.01–0.04), respectively; p<0.05 for all cases. The median percentages of naïve B cells (CD19+IgD+CD27-) were significantly higher in ERA pts compared to LORA pts and healthy donors: 75.3% (69.7–83.6) vs 54.3% (39.7–69.1) and 64.7% (57.6–72.4), respectively, p<0.05 for both cases.In ERA pts, we found a significant correlation between absolute numbers of non-switched memory B cells (CD19+IgD+CD27+), RF and anti-CCP: r=0.50 and 0.45, respectively, p<0.05 for both cases. In LORA pts, the percentage of memory B cells (CD19+CD27+) correlated positively with ESR (r=0.58), p<0.05; a positive association was observed between the absolute numbers of switched memory B cells (CD19+IgD-CD27+), CRP (r=0.48) and RF (r=0.49), p<0.05 for all cases.Conclusionsin ERA pts, immunophenotyping showed increased frequencies of naïve B cells and decreased frequencies and absolute numbers of switched memory B cells compared to LORA cohort.References Fedele et al. BMC Immunology 2014, 15:28. doi: 10.1186/s12865-014-0028-1. Memory B cell subsets and plasmablasts are lower in early than in long-standing Rheumatoid Arthritis.Moura RA et al. Rheumatology 2010 Jun;49(6):1082–92. doi: 10.1093/rheumatology/keq029. Alterations on peripheral blood B-cell subpopulations in very early ar...

ObjectivesTo 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 analysisMethods45 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 groupResultsAfter 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<0.05Treg 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)ConclusionsMT therapy in early RA pts results in increased Treg suppressor activity according to growin...