BackgroundAlterations of B-cell subset distribution have been described in the peripheral blood of systemic lupus erythematosus (SLE) patients (pts), but data are still controversial and association between B-cell subsets and SLE activity remains unclear.ObjectivesTo examine B-cell subsets in peripheral blood of healthy donors and active SLE pts by flow cytometry before Rituximab (RTX) treatment, and to analyze the association between B-cell subsets and SLE activity.Methods23 active SLE pts (21F/2M); median age 36 years (range) (30-40); disease duration 6.7 years (2-8); SLEDAI-2K score 13.5 (8-19), SLEDAI-2K≥8 in 18 pts, SLEDAI-2K<8 in 5 pts; global BILAG index (calculated by i-BLIPS softwear) 20.5 (14.5-24) were assessed before RTX treatment for B-cell subsets and laboratory data: ESR, CRP, C3, C4, ANA, anti-dsDNA Ab. All pts were treated with prednisolone 17.5 (10-25) mg/day, mycophenolate mofetil (7pts), azathioprine (5pts), antimalarials (19pts), methotrexate (1pts). 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 memory B cells (CD19+CD27+), non-switched memory B cells (CD19+IgD+CD27+), naïve (CD19+IgD+CD27-), and transitional (CD19+IgD+CD10+CD38++CD27-) B cells were lower in SLE pts compared to healthy donors (N=27): 1.3% (1.0-2.2) vs 2.2% (1.6-3.3), 2.1% (1.6-5.9) vs 10.0% (6.4-12.7), 58.2% (40.2-66.9) vs 65.8% (55.1-73.4), and 0.1% (0-0.1) vs 0.1% (0.1-0.3), respectively, p<0.05 for all cases. The percentages of double-negative B cells (CD19+IgD-CD27-) in SLE pts were higher than in healthy donors: 29.6% (20.9-41.3) and 9.8% (8.4-12), respectively, p<0.0001. In total SLE cohort, we found a negative association of the frequencies (r=-0.59) and absolute numbers (r=-0.59) of memory B cells (CD19+CD27+) with anti-dsDNA Ab levels, p<0.05 for both cases. The frequencies of double-negative B cells (CD19+IgD-CD27-) correlated positively with SLEDAI-2K (r=0.55), BILAG (r=0.55), ESR (r=0.60), and daily prednisolon dosage (r=0.50), p<0.05 for all cases. In active SLE pts (N=18, SLEDAI-2K≥8) this correlation was even higher: for SLEDAI-2K: r=0.67, BILAG: r=0.75, ESR: r=0.79. A strong negative association of the frequencies of double-negative B cells (CD19+IgD-CD27-) with C3 and C4 levels was observed in 17 pts with high anti-dsDNA Ab titers (>50 IU/ml): r=-0.80 and r=-0.59, respectively, p<0.05 for both cases.ConclusionsImmunophenotyping showed the decreased frequencies of memory, non-switched, naïve, transitional B-cell and increased frequencies of double-negative B cells in our active SLE cohort. Positive correlation between the level of double-negative B cells and disease activity indexes (SLEDAI-2K, BILAG), ESR, as well as a strong negative association with C3 and C4 could suggest that IgD-CD27- ...

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...

ObjectivesB subsets. To study the correlation between the counts of B cell subsets and SLE activity, and their dynamics after treatment with rituximab.MethodsRituximab in doses from 500 to 2000 mg was administered to 8 SLE patients with high and moderate disease activity (1 male and 7 females), 6 patients had lupus nephritis. The relative counts of CD19 + B cells, the total population of memory B cells (CD19 + CD27 +), “preswitch” (CD19 + IgD + CD27 +) and “postswitch” (CD19 + IgD-CD27 +) memory B cells, naive (CD19 + IgD + CD27-) and transitional (CD19 + IgD + CD10 + CD38 ++ CD27-) B cells, plasmablasts (CD19 + CD38 +++ IgD-CD27 + CD20-), short-lived plasma cells (CD19 +, CD38 +), long-lived plasma cells (CD19 + CD138 +), and double negative B-cells (CD19 + CD27-IgD-) were measured. All B cell subsets were determined by multi-color flow cytometry using a panel of monoclonal antibodies to B-lymphocytes' surface membrane markers. Statistical analysis was performed using the U Mann-Whitney test.ResultsThere was no statistically significant correlation between SLE activity and B-cell subsets counts before initiation of rituximab, most likely due to a small sample size. 2-12 scores decrease by SLEDAI2K scale in SLE activity from average 19±5 score “before” to average 11±4 scores “after” was documented already in 3 months after initiation of rituximab. This was accompanied by reduction of CD 19+ levels (median 6.25% (4.2 -10,5); 0,1% (0,05-1,5), (p =0,004;)), count of short-lived plasma cells (CD19 +, CD38 +) (median 5.5% (2,2-7,15); 0,15% (0-1,3), (p =0,008)), memory B cells (CD19 + CD27 +) (median 1% (0,8-3); 0,05% (0-0,4), (p =0,01)), and naive (CD19 + IgD + CD27-) B cells (median 42.6% (22,9-49,75); 14% (2,15-21,2), (p =0,01)). Residual CD19 + cells during severe depletion phase were mostly represented by “preswitch” (CD19 + IgD + CD27 +) and “postswitch” (CD19 + IgD-CD27 +) memory B cells, and double negative memory B cells (CD19 + CD27-IgD -).ConclusionsReduction of SLE clinical activity is associated with accompanying decrease in the counts of short-lived plasma cells (CD19 +, CD38 +), memory B cells (CD19 + CD27 +), naive (CD19 + IgD + CD27-) B-lymphocytes, which most likely indicates that these cells are major targets for anti-B cell therapy, as well as key players in autoimmune process development and progression.Disclosure of InterestNone declared

ObjectivesTo study B-cell subpopulation dynamics in SLE patients following Rituximab (RTX) therapy.MethodsThe study included 31 SLE pts (3m/28f) with high (SLEDAI2K≥10–28 pts.) and moderate (SLEDAI2K<10–3 pts.) disease activity; out of them 12 pts with SLE nephritis, 5 pts with neurolupus and 8 with vasculitis. RTX was administered to pts who failed to respond to glucocorticoids (GCs) and cytostatics (CTs). B-cells subpopulations were assessed before RTX administration (Mo0), and at Mo3 and Mo6 of RTX therapy. RTX was administered at 500 to 2000 mg doses depending on disease activity. The absolute counts of CD19+ B-cells, the total population of memory B-cells (CD19+CD27+), “preswitch” (CD19+IgD+CD27+) and “postswitch” (CD19+IgD-CD27+) memory B-cells, “naive” (CD19+IgD+CD27-), plasma cells (CD19+CD38+) and double negative B-cells (CD19+CD27-IgD) were measured. All B cell subsets were analyzed with multicolor flow cytometry using a panel of monoclonal antibodies to B-lymphocytes' surface membrane markers.ResultsFollowing initiation of RTX SLE clinical and lab activity indices have decreased in all 31 pts by Mo3 and Mo6 of follow up (SLEDAI-2K Mo0–Me 15 [12;18], Mo3-Me 6 [4;10], Mo6–Me 4 [2;8]), as well as absolute count CD19+ B-cell population (Mo0–Me 0,119x109/l [0,05;0,26], Mo3–Me 0x109/l [0;0,003], Mo6-Me 0,004x109/l [0;0,02]). B-cell repopulation by Mo6 in 15 out of 31 pts without signs of relapse and 4 pts with earlier relapse SLE was dependent on “naïve” B-cells (Me 0,0015x109/l [0,0002;0,01] vs Me 0,006x109/l [0,0033;0,008]), double negative (Me 0,001x109/l [0,0002;0,002] vs Me 0,0035x109/l [0,0018;0,005]) “postswitch” (Me 0,0005x109/l [0,00008;0,003] vs Me 0,0012x109/l [0,0003;0,0035]) and “preswitch” memory B-cells (Me 0,0006x109/l [0,00007;0,001] vs Me 0,0023x109/l [0,0005;0,005]).ConclusionsDecrease in clinical and lab SLE activity was documented in all 31 pts by Mo3 after one course of RTX therapy. In 4 pts with earlier relapse SLE at Mo6 B-cell was found repopulation and significant increase “naïve” B-cells, double negative, “postswitch” and “preswitch” memory B-cells compared with the group without relapse.Disclosure of InterestNone declared

Objectivesto study B-cell subsets dynamics in SLE patients receiving rituximab (RTX) therapy with following 6 months observation.MethodsThe study included 16 SLE pts (1m/15f) with high (SLEDAI2K>10 - 13 pts.) and moderate (SLEDAI2K<10- 3 pts.) disease activity. Out of them 7 pts with lupus nephritis and 3 pts with neurolupus. RTX was administered to pts who failed to respond to glucocorticoids (GCs) and cytostatics (CTs). B-cells subsets were assessed before RTX administration (month 0), and at month 3 and month 6 of RTX therapy. RTX was administered at 500 to 2000 mg doses depending on disease activity. We investigated the absolute and relative number of CD19+ B cells, the total population of memory B cells (CD19+CD27+), “preswitch” (CD19+IgD+CD27+) and “postswitch” (CD19+IgD-CD27+) memory B cells, naive (CD19+IgD+CD27-) and transitional (CD19+IgD+CD10+CD38++CD27-) B cells, plasmablasts (CD19+CD38+++IgD-CD27+CD20-), short-lived plasma cells (CD19+CD38+), long-lived plasma cells (CD19+CD138+), and double-negative B-cells (CD19+CD27-IgD) were measured. All B cell subsets were analyzed by technique of multicolor flow cytofluorometry using panel of monoclonal antibodies to surface membrane markers of B-lymphocytes.ResultsFollowing initiation of RTX SLE clinical and lab activity indices have decreased in all 16 pts by month 3 and month 6 of follow up (SLEDAI-2K 0 month –Me 15 [11;19], 3 month -Me 7 [3;10], 6 month –Me 5 [4;9], p<0.05), as well as decrease of absolute count CD19 B-cell subpopulation (0 month – Me 0,11 [0,0741; 0,2], 3 month - Me 0,0017 [0; 0,003], 6 month - Me 0,0045 [0,0005; 0,01], p<0.05). B-cell repopulation by 6 month in 11 out of 16 pts was due to “naive” B-cells Me 0,00085 [0; 0,008], double negative Me 0,001 [0; 0,0025] and “postswitch” memory B-cells Me 0,0013 [0,0001; 0,003]. SLE exacerbation by 6 month was documented in 5 pts, in 3 out of them B-cell repopulation occurred. A correlation between baseline high SLE activity before RTX infusions (SLEDAI-2K 5 pts – Me 20 [18;24], 11 pts. Me 12 [8;12]) and SLE exacerbation at 6 month after initiation of RTM therapy (p<0,05, r=0,54) was found.ConclusionsDecrease in clinical and lab SLE activity was documented in all 16 pts by 3 month after one course of RTM therapy. In 3 out of 5 exacerbated pts at 6 month B-cell repopulation was found, but without significant correlation with counts of B-cells subpopulations during the whole follow up period.Disclosure of InterestNone declared