THU0092 Intra-cytoplasmic cytokines in cd20 positive lymphocyte subpopulations in rheumatoid arthritis (RA) patients
Authors: S. Aamar, A. Kerpel, R. Rabinowitz, P. Levy, O. Zelig, N. Schlesinger, M. Schlesinger
Journal: Annals of the Rheumatic Diseases (2018)

<h3>Background</h3> The administration of biological drugs inactivating various elements of the immune system such as cytokines and immune cells, induces a significant ameliorating effect on rheumatoid arthritis (RA) patients. The mechanisms involved in the pathogenesis of RA are unclear, and the precise role of different cellular elements of the immune system in its pathogenesis remain to be elucidated. <h3>Objectives</h3> In the present study we concentrated on one cellular target for a biologic treatment in RA (Rituximab), the CD20 positive lymphocytes. Various types of lymphocytes are positive to CD20. Based on its expression, these lymphocytes include dim and bright CD20 +types. These cells' capacity to secrete various cytokines was determined among lymphocytes of healthy controls (HC) and RA patients. <h3>Methods</h3> Peripheral blood lymphocytes (PBL) of RA patients (n=20) and HC (n=15) were isolated using Ficoll Hypaque gradient columns. To 5×10<sup>5</sup> cells in 0.5 ml RPMI-1640 medium, 1.25 ng PMA, 0.5 µg Ionomycin and 5 µg Brefeldin were added for 5 hours of activation after which cells were incubated for 30 min at 4°C with Phycoerythrin-(PE) conjugated anti-CD20 monoclonal antibody (mab). Thereafter, suspensions were incubated in fixation buffer for 45 min, washed and re-suspended in 100 µl of permeabilization buffer and 20 µl of fluorescent (FITC) conjugated anti-cytokine mabs, and were incubated at 4°C for 30 min. A t-test for independent samples, was performed. <h3>Results</h3> Intra-cytoplasmic detection of various key-role cytokines, such interleukins 4 (IL-4), 17 (IL-17),10 (IL-10) and interferon gamma (INF-γ) was determined. The percentage of IL-4 positive lymphocytes among CD20 +cells in HC was higher in CD20 +dim cells (4.5%) than in CD20 +bright cells (1.2%) (p value 0.0001). Among lymphocytes of RA patients the proportion of IL-4 positive cells was much higher than in HC (p&lt;0.0002) and was comparable between dim (54.5%) and bright (48.6%) CD20 +lymphocytes. The proportion of IL-10 positive cells among both dim and bright CD20 +lymphocyte subpopulations was significantly lower in RA patients than in HC (p&lt;0.0002). While in HC, 56% of CD20 +dim and 61% of CD20 +bright lymphocytes, showed IL-10 positivity, among RA lymphocytes it constituted only 17.8% and 9.3% of the CD20 +dim and bright lymphocytes, respectively. Staining with anti-IL-17 mab yielded in low proportions of IL-17 positive cells in all CD20 +populations tested, in both HC and RA. Among dim CD20 +lymphocytes, IL-17 positive cells constituted 1.4% (HC) and 3.6% (RA). IL-17 staining appeared on only 0.74% of CD20 +bright lymphocytes in HC and similarly in RA (0.54%). While the proportion of INF-γ positive lymphocytes was similar in HC and RA, it was significantly higher among dim (11.8% HC, 9.5% RA) than in bright CD20+ (1% HC, 1.4% RA) indicating a different cell pattern (p=0.0001). <h3>Conclusions</h3> The present study demonstrates that CD20 +lymphocytes, targeted in RA treatment, expressing different levels of CD20, in RA differ from HC in their capacity to produce various cytokines. Although there was no difference in IL-17 and INF-γ they may modulate the immune response in RA due to higher levels of IL-4 and reduced IL-10 levels produced by both CD20 +cell populations; dim and bright lymphocytes. <h3>Disclosure of Interest</h3> None declared