Background/Purpose . Memory B cells have been shown to play important roles in the pathogenesis of rheumatoid arthritis (RA). With the advent of B cell targeted therapies the modulation of memory B cells seems to be a prime target. Human peripheral memory B cells can be classified into three major subtypes by the phenotypic expression of CD27 and IgD: CD27+IgD+ preswitch, CD27+IgD- postswitch and CD27-IgD- double negative (DN) memory B cells. We aimed in this study to analyze different subsets of memory B cells in patients with RA under cytokine inhibition. 

Methods . Memory B cell subsets were phenotypically analyzed for activation (expressions of CD95 and ki-67) and their isotype profile using 10 color flow cytometry at baseline, week 12 and week 24 during cytokine inhibition. Single B cell PCR approach was used to study isotypes specific Ig-receptors. Mann-Whitney U test was used for statistical analysis by using GraphPad Prism 5. 

Results . Surface and intracellular staining of memory B cell subsets showed a significantly higher percentage of CD95 and ki-67 expression in RA (n=60) compared to healthy donors (n=20). During cytokine (IL-6 or TNF-α) inhibition, both CD95 and ki-67 expression was significantly reduced at week 12 and 24 in all 3 types of memory B cells. RA patients showed a significant relative expansion of DN IgD-/CD27- B cells with a mixture of IgA, IgG and IgM expression dominated by the IgG phenotype. During IL-6 receptor inhibition, IgA+ DN B cells decreased significantly from 25.1 (8.0-54.2) percent median (range) to 19.0 (4.8-51.1) at week 12 (P= 0.0016) and 20.5 (4.6-33.8) at week 24 (P=0.0008) respectively without any remarkable change in relative IgG+ and IgM+ B DN B cells. In the postswitch compartment, IgA+ and IgG+ postswitch B cells were not influenced during IL-6R inhibition. Isotype specific analysis of rearranged Ig-R sequences from DN B cells revealed that mutational frequencies were highest in IgA+ B cells followed by IgG+ and IgM+, respectively. During IL-6R inhibition, significantly reduced mutational frequencies in Ig-receptors of all DN isotypes at week 12, 24 and 1year (p<0.0001) were observed. Accordingly hotspot motif targeting was also decreased whereas CDR3 length increased during therapy.

Conclusion . Our study suggests that all three major memory B cell subsets are activated in RA which can be significantly reduced by IL-6R and TNF-α inhibition in vivo. DN B cells are expanded in RA and IL-6R inhibition resulted in a reduction of particularly IgA+ DN B cells. IL-6 inhibition leads to a reduced mutational frequency and hot spot targeting in IgA, IgG as well IgM DN Ig receptors. The results are in accordance with a specific effect on DN memory B cells by in vivo IL-6 receptor inhibition.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/memory-b-cell-subtype-modulation-in-patients-with-rheumatoid-arthritis/