Phenotype analysis of CD27-IgD- B cells in RA patients and their relation to EULAR response. (A) Representative FACS plot. Characterization of (CD27-IgD-) DN B cells, PS = post-switch (CD27 + IgD-), Pre = pre-switch (CD27 + IgD+) and naïve (CD27-IgD+) B cells. (B) Comparison of DN B cells in RA patients and HD. DN B cells in RA patients (n = 44) and HD (n = 45) show a significantly higher percentage of the frequency of DN B cells in RA patients (P <0.0001). (C) EULAR response to IL-6R inhibition. Week 12 EULAR good responders (BL DAS28 = 5.1 ± 0.3) to TCZ have significantly (P = 0.006) lower frequency of DN B cells at baseline compared to EULAR moderate responders (BL DAS28 = 5.3 ± 0.3). (D) EULAR responses to IL-6R inhibition (absolute cell numbers). Week 12 EULAR good responders have significantly (P = 0.05) lower absolute DN B cell numbers at baseline compared to moderate responders. Data shown in box-whisker plot where boxes represent 25th to 75th percentiles and the lines within the boxes represent the median. P values were determined by Mann-Whitney U test using GraphPad Prism 5. (***
P <0.0001, **
P <0.001 and *
P <0.05). BL DAS28, baseline disease activity score 28; DN, double-negative; EULAR, European League Against Rheumatism; HD, healthy donor; Ig, immunoglobulin; IL-6R, interleukin-6 receptor; RA, rheumatoid arthritis; TCZ, tocilizumab.