Therapeutic Cytokine Inhibition Modulates Activation and Homing Receptors of Peripheral Memory B Cell Subsets in Rheumatoid Arthritis Patients

Abstract

Memory B cells have known to play an important role in the pathogenesis of rheumatoid arthritis (RA). With the emergence of B cell-targeted therapies, the modulation of memory B cells appears to be a key therapeutic target. Human peripheral memory B cells can be distinguished based on the phenotypic expression of CD27 and IgD, characterizing the three major B cell subpopulations: CD27+IgD+ pre-switch, CD27+IgD- post-switch, and CD27-IgD- double-negative memory B cells. We evaluated different memory cell populations for activation markers (CD95 and Ki-67) and chemokine receptors (CXCR3 and 4) expressing B cells in active RA, as well as under IL6-R blockade by tocilizumab (TCZ) and TNF-α blockade by adalimumab (ADA). Memory B cells were phenotypically analyzed from RA patients at baseline, week 12, and week 24 under TCZ or ADA treatment, respectively. Using flow cytometry, surface expression of CD95, intracellular Ki-67, and surface expressions of CXCR3 and CXCR4 were determined. Compared with healthy donors (n = 40), the phenotypic analysis of RA patients (n = 80) demonstrated that all three types of memory B cells were activated in RA patients. Surface and intracellular staining of B cells showed a significantly higher percentage of CD95+ (p < 0.0001) and Ki-67+ (p < 0.0001) cells, with numerically altered CXCR3+ and CXCR4+ cells in RA. CD95 and Ki-67 expressions were highest in post-switch memory B cells, whereas CD19+CXCR3+ and CD19+CXCR4+ expressing cells were substantially higher in the pre-switch compartment. In all subsets of the memory B cells, in vivo IL-6R, and TNF-α blockade significantly reduced the enhanced expressions of CD95 and Ki-67. Based on our findings, we conclude that the three major peripheral memory B cell populations, pre-, post-switch, and double-negative B cells, are activated in RA, demonstrating enhanced CD95 and Ki-67 expressions, and varied expression of CXCR3 and CXCR4 chemokine receptors when compared with healthy individuals. This activation can be efficaciously modulated under cytokine inhibition in vivo.

Keywords: B cells; adalimumab; inflammation; memory B cells; rheumatoid arhritis; tocilizumab (IL-6 inhibitor).

FIGURE 1

Activated B cells are expanded in RA. In patients with RA patients, B cells show a significantly higher percentage of surface CD95+ (A) and intracellular Ki-67+ (B) expressing B cells when compared with healthy donors (HD). Data are shown in box-whisker plots showing individual dots, where boxes represent 25th to 75th percentiles and the lines within the boxes represent the median. P values were determined with the Student’s unpaired t-test using GraphPad Prism 6 (***P < 0.0001). n = number of individuals.

FIGURE 2

Modulation of CD95 expression on B cell subsets during IL-6R and TNF-α inhibition therapy. For CD19+CD95+ expressing cells from all B cell subsets, including total B cells, pre-switch memory, and post-switch memory B cells, are significantly reduced during IL-6R inhibition (A) and TNF-α inhibition (B) therapy. CD19+Ki-67+ expressing cells from all B cell subsets are significantly reduced during IL-6R inhibition (C) and TNF-α inhibition (D) therapy. Values were consistently compared with baseline levels by using the Student unpaired t-test (***p < 0.0001, **p < 0.001, and *p < 0.01). BL = baseline, W12 = week 12, and W24 = week 24.

FIGURE 3

Correlation between activated B cells and disease activity. A significant correlation can be observed between activated B cells, identified by their CD95+ or Ki-67+ expression, and DAS28 as a measure of RA disease activity (A). Among B cell subsets, post-switch exhibits a positive correlation, pre-switch a negative correlation, while double-negative (DN) B cells fail to demonstrate any correlation with DAS28 (B). The relationship between variables was evaluated using the Pearson linear correlation test. A two-sided p-value of <0.05 was considered statistically significant.

FIGURE 4

Enhanced expression of chemokine receptor CXCR3 and CXCR4 on B cells during active RA. CD19+CXCR3+ and CD19+CXCR4+ expressing B cells are elevated in patients with active RA when compared with healthy donors (HD).

FIGURE 5

Expression of CXCR3 and CXCR4 in B cell subsets during IL-6R and TNF inhibition therapy. CXCR3 expression on B cell subsets increases in some subsets during IL-6R inhibition (A) and decreases during TNF-α inhibition (B) therapy. CXCR4 expression on B cell subsets reduces in certain subsets during IL-6R inhibition (C) and TNF-α inhibition (D) therapy. Values were consistently compared with baseline levels using the Student unpaired t-test (***p < 0.0001, **p < 0.001, and *p < 0.01). BL = baseline, W12 = week 12, and W24 = week 24.