CD27-IgD- memory B cells are modulated by in vivo interleukin-6 receptor (IL-6R) blockade in rheumatoid arthritis

Abstract

Introduction: Enhanced B cell activity, particularly memory B cells have gained interest in evaluating response during therapies with biologics. CD27-IgD- double-negative (DN) B cells lacking the conventional memory marker CD27 are reported to be part of the memory compartment, however, only scarce data is available for rheumatoid arthritis (RA). We therefore focused on DN B cells in RA, studied their isotypes and modulation during interleukin-6 receptor (IL-6R) inhibition by tocilizumab (TCZ).

Methods: DN B cells were phenotypically analyzed from 40 RA patients during TCZ at baseline week 12, week 24 and 1 year. A single B cell polymerase chain reaction (PCR) approach was used to study Ig receptors, VH gene rearrangements and specific isotypes.

Results: Phenotypic analysis showed a significantly expanded population of DN B cells in RA which contain a heterogeneous mixture of IgG-, IgA- and IgM-expressing cells with a clear dominance of IgG+ cells. DN B cells carry rearranged heavy chain gene sequences with a diversified mutational pattern consistent with memory B cells. In contrast to tumor necrosis factor alpha (TNF-α) inhibition, a significant reduction in mutational frequency of BCR gene rearrangements at week 12, 24 and 1 year (P <0.0001) was observed by in vivo IL-6R inhibition. These changes were observed for all BCR isotypes IgG, IgA and IgM at week 12, 24 and 1 year (P <0.0001). IgA-RF, IgA serum level and IgA+ DN B cells decreased significantly (P <0.05) at week 12 and week 24 during TCZ. Patients with a good European League Against Rheumatism (EULAR) response to TCZ had less DN B cells at baseline as compared to moderate responders (P = 0.006). Univariate logistic regression analysis revealed that the frequency of DN B cells at baseline is inversely correlated to a subsequent good EULAR response (P = 0.024) with an odds ratio of 1.48 (95% confidence interval as 1.05 to 2.06).

Conclusions: In RA, the heterogeneous DN B cell compartment is expanded and dominated by IgG isotype. TCZ can modulate the mutational status of DN Ig isotype receptors over 1 year. Interestingly, the frequency of DN B cells in RA may serve as a baseline predictor of subsequent EULAR response to TCZ.

Figure 1

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.

Figure 2

Surface expression of immunoglobulin isotypes during IL-6R inhibition. (A) Representative FACS plots showing the expression of immunoglobulin isotypes, IgA, IgG and IgM on gated DN and post-switch B cells. (B) During IL-6R inhibition, IgA+ DN B cells decreased significantly from 24.7 (10.0 to 64.2) percent median (range) to 18.4 (4.8 to 34.7) at week 12 (P = 0.004) and 20.5 (4.6 to 33.8) at week 24 (P = 0.04) respectively. There were no remarkable changes in relative IgG+ and IgM+ DN B Cells. (C) IgA+ and IgG+ post-switch B cells were not influenced during IL-6R inhibition. BL, baseline, W12, week 12 and W24, week 24. DN, double-negative; Ig, immunoglobulin; IL-6R, interleukin-6 receptor.

Figure 3

Ig-receptor somatic hypermutation of gene rearrangements during IL-6R and TNF-α inhibition in DN B cells. (A) Ig-V_H3 gene rearrangements during IL-6R inhibition. Reduction in mutational frequency of Ig-V_H3 gene rearrangements of DN B cells from the peripheral blood of RA patients during TCZ therapy. The mutational frequency was significantly reduced at week 12, 24 and 1 year (^*** P <0.0001 compared to BL). (B) Ig-receptor somatic hypermutation of V_H3 gene rearrangements during TNF-α inhibition. During TNF-α inhibition by adalimumab, a comparable mutational frequency of V_H gene rearrangements of DN B cells from the peripheral blood of RA patients were observed at all time points. (C) Ig-receptor somatic hypermutation of isotype-specific IgA+, IgG+ and IgM+ gene rearrangements during IL-6R inhibition in DN B cells. At the BL, the mutational frequency of IgA+ DN B cells is significantly higher compared to IgG+ and IgM+ DN B cells. During TCZ therapy, all isotypes IgA+, IgG+ and IgM+ DN B cells showed a significantly reduced mutational frequency (^*** P <0.0001,^** P <0.001). In a scatter plot, the line represents mean of all values and each dot depicts the mutational frequency of a single sequence. P values were determined by Wilcoxon test using GraphPad Prism 5. (BL, baseline; W12, week 12; W24, week 24; n, number of individuals; s, number of sequence analyzed). DN, double-negative; HD, healthy donor; Ig, immunoglobulin; IL-6R, interleukin-6 receptor; RA, rheumatoid arthritis; TCZ, tocilizumab; TNF-α, tumor necrosis factor alpha.

Figure 4

CDR3 length during IL-6R and TNF-α inhibition. (A) Significant increase in the CDR3 length of Ig-V_H gene rearrangements of DN B cells from the peripheral blood of RA patients during IL-6R inhibition. (^*** P <0.0001). (B) During TNF-α inhibition, a comparable CDR3 length of Ig-V_H gene rearrangements of DN B cells were observed at all time points. (C) There is significant increase in the length of the CDR3 in all three isotypes specific to DN B cells during IL-6R inhibition. P values were determined by Wilcoxon test using GraphPad Prism 5. CDR3, the length of the third complementary determining region; DN, double negative; IL-6R, interleukin-6 receptor; RA, rheumatoid arthritis; TNF-α, tumor necrosis factor alpha.

Figure 5

Frequency of targeted mutations within hotspot motifs during IL-6R and TNF-α inhibition. (A) A significant reduction in frequency of targeted mutations within RGWY/WRCY is found in Ig-V_H gene rearrangements of DN B cells during TCZ therapy (^* P = 0.046, ^** P = 0.004). (B) During TNF-α inhibition, a comparable mutational hotspot targeting of V_H gene rearrangements of DN B cells were observed at all time points. (C) A significant reduction in frequency of targeted mutations within RGWY/WRCY is found in all three isotypes specific to DN B cells during IL-6R inhibition. P values were determined by Wilcoxon test using GraphPad Prism 5. DN, double negative; Ig, immunoglobulin; IL-6R, interleukin-6 receptor; TCZ, tocilizumab; TNF-α, tumor necrosis factor alpha.