Background/Purpose: Repeat treatment with rituximab (RTX) predisposes some patients with rheumatoid arthritis (RA) to develop low levels of serum immunoglobulins (Igs). Understanding B cell function in relation to preserving immunity and a sustained clinical response is thus essential. We have investigated the relationships between B cell phenotype and in vitro function in patients RTX-naïve and in those maintaining normal serum Igs (IgM>0.4g/L and IgG>7g/L) or developing low Igs after RTX.

Methods: Serum and peripheral blood mononuclear cells (PBMC) were collected from 56 RA patients (12 pre- and 34 post-RTX). Flow cytometry (% and Mean Fluorescence intensity–MFI) was used to determine naïve and memory B cell subsets (IgD/CD27) and additionally BAFFR, CD32, CD5 and IgM. Post-RTX all patients had evidence of IgD+CD27- naïve B cell reconstitution (>25%) after a period of sustained depletion (<5 CD19+B cells/ml). Serum Igs were analysed in relation to phenotypes, BAFF and soluble CD23 (sCD23; released on differentiation to CD27+ status; normal range 1024-5025pg/ml). PBMC were cultured for 7 days with T cell dependent (TD) (anti-IgM+anti-CD40+IL4+IL21) and T cell independent (TI) (CpG+IL2) stimuli. Classes of Igs, sCD23 and anti-cyclic citrullinated peptides (anti-CCP) were measured in supernatants.

Results: After a median 5 cycles of RTX, 17 patients had normal serum Igs and 17 had developed either low IgM (n=11) or IgG (n=9) or both (n=3). Significant differences between low and normal Igs groups were noted in disease duration (median 25 vs 15 years, p=0.005) and sCD23 levels (median 908 vs 3702pg/ml, p=0.002). Median number and % CD19+B cells, RTX cycles, time from last cycle and serum BAFF were similar. Patients with low Igs also had lower %BAFFR+ pre-switch B cells (IgD+CD27+;p=0.008), higher %CD32B+ switched B cells (IgD-CD27+;p=0.02) and a negative correlation of serum IgM levels and %CD32B+ switched B cells (R²=0.44;p=0.004). In vitro, IgM, IgG or IgA and sCD23 production (normalized for B cell count) was similar between groups. IgM-CCP was only produced after TD and TI stimulus but IgG-CCP was produced spontaneously in all cultures. Significantly higher levels of both IgM- and IgG-CCP antibodies were detected in supernatants from low Ig group.

Conclusion: In patients developing low serum Igs, defective B cell maturation to memory phenotype was suggested by significantly lower levels of serum sCD23. Low serum Igs were also associated with possible reduced BAFFR-mediated anti-apoptotic signaling in IgD+CD27+ B cells and a greater %CD32B+ on memory B cells, resulting in higher thresholds for differentiation to Ig production. In vitro, however, similar amounts of Ig were present in cultures from those with normal or low serum Igs, suggesting a lack of underlying, intrinsic B cell defects. The lack of efficient B cell differentiation in individual patients with low Igs may relate to B cell interactions with immune complexes or with different T cell subsets, possibly resulting from disturbances in RTX-induced germinal centre structure. Our results further suggest increased selection and/or survival properties of autoreactive B cells in patients with low serum total Igs evident in a more robust production of anti-CCP antibodies.

« Back to 2016 ACR/ARHP Annual Meeting

ACR Meeting Abstracts - https://acrabstracts.org/abstract/b-cell-phenotype-and-in-vitro-function-in-patients-with-rheumatoid-arthritis-developing-low-serum-immunoglobulins-after-multiple-cycles-of-rituximab/