Induction of long-term B-cell depletion in refractory rheumatoid arthritis patients preferentially affects autoreactive more than protective humoral immunity

Abstract

Introduction: B-cell depletion has become a common treatment strategy in anti-TNF-refractory rheumatoid arthritis (RA). Although the exact mechanism of how B-cell depletion leads to clinical amelioration in RA remains to be elucidated, repetitive treatment with B-cell-depleting agents leading to long-term B-cell depletion has been reported to be beneficial. The latter has led to the hypothesis that the beneficial effects of B-cell depletion might act through their influence on pathogenic autoreactive plasma cells.

Methods: In this study, we investigated the effects of a fixed retreatment regimen with anti-CD20 mAbs on the humoral (auto)immune system in a cohort of therapy-refractory RA patients.

Results: Fixed retreatment led to long-term B-cell depletion in peripheral blood, bone marrow and, to a lesser extent, synovium. Also, pathologic autoantibody secretion (that is, anticitrullinated peptide antibodies (ACPAs)) was more profoundly affected by long-term depletion than by physiological protective antibody secretion (that is, against measles, mumps and rubella). This was further illustrated by a significantly shorter estimated life span of ACPA-IgG secretion compared to total IgG secretion as well as protective antibody secretion.

Conclusion: By studying plasma cell function during an extensive 2-year period of B-cell depletion, autoantibody secretion was significantly shorter-lived than physiologically protective antibody secretion. This suggests that the longevity of autoreactive plasma cells is different from protective long-lived plasma cells and might indicate a therapeutic window for therapies that target plasma cells.

Figure 1

Long-term B-cell depletion in peripheral blood and bone marrow in rheumatoid arthritis patients treated with a fixed regimen of 6-monthly anti-CD20 monoclonal antibodies with a follow-up of 24 months. (A) Absolute numbers of CD3^-CD19⁺ B cells measured in peripheral blood every 3 months presented as Tukey box-and-whisker plots. (B) Percentages of CD3^-CD19⁺ B cells in bone marrow measured at baseline, 3 and 21 months presented as Tukey box-and-whisker plots. (C) Serum B lymphocyte-activating factor (BAFF) concentrations before every rituximab course presented as Tukey box-and-whisker plots. (D) Disease Activity Score in 28 joints (DAS₂₈) of the 11 individual patients (grey lines) and overall median DAS₂₈ scores (black line) measured every 3 months. A DAS₂₈ score below 3.2 (dotted line) indicates low disease activity.

Figure 2

Immunoglobulin secretion from CD3^-CD38^bright cells in bone marrow. (A) Fresh bone marrow-derived mononuclear cells (BMMCs) were stained with PerCP cyanin 5.5 dye for flow cytometric cell sorting (FL2H) according to immunoglobulin D (IgD) phycoerythrin and CD38 expression, resulting in four populations: CD38^low cells, IgD⁺CD38^high cells, IgD^-CD38²⁺ cells and IgD^-CD38^bright cells. These cell populations were incubated on precoated ELISPOT plates as described in the Methods section. (B) Using the enzyme-linked immunosorbent spot assay, we detected the number of spot-forming units (SFUs) and quantified them for the IgG, IgM and IgA isotypes. PBS = phosphate-buffered saline. Bars represent means ± SEM of five experiments. (C) Calculated total number of SFUs for total immunoglobulin (total Ig) secretion. (D) 2 × 2 table showing the sensitivity and specificity of the population of CD3^-CD38^bright cells to secrete immunoglobulins. The sensitivity was 92.9%, and the specificity was 53.6%. FACS = fluorescence-activated cell sorting.

Figure 3

Comparison of relative effects of long-term B-cell depletion between total immunoglobulin secretion, autoantibody secretion and protective antibody secretion. Patients were treated with a fixed regimen of 6-monthly anti-CD20 monoclonal antibodies at baseline, 6, 12 and 18 months with a follow-up of 24 months. (A) Median percentage change from baseline serum concentrations of total immunoglobulin G (total IgG), rheumatoid arthritis (RA)-specific anticitrullinated protein autoantibodies of the IgG isotype (ACPA-IgG) and IgG-isotype antibodies against measles (measles-IgG), mumps (mumps-IgG) and rubella (rubella-IgG). (B) Median percentage change from baseline serum concentrations of total IgM and RA-specific ACPA-IgM. (C) Median percentage change from baseline serum concentrations of total IgA and RA-specific ACPA-IgA. Brackets in (A) through (C) represent the nonparametric statistical comparison at 24 months between the represented (auto)antibodies and total IgG/IgA. * P < 0.05. For clear representation, log₂ scales are used for the y-axes.