Immunohistochemical analysis as a means to predict responsiveness to rituximab treatment

Abstract

Anti-CD20-mediated B cell depletion with rituximab is a new and effective therapy for rheumatoid arthritis (RA). Although B cells in peripheral blood (PB) are consistently depleted in all patients, the clinical effects are more heterogeneous, possibly related to differences in the depleting effects of lymphoid or solid tissues. The aim of this study was to investigate B cell depletion in different compartments (PB, bone marrow, and synovium) and determine predictive variables for responsiveness to rituximab therapy. Before and 12 weeks after rituximab treatment, samples of PB, bone marrow, and synovium were collected from 25 patients with RA refractory to disease-modifying antirheumatic drugs and tumor necrosis factor-blocking agents. CD19+ and CD20+ B cells in PB and bone marrow were measured by flow cytometric analysis, whereas CD79a+ and cytoplasmic CD20+ B cells in the synovium were stained by immunohistochemistry. The effects of rituximab on serum Ig and autoantibodies were measured by enzyme-linked immunosorbent assay. Rituximab effectively depleted the CD20+ subset of B cells in the PB, bone marrow, and synovium of RA patients. Rituximab significantly reduced autoantibody production (anti-citrullinated protein antibodies [ACPAs] and rheumatoid factor [RF]), in part due to a nonspecific decrease in total Ig production. Importantly, positivity for circulating ACPA IgM, in combination with a high infiltration of CD79a+ B cells in the synovium, but not of CD138+ plasma cells, was a predictor of clinical outcome after rituximab treatment. ACPA IgM titers were independently associated with synovial infiltration of CD20-,CD79a+ B cells, but not with CD138+ plasma cells. These data provide novel insights into the mechanisms of CD20-mediated B cell depletion in the lymphoid and solid tissues of RA patients and suggest a pivotal role for ACPA IgM-producing plasmablasts in RA.