B cell depletion with rituximab in patients with rheumatoid arthritis: Multiplex bead array reveals the kinetics of IgG and IgA antibodies to citrullinated antigens

The serology of patients with Rheumatoid arthritis (RA) is characterized by persistently raised levels of autoantibodies: Rheumatoid Factors (RhF) against Fc of IgG, and to citrullinated (Cit) protein/peptide sequences: ACPA, recognizing multiple Cit-sequences. B cell depletion therapy based on rituximab delivers good clinical responses in RA patients, particularly in the seropositive group, with responses sometimes lasting beyond the phase of B cell reconstitution. In general, ACPA levels fall following rituximab, but fluctuations with respect to predicting relapse have proved disappointing. In order to identify possible immunodominant specificities within either IgG- or IgA-ACPA we used a Multiplex bead-based array consisting of 30 Cit-peptides/proteins and 22 corresponding native sequences. The kinetics of the serum ACPA response to individual specificities was measured at key points (Baseline, B cell depletion phase, Relapse) within an initial cycle of rituximab therapy in 16 consecutive patients with severe, active RA. All had achieved significant decreases in Disease Activity Scores-28 and maintained B cell depletion in the peripheral blood (<5 CD19+cells/μl) for at least 3 months. At Baseline, mean fluorescence intensity shown by individual IgG- and IgA-ACPA were strongly correlated (R2 = 0.75; p < 0.0001) but IgA-ACPA were approximately 10-fold lower. Data were Z-normalised in order to compare serial results and antibody classes. At Baseline, a total of 68 IgG- and 51 IgA-ACPA had Z-scores ≥ 1 (above population mean) were identified, with at least one Cit-antigen identified in each serum. ACPA to individual specificities subsequently fluctuated with 3 different patterns. Most 51/68 (75%) IgG- and 48/51 IgA-ACPA (94%) fell between Baseline and Depletion, of which 57% IgG- and 65% IgA-ACPA rebounded pre-Relapse. Interestingly, 17/68 IgG-ACPA (25%) and some IgA-ACPA (3/51; 6%) transiently increased from Baseline, subsequently falling pre-Relapse. Individual responses to particular Cit-epitopes were not linked to particular patterns of fluctuation, but IgG- and IgA-ACPA to individual Cit-antigens often followed similar courses. Some new IgG- and IgA-ACPA, generally to different Cit-antigens however, arose at Relapse in 4 patients. The complexities of the ACPA response after rituximab may therefore reflect its ability to deplete or modify the function of parent B cell clones, which varies between patients. Although relapse following rituximab invariably follows naïve B cell exit from the bone marrow, these studies show that interactions between both 'new' and residual autoreactive memory B cells may be key to resumption of symptoms. The lack of identification of any immunodominant specificity suggests that the process of citrullination, rather than any particular Cit-antigen drives the autoimmune response in RA patients.