Plasmablasts generated during B-cell depletion secrete antimicrobial IgA. (A) Spontaneous in vitro production of IgA from PBMCs of RTX-treated patients with RA (RA/RTX; open box) and healthy controls (HCs; gray box). Dotted line indicates the detection limit. Box plots depict minimum, maximum, and median values and upper and lower quartiles. IgA produced patient samples (B) analyzed (5%), whereas all samples bound to ≥ 1 of the 4 microbes and microbial antigens tested (C). PBMC supernatants were subjected to enzyme-linked immunosorbent assay at different dilutions with the indicated antigens for coating and IgA-specific detection. Frequencies of samples from RTX-treated patients (n = 37; black lines) yielding optical densities exceeding negative control samples at the highest concentration (undil) are indicated and reflect proportions of samples that showed binding to a particular antigen. Almost all samples tested showed concentration-dependent binding. Binding to irrelevant protein antigen (tetanus toxoid) was used as a control assay on the same samples, yielding infrequent binding of the secreted IgA compared with microbial antigens. Colostrum IgA isolated from breast milk was used as an internal positive control, pooled blood sera depleted of IgA, and medium cultured without PBMCs as internal negative controls. All PBMC supernatants from B-cell depleted and control donors bound to ≥ 1 of the bacteria or microbial antigens. Dotted black lines indicate pooled data from 8 healthy controls.