Effects of TACI siRNA and heparitinase on APRIL- and BAFF-induced B-cell responses. (A) Effects of TACI siRNA and heparitinase treatment on B-cell proliferation (top), IgA production (middle), and IgG production (bottom) in response to CD40L, BAFF, APRIL, and agonistic anti-TACI mAb. IgA- or IgG-negative B cells treated with control siRNA, TACI siRNA, or heparitinase (10 U/mL) were cultured with anti-BCR antibodies (anti-Igκ and anti-Igλ, 0.5 μg/mL each), CD40L (2 μg/mL), BAFF (4 μg/mL), APRIL (8 μg/mL), anti-TACI mAb (11H3; 5 μg/mL) or control mouse IgG2a (5 μg/mL) in the presence or absence of IL-4 (20 U/mL) and TGF-β (1 ng/mL). [³H]-Thymidine incorporation in B cells was measured during the last 18 hours of 72-hour culture (top). IgA (middle) and IgG (bottom) secretion were measured by ELISA after 10-day culture. Data are mean ± SD. *P < .05. (B) Ratios of IgA/IgM and IgG/IgM production in response to CD40L, BAFF, APRIL, and agonistic anti-TACI mAb. After incubation, as described in panel A, IgM secretion and viable cell number were determined after 10-day culture (Table S1). Per cell IgA, IgG, or IgM production was calculated based on data shown in panel A and Table S1. Then the ratios of IgA/IgM and IgG/IgM were determined. Data are mean ± SD. *P < .05. (C) Flow cytometric analysis of IgA-positive B cells. After incubation as described in panel A, cells were stained with PE-labeled anti-CD19 mAb and FITC-conjugated anti-IgA antibody and were analyzed in living cells only. The percentage of CD19⁺ IgA⁺ cells is indicated in each plot. Data are representative of 3 independent experiments with similar results.