Microbiota imprint LDCs with the capacity to mediate IgA CSR. (A–E) Flow sorted LDCs (defined as MHCII^hiCD11c⁺Siglec-F⁻CD64⁻ cells) were cultured with CD43^negCD19⁺IgM⁺ B cells and IgA⁺ B cells were quantified. Data from three experiments with seven mice pooled per experiment are shown. Statistical comparisons are made to SPF mice. (A) Representative flow cytometry plots. (B) Cumulative data. (C) ELISA to quantify the levels of IgA secreted by B cells in culture. (D and E) qRT-PCR of mRNA isolated from B cells stimulated for 2 d with the respective DC populations for AICDA (D) and GLT-α (E). Data are normalized to GAPDH and expressed in relative units. (F) Microarray analysis comparing LDCs from SPF and GF mice. Heat map showing the differential expression of genes in the IgA CSR pathway. Colors correspond to significant fold change expression. Red, high expression; blue, low expression. (G) LDCs from SPF mice, mice treated with oral antibiotics for 12 wk + oral PBS for 5 d, and mice treated with oral antibiotics for 12 wk + oral LPS for 5 d were cultured with CD43^negCD19⁺IgM⁺ B cells and IgA⁺ B cells were quantified. Cumulative data from two independent experiments with seven mice pooled per experiment are shown. Statistical comparisons are made to the antibiotics + PBS group. (H and I) LDCs from SPF mice, GF mice, and GF mice conventionalized with the cecal contents of SPF mice were cultured with CD43^negCD19⁺IgM⁺ B cells and IgA⁺ B cells were quantified. (H) Representative flow cytometry plots. (I) Cumulative data from two independent experiments, with five mice pooled per experiment. Statistical comparisons are made to the SPF group. **, P < 0.01; ***, P < 0.001.