LDC CD103⁺ and LDC CD24⁺ mediate IgA class switching in an RA- and TGF-β–dependent manner. (A) Microarray analysis of lung APC subsets. 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. (B) qRT-PCR of mRNA isolated from flow-sorted LDC CD103⁺, LDC CD24⁺, LMφ CD64⁺, and SpDC, respectively. The expression of Integrin β8 mRNA was normalized to actin and expressed in relative units. (C) qRT-PCR of mRNA isolated from flow-sorted LDC CD103⁺, LDC CD24⁺, LMφ CD64⁺, and SpDC, respectively. The expression of mAldha2 was normalized to GAPDH and expressed in relative units. (D–F) Flow-sorted LDC CD103⁺ or LDC CD24⁺ were cultured with naive B cells in the absence or presence of the RAR-β inhibitor LE540, and IgA⁺ B cells were quantified. (D) Representative flow cytometry plots. (E) Cumulative data from three different experiments with seven mice pooled per experiment. (F) Escalating doses of LE540 (25 nM, 250 nM, and 2.5 µM) were added to the DC–B cell culture on day 0. Cumulative data from two independent experiments is shown (with seven mice pooled per experiment). Statistical comparisons are made with (–) LE540 condition. (G– I) Flow-sorted LDC CD103⁺ or LDC CD24⁺ were cultured with naive B cells in the absence or presence of α-TGF-β neutralizing antibody, and IgA⁺ B cells were quantified. (G) Representative flow cytometry plots. (H) Cumulative data from three different experiments with seven mice pooled per experiment. (I) Escalating doses of α-TGF-β neutralizing antibody (8.75, 17.5, 35, and 70 µg/ml) were added to the DC–B cell cultures on days 0 and 3. Cumulative data from two independent experiments (with seven mice pooled per experiment) is shown. Statistical comparisons are made with (–) α-TGF-β condition. *, P < 0.05; **, P < 0.01; ***, P < 0.001.