Figure 5.

AhR deficiency in B cells impaired Breg differentiation ex-vivo. CD19⁺ B cells were negatively selected from whole splenocytes of wild-type (WT), and AhR^-/-, mice were disaggregated and seeded with LPS (10μg/ml), and increasing concentrations of L-Kyn (50µM and 100μM) for 72 hrs. Bregs were identified as CD19⁺CD5⁺CD1d^hiIL-10⁺ by flow cytometry. (A) AhR deficiency inhibited L-Kyn induced Breg differentiation following stimulation of B cells with LPS+L-Kyn. Data shown here are representative of three independent experiments with three replicates for each group and represent the mean ± STDEV values. (B) AhR deficiency altered the proliferation of Breg. CD19⁺ cells were negatively selected from whole splenocytes of WT, and AhR^-/- mice and labeled with CFSE and then stimulated with LPS and increasing concentrations of L-Kyn for 72 hrs. Total percentages of Breg⁺CFSE^low (CD19⁺CD5⁺CD1d^hiIL-10⁺CFSE^low) cells were determined by flow cytometry analysis. Data shown here are representative of two independent experiments with 3 technical replicates in each condition. (C) Breg precursors (CD19⁺CD1d^hiCD5⁺) induced by LPS+L-Kyn were not impaired in AhR^-/-. Total percentages of CD19⁺CD1d^highCD5 in AhR^-/- were determined by flow cytometry analysis. Data shown here are pooled of three independent experiments with 3 technical replicates in each condition. *p < 0.05, **p < 0.001, ***p < 0.0001, ****p < 0.00001.