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. 2021 Nov 19;12:747780. doi: 10.3389/fimmu.2021.747780

Figure 3.

Figure 3

Trp metabolite L-Kyn promoted Breg differentiation through AhR pathway. CD19+ B cells were negatively selected from whole splenocytes of WT mice, disaggregated and seeded with LPS (10μg/ml), increasing concentrations of L-Kyn (50µM and 100μM) and LPS + L-Kyn + CH 223191(10μM) an aryl hydrocarbon receptor antagonist (AhRA) for 72 hrs. Bregs were identified as CD19+CD5+CD1dhiIL-10+ by flow cytometry. (A) Total percentage of Bregs (CD19+CD5+CD1dhiIL-10+) in ex-vivo experiments performed with negatively selected purified B cells following stimulation with no LPS, LPS, LPS+L-Kyn, LPS+AhRA+L-Kyn, and LPS+AhRA. Data shown here are pooled from six independent experiments with 3 technical replicates in each condition. (B) IL-10 levels in the media collected from above described experiments. (C) Representative figure of FACS analyses of splenic tissue confirmed the reduction of Breg (CD19+CD5+CD1dhiIL-10+) in tumor-bearing AhR-/- on day 9 post tumor implant. (D) Total percentage of Bregs (CD19+CD5+CD1dhiIL-10+) in spleens of tumor-bearing WT and AhR-/- mice on Day 9 post tumor implant (n = 4-6 mice per group). *p < 0.05, **p < 0.001, ***p < 0.0001, ****p < 0.00001.