Fig. 3

Promotion of Treg cells by AHR activated macrophages through PD-L1. a Representative immunofluorescent images showing F4/80 and PD-L1 staining in the lungs of Ahr^flflLyz2^Cre−/− (Control) and Ahr^flflLyz2^Cre+/− mice inoculated with 4T1 cells for 14 days (n ≥ 15 for each group). Scale bar, 60 μm. b Schematic of lung macrophage isolation from mice bearing 4T1 tumors at indicated timepoints (created with BioRender.com). Correlation analysis between Pdl1 and Ahr mRNA expressions in F4/80⁺ cells from the PMN (n = 15 for each group). c Pdl1 mRNA expression in peritoneal macrophages, with or without addition of serum from 4T1 tumor bearing mice (n ≥ 3 for each group, repeated twice). d PD-L1 expression on peritoneal macrophages of wild-type (WT) and Ahr^−/− mice, with or without 4T1-CM treatment for 12 h (n ≥ 3 for each group). e PD-L1 expression on peritoneal macrophages from Ahr^flflLyz2^Cre−/− (Control) and Ahr^flflLyz2^Cre+/− mice, with or without 4T1-CM (n ≥ 3 for each group, repeated twice). f Flow cytometry analysis of Treg cell differentiation when co-cultured with normal medium or 4T1-CM pretreated macrophages from WT mice and Ahr^−/− mice in the presence of isotype control or anti-PD-L1. BMDMs from WT mice (Control) or Ahr^−/− mice were pretreated with normal medium (Control) or 4T1-CM for 2 days. Naïve CD4⁺ T cells were co-cultured with pretreated BMDMs for 3 days under the Treg cell differentiation conditions, with the addition of PD-L1 neutralizing antibody or Isotype control. g Bar graph showing quantification of Treg cell differentiation (n = 4 for each group). h ChIP analysis of AHR recruitment to the Pdl1 promoter in peritoneal macrophages stimulated with 4T1-CM (n = 4 for each group). Data are analyzed by unpaired two-tailed t-test (a, c, d, e, g, h) or Pearson correlation analysis (b) and presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, no significance