Fig 2: BC cell-intrinsic PD-L1 promotes mTOR signals. Western blot of control and PD-L1KO MB49 cells in serum replete (A, C) and starved conditions (B, C) ±incubation with 2.5 nM rapamycin for 6 h and blots from RT4 and PD-L1KO RT4 cells in serum replete and starved conditions (D). Western blot of control and PD-L1lo UM-UC-3 (E) and UM-UC-14 cells (F) in serum replete conditions. Quantification of selected proteins shown in (A) and (C). All blots were run on the same membranes but separated spatially. Lanes are unedited but have been reorganized for presentation clarity. (G,H) MTT assay of control or PD-L1KO MB49 cells after treatment with 2.5 nM rapamycin for 72 h. Veh, vehicle. Rap, rapamycin. P, (C-E,G,H) unpaired t-test

Fig 3: Expression of PD-L1 and CSF1R in mouse myeloid cells and human blood CD11c+ cells after Lalistat2 treatment.(A) LAL enzymatic activity in HD1A myeloid cells after incubation with 10 µM, 50 µM, 100 µM, and 200 µM Lalistat2 or DMSO (S) for 72 hours. (B) Murine HD1A myeloid cells were incubated with 10 µM, 50 µM, 100 µM, and 200 µM Lalistat2 or DMSO (S) for 72 hours. Percentages of CD11c+, PD-L1+, and CSF1R+ cells in HD1A myeloid cells were analyzed by flow cytometry. (C) Expression of PD-L1 in HD1A myeloid cells after Lalistat2 or DMSO treatment for 72 hours by Western blot analysis. Representative blots are shown. (D) Human white blood cells from healthy individuals were incubated with 10 µM Lalistat2 (L) or DMSO (S) for 24 hours. Percentages of CD11c+ cells in the whole white blood cells were analyzed by flow cytometry. (E) Percentages of PD-L1+ and CSF1R+ cells in blood CD11c+ cells of healthy individuals treated with Lalistat2 (L) versus DMSO (S). Data are expressed as mean ± SD. Experiments were independently repeated, n = 4 for A and B, n = 3 for C, n = 6 for D, n = 5 for E. *P < 0.05, **P < 0.01, 1-way ANOVA.

Fig 4: AhR inhibitor exhibits anti-lung cancer activity in vivo. a C57BL/6 mice were intravenously injected with LLC (5 × 105) cells, and 3 days later the mice were randomized to receive vehicle or ANF treatment. Micro-CT scanning images and HE staining of lung sections are shown. Scale bar = 500 µm. b IHC assays of ANF-treated mice’ lung tumor tissues using indicated antibodies. Scale bar = 500 µm. c Flow cytometry analysis of CD8+, CD4+, CD45+CD3+, and CD45+B220+ cells in the lung tissues. d The expression of PD-L1 in lung tissues was detected by real-time PCR. e The expression of TNFa and IFN? in the lung tissues was detected by real-time PCR. f Life span of the mice. g, h The C57BL/6 mice were intravenously injected with LLC (5 × 105) cells, and 3 days later randomized to receive vehicle or CH223191 treatment. Micro-CT scanning images (g) and life span of the mice (h) are shown. i, j The NOD/SCID mice were intravenously injected with LLC (5 × 105) cells, and 3 days later randomized to receive vehicle or ANF treatment. Micro-CT scanning images (i) and life span of the mice (j) are shown. k C57BL/6 mice were injected intravenously with LLC (5 × 105) cells and treated with ANF and/or anti-PD-L1 antibody. Micro-CT scanning images, HE and IHC staining of lung sections of the mice are shown. Scale bar = 500 µm. l Flow cytometry analysis of CD8+, CD4+, and CD3+ cells in the lung tissues. m, n The expression of IFN? (m) and TNFa (n) in the lung tissues was detected by real-time PCR. o Life span of the mice. P values in c-e, l-n, Student’s t test; P values in f, h, j, o, Log-rank test. Error bars, sd

Fig 5: A threshold level of AhR is critical to increased PD-L1 expression. a The expression of PD-L1 and AhR was detected by IHC assay in NSCLCs. b Immunofluorescence assays of smoker patients’ formalin-fixed paraffin embedded (FFPE) 5-µm sections using antibodies against PD-L1 and AhR, and DAPI. Arrow, cancer cells; *, non-cancerous cells. c Western blot analyses of lysates of tumor (T) and adjacent normal (N) lung tissues harvested from NSCLCs (n = 62). d Quantification of the ratios of PD-L1 in tumors to PD-L1 in counterpart normal lung tissues in smoker and nonsmoker patients. Determined by densitometry analyses of immunoblot bands in (c). NS, nonsmoker; S, smoker. P value, Student’s t test. e Overall survival of the 62 patients. P value, log-rank test. f PD-L1 expression in an Oncomine report. g The expression of PD-L1 and AhR in DNA microarray data of TCGA datasets. AhR z score was calculated as: (RNA-Seq by Expectation Maximization (RESM) in tumor - mean of RESM values in normal)/Standard deviation of RESM values in normal). P value, Mann-Whitney test. h The association between PD-L1 and AhR in patients. Data are from TCGA datasets. i The smoking status, PD-L1 expression, and AhR Z score in NSCLCs. j–l The expression of PD-L1, AhR Z score, and TP53 (j), KRAS (k), or STK11 (l). P values in i-l, Mann-Whitney test. WT wild type, M mutant, H heavy smoker, L light smoker