Mice and human subjects

All human tissues were collected with informed consent, following approval by the Ethics Committee of the First Affiliated Hospital of Soochow University (no. SUFAH-2019-141). There were 5 breast cancer patient samples and 3 control samples. Clinical details were recorded in Supplementary Table 4.

Mice were maintained in a specific pathogen-free (SPF) facility of the Shanghai Institute of Nutrition and Health of the Chinese Academy of Sciences and used in accordance with the ethical guidelines of the Institutional Animal Care and Use Committee.

Wild-type (WT) mice, with a BALB/c background, aged 8–10 weeks, were obtained from the Shanghai Laboratory Animal Center of the Chinese Academy of Sciences. C57BL/6 strain Lyz2Cre mice and C57BL/6 strain Ahrfl/fl mice were purchased from the Jackson Laboratory, USA. C57BL/6 strain Ahr–/– mice were generated previously.58 C57BL/6 strain Ahrfl/flLyz2Cre mice were generated through the crossing of C57BL/6 strain Ahrfl/fl mice with C57BL/6 strain Lyz2Cre mice. BALB/c strain Ahr−/− mice were produced by backcrossing C57BL/6 strain Ahr−/− mice with BALB/c wild-type mice for 8 generations. BALB/c strain Ahrfl/flLyz2Cre mice were generated by further crossing C57BL/6 strain Ahrfl/flLyz2Cre mice with BALB/c wild-type mice for 8 generations.

Cell isolation and culture

The 4T1 cell line (cat. no. CRL-2539) was purchased from ATCC. Peritoneal macrophages were harvested from BALB/c mice i.p. injected with 2 mL of 5% (wt/vol) thioglycollate broth media (BD, B11716) for 5 days. Bone marrow-derived macrophages were generated from femur and tibia bone marrows of BALB/c mice. Bone marrow cells were cultured in medium containing 20 ng/mL M-CSF (GenScript, Z02930-50) for 6 days. Mouse CD4+ T cells were isolated from splenocytes using immunomagnetic separation beads (Miltenyi Biotec, 130-104-454). F4/80+ cells were isolated from lung tissue suspension using immunomagnetic separation beads (Invitrogen, 8802-6863-74). All cells were grown in RPMI 1640 (C11875500CP) complete medium, containing 10% FBS (EallBio, 03.A16001DC), 2 mM glutamine (Gibco, 35050061), and 100 U/mL penicillin-streptomycin (Gibco, 15140122). The Py8119 cell line was kindly provided by Dr. Guohong Hu of Shanghai Institute of Nutrition and Health of Chinese Academy of Sciences. Py8119 cells were cultured in DMEM medium (Gibco,11995073), containing 10% FBS (EallBio, 03.A16001DC), 100 U/mL penicillin-streptomycin (Gibco, 15140122), and 2 mM glutamine (Gibco, 35050061).

Mouse 4T1 tumor models

For the pulmonary metastatic model, 5 × 105 to 8 × 105 4T1 cells suspended in 100 µL PBS were injected into the right mammary fat pad of BALB/c mice. Experiments were typically ended after 5 weeks unless otherwise stated. The serum and lungs were collected and processed following the ex vivo methods described. To inhibit STAT5 in vivo, STAT5 inhibitor (MCE, HY-452 101853-5 mg) or DMSO (Sigma, D2650) was diluted in 100 μl PBS and injected i.p. administered at a dose of 200 μg/mouse on day −3, −1, 1, 3, 5, 7, 9, 11, and 13 before and after mice inoculating with 4T1 cells into the fat pad. STAT5 inhibitors were prepared in a DMSO stock solution and further mixed with PBS prior to in vivo administration.

For the Py8119 tumor model, 5 × 104 to 1 × 105 Py8119 cells were mixed with Matrigel (Corning biocoat, 356237) and injected into the mammary fat pads of 8-week-old male mice. Surgical resection of the tumor was performed 4 weeks later, experiments were typically ended after another 3 weeks.

RNA isolation and RT-qPCR

Total RNA was extracted using the Trizol reagent (Invitrogen, 15596018) and RNAprep pure cell/bacteria kit (TIANGEN, DP430), following the manufacturers’ instructions. cDNA was synthesized using the RT Master Mix kit (Takara, RR036A). Relative mRNA expression was determined by real-time qPCR. The FastStart Universal SYBR Green Master kit (Roche, 4913914001), cDNA, and primers were performed. Primer sequences are as follows: mouse Actb : forward 5′- TTCCAGCCTTCCTTCTTGGG-3′ reverse 5′- TGTTGGCATAGAGGTCTTTACGG-3′; mouse Ahr : forward 5′- CCGAAGCACACGCAAATCAA-3′ reverse 5′- CCCTTCCAGGGAAGTCCAAC-3′; mouse Nqo1: forward 5′-AGGATGGGAGGTACTCGAATC′ reverse 5′- AGGCGTCCTTCCTTATATGCTA-3′; mouse Cyp1a1: forward 5′- ACAGACAGCCTCATTGAGCA-3′ reverse 5′- GGCTCCACGAGATAGCAGTT-3′; mouse Cyp1b1: forward 5′- CACCAGCCTTAGTGCAGACAG-3′ reverse 5′- GAGGACCACGGTTTCCGTTG-3′; mouse Tsg6: forward 5′- GGGATTCAAGAACGGGATCTTT-3′ reverse 5′- TCAAATTCACATACGGCCTTGG-3′; mouse Pdl1: forward 5′- GCTCCAAAGGACTTGTACGTG-3′ reverse 5′- TGATCTGAAGGGCAGCATTTC-3′.

Western blot and immunoprecipitation

Cells were rinsed with PBS and then lysed in RIPA buffer (Beyotime, P0013B) containing PI (Roche, 04693116001) and PMSF on ice for 30 min. The protein concentration of each sample was determined using a BCA Protein Assay kit (Thermo Scientific, 23225). Protein samples (20–30 μg) were loaded onto SDS–PAGE gels and transferred onto a nitrocellulose membrane. After blocking with 5% fat-free milk or 5% BSA in Tris-buffered saline with 0.1% (w/v) Tween 20 at room temperature for 1 h, nitrocellulose membranes were incubated with the indicated primary and secondary antibodies, following the manufacturer’s protocols.

For the Immunoprecipitation assay, samples were rotated with the antibody against AHR (Enzo, BML-SA210-0100) at 4 °C overnight. Protein G agarose beads (Roche, 11243233001) were then added, and incubation continued for 4 h. The beads (30 µl) were spun down and washed three times with cold PBS. Samples were reconstituted in 1× loading buffer and heated to 100 °C for 10 min.

For the ubiquitination assay, prior to RIPA collection, cells were incubated with 5 μM MG132 (Sigma, C2211-5MG) for 4 h. Samples were then incubated with an anti-Ubiquitin antibody (Abcam, ab140601) at 4 °C overnight, followed by washes and processed for western blot analysis.

For nuclear-cytoplasmic separation assay, cells were collected and washed 3 times with pre-cooled 1 × PBS to estimate the volume of cell precipitation. Five times the volume of pressure-reducing buffer was added and placed on ice for 10 min. The cells were subjected to centrifugation at 3000 × g for 5 min at 4 °C, the supernatant was collected and again subjected to centrifugation at 6000 × g for 10 min at 4 °C, The resulting supernatant was considered as the cytoplasmic portion. The precipitate portion of the first centrifugation was washed twice with hypotonic buffer. The precipitate was considered as the nucleus portion.

Hematoxylin and eosin staining

Lungs from 4T1 tumor-bearing mice were collected and fixed in 4% paraformaldehyde overnight. After fixation, the lungs were thoroughly washed with water for several hours. Subsequently, the lungs underwent a dehydration process involving sequential treatments with increasing concentrations of ethanol: 70% ethanol (overnight), 80% ethanol (2 h), 85% ethanol (2 h), 90% ethanol (45 min), 95% ethanol (20 min), and 100% ethanol (10 min, twice). Following ethanol treatment, the samples were immersed in xylene for 4 min, repeated twice. Finally, the specimens were embedded in paraffin, sectioned into 5 µm-thick slices, and stained using standard Hematoxylin & Eosin (H&E).

Macrophages in vitro treatment

Peritoneal macrophages or bone marrow-derived macrophages were seeded in 6-well plates at a density of 1.2–2 × 106 cells/well or in 96-well ultra-low attachment-bottom plates at a density of 2–4 × 105 cells/well. Macrophages were allowed to adhere to the wells and rest for 6 h. Subsequently, the macrophages were pre-treated with either DMSO, CH223191 (MCE, HY-12684), STAT5-IN-1 (MCE, HY-101853-5mg), or IQDMA (Abcam, ab141192) for 2 h. After pre-treatment, the macrophages were cultured in RPMI 1640 complete medium supplemented with the respective compounds (DMSO, CH223191, STAT5-IN-1, or IQDMA) and tumor-conditioned medium with the same compounds for continued culture. For MG132 (Sigma, C2211-5MG) or CHX (Sigma-Aldrich, C7698-1G) treatment, the respective compound was added for 4 h before cells were collected for analysis.

Flow cytometry and cell sorting

Single-cell suspensions were prepared. All antibodies were diluted at a 1:100 ratio. Samples were incubated at 4 °C, protected from light, for 30 min with the indicated fluorescent antibodies for surface molecular staining. After staining, samples were washed with PBS to remove excess antibodies. Prior to staining macrophages, incubate the cells with anti-mouse CD16/CD32 antibody (eBioscience, 14-0161-86) for 15 min at room temperature to prevent Fc receptor binding in the subsequent staining steps. For staining of the intranuclear protein Foxp3, cells were fixed and permeabilized overnight at 4 °C using the Intracellular Fixation & Permeabilization Buffer Set (Invitrogen, 88-8824-00), following staining of the proteins on the cell membrane surfaces. Subsequently, staining with the Foxp3 antibody was performed at 4 °C for 30 min. Comprehensive information on the antibodies can be found in Supplementary Table 1. For cell sorting, a Moflo Astrios EQ cell sorter (Beckman Coulter) and Beckman CytoFLEX SRT were employed. Flow cytometry data were acquired on a CytoFLEX LX (Beckman Coulter) and analyzed using FlowJo software.

Mouse serum preparation

Mice were anesthetized, and cardiac blood samples were collected using syringes. These samples were transferred into 1.5 mL Ep tubes and left at room temperature for more than 2 h. They were then centrifuged at 4000 rpm for 10 min at room temperature, and the top liquid layers were carefully extracted. This process was repeated once more, and the final top liquid layers were collected and stored at −80 °C.

Preparation of mouse lung homogenate

Lung tissue (~0.1 g) was taken directly (without washing with PBS), combined with magnetic beads, and flash-frozen in liquid nitrogen. For each sample, 500 µl of pre-cooled PBS containing PMSF was added, and the tissue was ground for 1 min. Subsequently, the samples were centrifuged at 12,000 rpm at 4 °C for 15 min, and the supernatant was carefully removed.

Preparation of 4T1-CM

4T1 cells were plated on 10 cm2 dishes and cultured in 10 ml RPMI 1640 complete medium. The supernatants were collected when cell confluency reached 90–95%. Supernatants were then centrifuged at 2000 rpm for 5 min and sterilized using 0.22 μm filters. To stimulate macrophages, 4T1-CM was mixed with RPMI 1640 complete medium at a 3:7 ratio.

Treg cell differentiation in vitro

For in vitro Treg cell differentiation, splenic CD4+ T cells were maintained for 3 days in RPMI 1640 supplemented medium, in the presence of anti-CD3 (5 µg/mL) and anti-CD28 (2 µg/mL) antibodies, IL-2 (50 ng/mL), and TGF-β (5 ng/mL). To test the influence of macrophages on Treg cell differentiation, macrophages were plated in 96-well flat-bottom plates at different densities (0.3125 × 105, 0.625 × 105, or 1.25 × 105 cells/well) and maintained in complete RPMI 1640 medium supplemented with LPS, IL-4, or 4T1-CM (30% v/v). Forty-eight hours later, macrophages were washed with PBS twice and co-cultured with CD4+ T cells (5 × 105 or 1 × 106/well) under Treg cell differentiation medium for 3 days, with anti-PD-L1 antibody or Isotype (Bio X cell, BE0101) added.

Immunofluorescence staining

Cells on slides were treated with 4% paraformaldehyde for 10 min at room temperature. Fixed cells were then incubated with PBS containing 0.1% TritonTM X-100 for 10 min to increase cell permeability. Subsequently, cells were blocked with PBS containing 2% BSA for 10 min. The AHR monoclonal antibody (Invitrogen, MA1-514) was diluted in PBS containing 0.1% BSA at a 1:200 ratio. The AHR antibody was added to the cells and incubated overnight at 4 °C. The AHR antibody was then aspirated, and the cells were washed 3 times with PBS for 5 min each time. Samples were then incubated with a fluorescent secondary antibody diluted at a 1:2000 ratio in PBS containing 0.1% BSA at room temperature for 45 min. After washing with PBS 3 times, slides were sealed using Antifade mountant with DAPI (Invitrogen, P36931).

For immunofluorescence staining of mouse lung tissue, samples were fixed in 4% paraformaldehyde overnight, sequentially dehydrated with different concentration of sucrose, and embedded in OCT (Sakura Tissue-Tek). Nonspecific binding of antibodies was blocked with 5% BSA for 30 min. The F4/80 monoclonal antibody (CST, 70076T) and PD-L1 monoclonal antibody (Invitrogen, 16-5983-82) were diluted in PBS containing 1% BSA at a 1:200 ratio. Sections were incubated with diluted antibodies overnight at 4 °C. The antibodies were then aspirated, and the sections were washed 3 times with PBS. Sections were then incubated with a fluorescent secondary antibody diluted at a 1:2000 ratio in PBS containing 1% BSA at room temperature for 45 min. After washing with PBS 3 times, slides were sealed using Antifade mountant with DAPI (Invitrogen, P36931).

For immunofluorescence staining of human lung tissue, paraffin sections were subjected to antigen retrieval in a citrate acid buffer at 95 °C for 20 min. Sections were incubated with 3% hydrogen peroxide for 10 min, and washed 3 times with PBS, then blocked with 5% BSA for 30 min. F4/80 monoclonal antibody (CST, 70076T) and AHR antibodies (NOVUS, NB100-128SS) were diluted in PBS containing 1% BSA at a 1:400 ratio. Sections were incubated with diluted antibodies at room temperature for 1 h and then washed 3 times with PBS. The HRP-labeled antibody was incubated at room temperature for 30 min. After washing with PBS three times, the slides were sealed using Antifade mountant with DAPI (Invitrogen, P36931).

Immunohistochemistry

For immunostaining, paraffin sections were subjected to antigen retrieval in a citrate acid buffer at 95 °C for 20 min. Sections were incubated with 3% hydrogen peroxide for 10 min, then washed 3 times with PBS and blocked with 5% BSA for 30 min. The sections were incubated with antibodies against Foxp3 (Thermo Fisher, 14-5773-82; 1:200) to detect Treg cells. Following this, sections were treated with suitable secondary antibodies. Mayer’s hematoxylin was used to counterstain the slides.

Bulk RNA-seq

Alveolar macrophages (AM) were purified, and total RNA was extracted using the Trizol reagent (Invitrogen). Sequencing libraries were generated using the NEBNext Ultra RNA library prep kit from Illumina (NEB), following the manufacturer’s instructions, and index codes were added to each sample to attribute sequences. Samples were then sequenced on an Illumina HiSeq platform. Hisat2 v2.0.5 was used to build the index of the reference genome and to align paired-end clean reads with the reference genome. FeatureCounts v1.5.0-p3 was used to count the number of reads mapped to each gene. The fragments per kilobase of transcript per million mapped reads (FPKM) of each gene were calculated based on the length of the gene and reads count mapped to this gene. Metadata are available at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE277589.

ChIP-qPCR

A total of 1 × 107 cells were prepared for each sample. Samples were then subjected to the subsequent steps according to the instructions provided in the ChIP-IT® PBMC kit (Active Motif, 53040). After obtaining ChIP-DNA, real-time fluorescence quantitative PCR was performed to assess AHR binding. The AHR binding region to the Pdl1 promoter was predicted using the JASPAR database. The sequences of primers utilized for ChIP-qPCR can be found in Supplementary Table 2.

CRISPR-KO cell line construction

For the stable transfection of 4T1 cells, the cells were electroporated with the lentiCRISPR v2 (Addgene, 52961) plasmids. In brief, 1 × 106 4T1 cells were suspended in 100 µl of electroporation buffer with 2 µg of the lentiCRISPR v2 plasmids. Subsequently, the 4T1 cells were electroporated and cultured in RPMI 1640 complete medium containing 3 μg/ml puromycin (MCE, HY-B1743A-10mg) for selection. After culturing with puromycin for 4 days, the knockout efficiency was confirmed by flow cytometry analysis. To establish stable 4T1 cells carrying GM-CSF deletion, 3 μg of Cas9-EGFP proteins (GenScript, Z03393-100) and 25 pmol of synthesized small guide RNAs (sgRNAs) were directly electroporated into 4T1 cells. After incubation for 48 h, a single EGFP-positive cell was selected using flow cytometry. All gRNA sequences used for CRISPR-KO experiments are listed in Supplementary Table 3. lentiCRISPR v2 (Addgene, 52961) plasmid was generously provided by Dr. Yuexiang Wang from the Shanghai Institute of Nutrition and Health, University of Chinese Academy of Sciences.

ELISA assay

The concentration of mouse GM-CSF in the homogenate supernatant from the lungs or serum of 4T1 tumor-bearing mice was determined using the ELISA kit (eBioscience, BMS612) following the manufacturer’s protocol.

Overall survival of breast cancer patients with AHR expression

Overall survival data of breast cancer patients with AHR expression were obtained from 2976 patients curated by Kaplan-Meier Plotter based on RNA-seq. For breast cancer patients, inclusion criteria did not impose limitations regarding lymph node involvement, ER expression, PR expression, HER2 expression, KI67 index, Nottingham histologic grading, or classification by PAM50 subtype. Overall survival data for triple-negative breast cancer (TNBC) patients were obtained from 126 TNBC patients. ER-negative, PR-negative, and HER2-negative statuses were used to restrict the analysis to TNBC patients. Patients were split using the auto-selected best cutoff.

Gene expression and infiltrating immune cell correlation analysis

Gene expression and infiltrating immune cell correlation analysis were performed using the TIMER2.0 online tool.59 The correlation of AHR expression with CD274 and the correlation of AHR expression or CD274 with Treg infiltration in breast cancer (n = 1100) and basal-like breast cancer (n = 191) were analyzed. The correlation of AHR expression or CD274 with Treg infiltration was assessed using the quanTIseq method.

Statistical analysis

Data were presented as mean ± SEM or symbols & lines, as specified in respective figure legends, and were analyzed using Prism 9. Statistical significance was assessed using various tests, including paired two-tailed t-test, unpaired two-tailed t-test, log-rank (Mantel-Cox) test, Pearson correlation analysis, ordinary one-way ANOVA or Mann Whitney test, as appropriate. Significant differences between or among groups are indicated as follows: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, no significance.