70 μm cell strainer

PD-L1 protein was purified from a PD-L1-transfected HEK-293 cells. The supernatant and cell lysates were collected and PD-L1 protein was purified using a HisTrap column (Cytiva, MA, USA); recombinant PD-L1 protein (R&D, MN, USA) was used as the positive control. Pan T cells from spleen were filtered through a 70 μm cell strainer (Miltenyi, Germany) and extracted by a T cell isolation kit (Miltenyi Biotec, Germany). T cells were cultured in RPMI medium 1640 with 2 mM L-Glutamine, 10% FBS, and 100 U/mL penicillin/streptomycin. T cells were stained with CellTrace Violet dye as indicated by the CellTrace™ Violet Cell Proliferation Kit (Thermo Fisher, Waltham, Massachusetts, US), then incubated with 2x10⁵ anti-CD3/CD28 beads (Thermo Fisher, Waltham, Massachusetts, US) and 10U/ml IL-2 (R&D, MN, USA) with or without 5 μg/ml of purified PD-L1. Recombinant PD-L1 and PBS were used as positive and negative controls, respectively. T cells cultured with no anti-CD3/CD28 beads were also designed. After 72h, T cells were collected, and the percentage of proliferating cells from each group were determined by Attune Flow Cytometer (Thermo Fisher, Waltham, Massachusetts, US) with an emission of 405/445nm. T cell apoptosis rate was also detected by anti-7AAD antibody (abcam, Cambridge, UK) with an emission of 550/650nm.

We harvested inguinal lymph nodes at 2 weeks after ACLT and digested the lymph nodes with a mixture of type II collagenase (Yeasen, Shanghai, China) and DNase I (Yeasen, Shanghai, China) for 20 min at room temperature. The digest was filtered through a 70 μm cell strainer (Miltenyi, Germany) and then washed with 1× PBS. For staining of cytokine interleukin 17a (IL-17a), cells were stimulated with the leucocyte activation cocktail BD Pharmingen (BD Biosciences, Franklin Lakes, New Jersey, U.S.) for 4 hours at 37°C. Then, the cells were incubated with antibodies against surface markers on ice for 30 minutes in the dark. After fixation and permeabilization with a BD CytoFix/CytoPerm Kit (BD Biosciences, Franklin Lakes, New Jersey, U.S.), cells were stained with IL-17a fluorescent antibodies on ice for an additional 30 minutes in the dark. The T cell panel included the following antibodies: Zombie NTR Fixable Viability APC-Cy7 (Biolegend, San Diego, California, U.S), CD25 PE-Cy7 (Biolegend), CD3 FITC (Biolegend) and IL-17a PE (Biolegend). Analyses were performed using FlowJo software.

Placental chorionic villi were identified microscopically. Placental villi from the first-trimester placenta were separated from the chorionic membrane, villi from the second-trimester placenta were separated at the base of villi near the chorionic plate, and then the decidua and chorionic membrane were removed from the villous tissue. The remaining freshly floating and anchoring chorionic villi were washed with cold phosphate-buffered saline (PBS) 3–5 times and then cut into approximately 2–4 mm pieces. Tissues were mechanically and enzymatically dissociated using a human tissue dissociation kit (Miltenyi Biotec, Bergisch Gladbach, Germany, #130-095-929) on a gentleMACS™ Dissociator (Miltenyi Biotec). After dissociation, the homogenate was filtered using a 70-μm cell strainer (Miltenyi Biotec, #130-098-462) and a 40-μm cell strainer (Corning, Coring, NY, USA, #352340). To remove red blood cells, Red Blood Cell Lysis Solution (Miltenyi Biotec, #130-094-183) was used. Subsequently, the cells were washed once and resuspended in 100 μL of Ca²⁺ and Mg²⁺ free PBS containing 0.04% bovine serum albumin (BSA). Cell concentration and cell viability were determined by a hemocytometer and trypan blue staining.

Kidney single‐cell suspension was prepared as described previously. Briefly, the kidney was harvested from mice that were anesthetized. The kidney tissue was cut into 1–2 mm³ small pieces with scissors and then digested with 1 mg/ml collagenase I and collagenase IV (#C0130, #C5138, Sigma Aldrich) at 37°C for 30 min. The digested cell solution was passed through a 70 μm cell strainer (Miltenyi Biotec, Bergisch Gladbach) and washed twice with ice‐cold PBS. Flow cytometry was performed using the single‐cell suspension prepared according to the manufacturer's protocols. The reagent and antibodies used were as follows: live‐dead‐7‐AAD (#420403, BioLegend), CD45‐APC‐Cy7 (#157617, BioLegend), F4/80‐FITC (#157309, BioLegend), CD86‐APC (#105011, BioLegend), CD206‐PE (#141705, BioLegend), as well as their isotype controls. FACS data were acquired using a Beckman Coulter cytometer (Cytomics FC 500).

Given that immune cells were reported to be overrepresented in lung tissues^{15 (link)} and epithelial cells are relatively vulnerable to freezing and thawing, we employed balancing of major cell groups of the lung using cell surface marker labeling followed by FACS sorting. Balancing of fresh-dissociated lung cells using FACS sorting has been reported by other groups^{15 (link)}. Specifically, the dissociated single-cell suspensions were thawed and filtered using a 70 μm cell strainer (Miltenyi Biotec) to remove debris. The filtered cells were labelled with cell viability marker (DAPI) and antibody markers of EPCAM, CD31, and CD45. Live single-cells (DAPI-negative) were sorted based on three gates: EPCAM⁺CD45⁻ (designated “epithelial”), EPCAM⁻CD45⁺ (designated “immune”), and EPCAM⁻CD45⁻ (designed “endothelial or stromal”). To enrich epithelial cells, which are considered to have key roles in lung cancer etiology, we collected all “epithelial” cells from EPCAM⁺CD45⁻ gates and balanced the ratios to 6:3:1 (“epithelial”: “immune”: “endothelial or stromal”).

Blood was taken from either the left or the right caudal vein with 1 ml syringe and rapidly transferred to heparin saline anticoagulant tubes. For each rat, 0.5–1 ml of venous blood was harvested. Spleen single-cell suspensions were obtained by grinding followed by filtration through a nylon mesh. For leukocyte retrieval, the samples from the blood and spleen were centrifuged at 3000 rpm for 4 min at 4 °C. The pellets were treated with the red blood cell lysis buffer (Beyotime Biotechnology Co. Ltd., Jiangsu, China) and washed twice with phosphate buffer solution (PBS, HyClone Laboratories Inc., Logan, UT) to remove red blood cells. For brain cell isolation, the contralateral and ipsilateral hemispheres were cut into small pieces, ground, and filtered through a 70-μm cell strainer (Miltenyi Biotec, Bergisch Gladbach, Germany) to obtain a single cell suspension. Cell pellets were resuspended in 70% Percoll (Solarbio, Beijing, China), transferred into 15 ml tubes, and then overlaid with 30% Percoll. The mononuclear cells were harvested after centrifuging at 2000 rpm for 25 min at room temperature [25 (link)]. The leukocytes were analyzed for membrane marker expression by flow cytometry.