Cd86 fitc

Mononuclear cells from liver and spleen and stromal vascular cells (SVC) from AT (epididymal and perirenal depots) were isolated as described previously (Stefanovic‐Racic et al. 2012). Cell suspensions (2 × 10⁶ cells/sample) were preincubated with anti‐CD16/32 (Fc “blocking” antibodies [Abs]) for 15 min at 4°C, then stained with either fluorescent‐labeled Abs or IgG isotype controls for 30 min at 4°C. The following Abs were used: CD8/PerCP and CD45/PerCP (BD Biosciences, San Jose, CA), CD4/FITC, CD62L/PE, NK1.1/PeCy7, B220/v450, CD3/APC, CD86/FITC, MHC2/PE, CD11b/PeCy7, B220/v450, CD11c/APC, and F4/80/Alexa780 (eBiosciences, San Diego, CA). Following incubation with Abs, liver and AT cells were incubated in Aquaporin dye (Invitrogen, Grand Island, NY) for 15 min, to distinguish live from dead cells, then fixed in 4% paraformaldehyde (Fisher, Waltham, MA) before being analyzed using a FACSCalibur flow cytometer and FACSDiva software (BD Biosciences). A proportion of up to 1% false‐positive events were accepted in the isotype control samples.

Splenocytes were treated with AECs for 48 h, collected in a 1.5‐ml centrifuge tube and then stained with primary antibodies, including anti‐rat CD3‐FITC, CD4‐PE, CD8‐APC, CD45‐RA+, CD161‐PE, CD80‐PE, CD86‐FITC, and CD103‐Alexa Flox@647 (eBiosciences, CA, USA). As reported, we defined the CD3⁺CD4⁺ population as helper T (Th) cells, CD3⁺CD8⁺ population as cytotoxic T (Tc) cells, CD3^‐CD45RA⁺ population as B lymphocytes, CD3^‐CD161⁺ population as natural killer (NK) cells, CD3⁺CD161⁺ population as natural killer T (NKT) cells, and CD80‐PE, CD86‐FITC, and CD103‐Alexa Flox@647 as dendritic (DC) cells (Ayako et al., 2018 (link); Chen et al., 2018 (link); Xu, Wusiman, et al., 2019 (link)). After incubation at 4°C for 30 min, the cells were washed twice and resuspended in PBS before they were transferred to fluorescence‐activated cell sorting (FACS) tubes and analyzed by flow cytometry (BD Biosciences, San Jose, CA, USA). The cells were gated using forward and side scatter for dead cell exclusion. In each sample, 10,000 events were measured, and data were analyzed using Flow Jo 7.6 software.

Lung tissue was chopped and digested with collagenase-D (1 mg/ml; Roche) and DNase I (10 mg/ml; Sigma-Aldrich) for 1 h at 37°C with agitation. To detect cytokines, brefeldin A (5 µg/ml) was added during the digest. Lungs or spleens were passed through a 70-µm cell strainer to a obtain single-cell suspension, followed by RBC lysis with ACK buffer. The cells were incubated with Fcγblock (anti-CD16/CD32 antibody, BD Biosciences) (1:100) to block IgG Fc receptors. Cells were incubated with LIVE/DEAD Aqua (Invitrogen), followed by surface staining with fluorochrome-conjugated anti-mouse Abs for various markers. The following surface Abs were used: CD69-FITC, CD3-APC-ef780, MHCII-APC, Ly6C-PerCP-Cy5.5, CD86-FITC, CD11b-APC-ef780, CD38-ef450, F4/80-PE-Cy5, CD49d-PerCP-ef710, CD3-AF700, CD8-APC-ef780, CD44-PE-Cy7, CD4-PE-Cy5 (eBiosciences), CD44-BV605, CD4-BV785, CD103-PE, Ly6G-BV605, CD80-PE-Dazzle594 (BioLegend), CD62L-PE-CF594, SiglecF-PE, CD103-BV786 (BD Biosciences). For detection of intracellular cytokines, cells were fixed in 2% PFA and permeabilized with 0.5% saponin (Sigma-Aldrich, Ireland), followed by staining with IL-17A–V450 (BD Biosciences). Fluorescence minus one samples were used as controls. Flow cytometric analysis was performed on an LSR Fortessa, and data were acquired using Diva software (BD Biosciences). The results were analyzed using FlowJo software (TreeStar).

Cells cultured in vitro or isolated from tumors or spleens were processed for surface labeling with appropriate antibodies. Propidium iodide was used to distinguish the live cells. Cells were further permeabilized using 70% ethanol or True-Nuclear™ transcription factor buffer set (cat# 424401, BioLegend) and stained for Ki-67 or FOXP3. Data were acquired using the Beckman CytoFLEX flow cytometer and analyzed using FlowJo software. Antibodies used for flow cytometry were purchased from Biolegend (anti-mouse CD16/32, cat#101320; CD8a Alexa Fluor^® 647, cat#100724; NK 1.1 Alexa Fluor^® 647, cat#108720; Ly-6G/Ly-6C (Gr-1) Alexa Fluor^® 488, cat#108417; CD19 FITC, cat#152404; CD45R/B220 Alexa Fluor^® 647, cat#103226; F4/80 Alexa Fluor^® 488, cat#123120; CD206 Alexa Fluor^® 647, cat#141712; FOXP3 Alexa Fluor^® 488, cat#126406; CD4 Alexa Fluor^® 647, cat#100530; CD11c Alexa Fluor^® 488, cat#117311; H-2K^b/H-2D^b FITC, cat#114606; I-A/I-E Alexa Fluor^® 647, cat#107618; CD80 Alexa Fluor^® 647, cat#104718; CD86 FITC, cat#105006; CSF-1R Alexa Fluor^® 488, cat#135511) and eBioscience (anti-mouse Ki-67 FITC, cat#11-5698-82; CD3ε FITC, cat#11-0031-82; CD11b eFluor 660, cat#50-0112-82).

Splenocytes were prepared as described above. Cells were then incubated with the appropriate antibody at 4°C for 30 min in the dark in PBS with 2% FBS. Flow cytometry was performed using a FACS Fortessa II flow cytometer (BD Biosciences), according to standard techniques. For the characterization of splenic cells, the monoclonal antibodies used were: CD3-AF700 (17A2, #100216), CD4-BV510 (GK1.5, #100449), CD8-AF647 (53-6.7, #100727), CD25-PE conjugated (PC61.5, #12-0251-81), B220-BV421 (RA3-6B2, #562922), CD62l-PeCy5 (MEL-14, #104410), CD44-BV605 (IM7, #103047), CD11b-PerCPcy5.5 (M1/70, #101228), CD80-PE (16-10A1, #09605B), F4/80-BV711 (BM8, #123147), CD86-FITC (GL-1, #105005), CD206-AF647 (MR5D3, #565250), CD43-BV421 (S7, #562958), CD11c-AF700 (N418, #117320), MHCII-EFluor480 (M5/114.15.2, #48-5321-82) from eBiosciences (Thermo Fisher Scientific, Villebon-Sur-Yvette, France). Data were analyzed with FlowJo software (Tree Star, Ashland, OR).