Cxcr6

The preparation of single‐cell suspensions was described previously (Fu et al., 2020 (link)). Antibodies specific for the following surface markers were used: Ly6G (1A8‐Ly6g), CD11b (M1/70), CD45 (30‐F11), CD205 (205yekta), and TLR2 (CB225) were purchased from eBioscience (San Diego, CA, USA); CCR3 (J073E5), CCR4 (2G12), CCR5 (HM‐CCR5), CCR6 (29‐2L17), CCR7 (4B12), CCR9 (CW‐1.2), CXCR2 (SA203G11), CXCR3 (CXCR3‐173), CXCR4 (L276F12), CXCR5 (L138D7), CXCR7 (8F11‐M16), and CX3CR1(SA011F11) were purchased from Biolegend (San Diego, CA, USA); CCR1 (R&D), CCR2 (R&D), CCR8 (1055C, R&D), CCR10 (R&D), CXCR1 (R&D), and CXCR6 (R&D) were purchased from R&D Systems (Minneapolis, MN, USA). All antibodies were used at the fold dilution described on the manufacturer's recommendation. Unstained cells, single stain, fluorescence minus one control, and isotype controls were used for setting laser voltages and for compensation. FCM data were obtained by FACSAria III (BD Biosciences, San Jose, CA, USA) and analyzed with FlowJo v. 10 (BD Biosciences, San Jose, CA, USA).

Migration and invasion studies were performed using BD Biocoat migration and Matrigel invasion chambers (Becton-Dickson Labware), respectively. Serum free DMEM was added to bottom and top chamber of inserts and allowed to hydrate for 2 h at 37°C with 5% CO₂. Next, 0.5 × 10⁵ cells were added to the top chamber of inserts and 100 ng/ml CXCL16 (Peprotech, NJ) was added as chemo-attractant in the bottom chamber. To determine if the migration and invasion of LuCa cells is mediated specifically by CXCR6-CXCL16 interaction, cells pre-incubated with 1.0 μg/ml anti-CXCR6 antibody (MAB699, R&D Systems) were added to the top chamber in one well of Matrigel or control inserts and allowed to migrate/invade under chemotactic gradient of CXCL16 for overnight at 37°C and 5% CO₂. After incubation, non-migrating cells on the upper surface of the membrane were removed with a cotton swab. Cells at the bottom surface of the insert were fixed with 100% methanol for 2 min, stained for 2 min with crystal violet (Fisher Scientific), and rinsed twice with de-ionized water. Migrated/invaded cells were counted by microscopy at 40× magnification. All experiments were repeated three times to validate the results.

Cells were harvested in PBS with 5 mM EDTA and washed in staining buffer (PBS without Ca²+ Mg²⁺, 0.5% BSA, 2 mM EDTA, 0.025% NaN₃). mAbs directly conjugated to PE and APC fluorochromes and specific for the following antigens were used: MHC class I (APC anti-mouse H-2Kb/H-2Db, BioLegend), CD1 (APC anti-mouse CD1d, clone 1B1, BioLegend), CD44(APC rat anti-mouse CD44, clone IM7, BD Pharmingen), PD-L1 (BD Pharmingen), CXCR6 (R & D systems). Corresponding isotypes were used for negative control. Immunostaining was performed with saturating amounts of Abs for 30 min at 4°C. Samples were acquired with a flow cytometer FACSCanto II (BD Biosciences) and data were elaborated using FlowJo 9.3.2 software (TreeStar).