Cxcl9

CD4+ T cell trafficking was assessed as previously described [28 (link)]. In brief, WT and Cat Z^−/− CD4+ T cells were isolated from spleens of WT and Cat Z^−/− mice using the EasySep Mouse CD4+ T Cell Enrichment Kit (StemCell Technologies), according to the manufacturer’s instructions. 3 × 10⁵ CD4 + T cells were transferred to the upper filter of a 5 μm Transwell support plate (Corning) pre-coated overnight with 3 μg/ml ICAM-Fc (R&D Systems). T cells were allowed to settle for 30 minutes before the upper filter was exposed to the bottom chamber containing vehicle (PBS) or 1 μg/ml CXCL9 (R&D Systems), followed by an incubation period of 1 h at 37 °C. The absolute numbers of cells that migrated through the Transwell filter were enumerated with a hemocytometer.

RPMI-1640 medium containing 10% fetal calf serum was routinely used for primary cell cultures. KnockOut Serum Replacement (Thermo Fisher, Waltham, MA, USA) was used for all migration assays involving S1P. Anti-CD4-PE-Cy7 (GK1.5), anti-CD4-FITC (RM4–5), anti-CD8-PE (53-6.7), anti-CD8-PECF594 (53-6.7), anti-CD69 PerCP-Cy5.5 (H1.2F3), anti-CD44-PE (IM7), anti-CD62L-APC (MEL-14), and anti-p-Stat3-Percp cy5 were purchased from BD PharMingen (San Diego, CA, USA). Anti-CCR2-PE-Cy7 (SA203G11), anti-CD45.2-FITC (104), anti-CD45.1-APC (A20), anti-Qa-2-biotin (695H1-9-9), anti-Qa-2-Alexa Fluor 647 (695H1-9-9), anti-Ki67-PE-Cy7, anti-CXCR3-APC, and anti-CXCR6-PE were purchased from BioLegend (San Diego, CA, USA). Anti-CCR2-APC (Catalog # FAB5538A) and anti-S1P1-PE (Catalog # FAB7089P) were purchased from R&D Systems (Minneapolis, MN, USA). Unlabeled antibodies against Akt, p-Akt (Ser473), STAT3, p-STAT3 (Tyr705), FoxO1 (C29H4), and β-actin were purchased from Cell Signaling Technology (Beverly, MA, USA). Recombinant mouse CCL2, CXCL9, and CXCL16 were purchased from R&D Systems (Minneapolis, MN, USA). Stat3 inhibitor Stattic was purchased from Selleck.

Matrigel was diluted 1:4 in NK media. 50μL of this mixture was plated on the underside of a 5μm pore transwell insert (Corning, cat#CLS421). This was allowed to solidify for 20 minutes at room temperature. 2×10⁵ cells in 200μL media were plated in the top well of the plate. 100ng/mL CXCL9 (R&D systems, cat# 392-MG) was added to 400μL media plated in the lower well of the plate. The cells were allowed to migrate for 24 hours and the number of cells in the bottom well was counted using a hemocytometer.

ELISA kits were used to determine the levels of serum IL-21 (Creative Diagnostics, NY, USA), IL-18 (MBL, Nagoya, Japan), CCL20, CXCL9 (R&D, Minneapolis, USA), CCR6 (CUSABIO, Hubei, China), and CXCR3 (Cloud-clone, Houston, TX, USA), according to the manufacturer’s instructions.

Transwell assays were performed to determine the migration ability of NK cells through 3‐µm transwell filters (Corning, USA; Cat #3415) from the upper chambers to the medium in the lower chambers, with treatments of interest. Briefly, peripheral NK cells (CD3⁻CD56⁺) were isolated from PBMCs using FACS. After overnight stimulation with IL‐2, NK cells (1 × 10⁵) were placed into the upper chambers for migration test. DPP4 protein (Biolegend; Cat #764102) and the supernatants from NKTCL cells (YT and NK‐92) were incubated with DPP4 inhibitor (Linagliptin; Selleck; Cat #S3031) or DMSO (Sigma‐Aldrich; Cat #D4540) as control for 1 h, which were then incubated with culture medium without chemokines (PBS) or containing 100 ng mL⁻¹ of chemokine mixture with CXCL2 (R&D Systems, USA; Cat #276‐GB‐010), CXCL9 (R&D Systems; Cat #392‐MG‐010), and CXCL10 (R&D Systems; Cat #266‐IP‐010) for a 6‐h pretreatment and subsequently added into lower chambers. Migrated NK cells were collected for cell counting after incubation at 37 °C and 5% CO₂ for 3 h. Transwell assays were performed in seven independent replicates.

Cytokine levels were measured by commercially available enzyme-linked immunosorbent assay (ELISA) kit according to manufacturer instructions: CXCL9, IL-6, TNF-α, and CCL-17 (R&D Systems, Minneapolis, MN).