Recombinant mouse il 15

Total 96-well flat-bottom plates were coated overnight with 1 μg mL⁻¹ purified anti-mouse CD3ε (Tonbo Biosciences, 145-2C11, Cat#70-0031-M001) and 60 pmoles of mouse Btnl2-Fc, PDL1-Fc or mFc (Adipogen Life Sciences) at 4 °C and washed twice with DPBS before adding IELs to the cultures. Freshly isolated IELs were labeled with CellTrace CFSE Cell Proliferation dye according to the manufacturer’s instructions (Thermo Fischer Scientific, Cat#C34554). CFSE-labeled IELs were plated at 200,000 cells per well in RPMI 1640 supplemented with 10% FCS, 1% Pen/Strep, 2% HEPES, 1% Glutamine, 1% nonessential amino acids, 1% sodium pyruvate, 0.1% β-mercapto-ethanol (Gibco), recombinant mouse IL-7 (10 ng mL⁻¹, R&D), recombinant mouse IL-15 (10 ng mL⁻¹, R&D) and recombinant human IL-2 (10 ng mL⁻¹, Peprotech). Cells were incubated for 72–96 h at 37 °C in 5% CO₂ prior to analysis.

For cytokine stimulation assays with murine B lymphocytes, cells were seeded at a density of 2.5×10⁶/mL in 96-well plates, with a well solution volume of 200 µl. Cells were starved in serum-free medium for 2 hours, then were stimulated with either 0.1 nM of GM-CSF, 0.1 nM of IL-15, or both cytokines, or 0.1 nM of GIFT15 for 1 h at 37°C. Cells were collected, washed once with ice-cold PBS, and lysed in buffer supplemented with protease inhibitors and phosphatase inhibitor. Cell lysates were separated by SDS-PAGE, and Western blot analysis was performed with α-phosphorylated STAT3 and STAT5 antibodies (Cell Signaling, Danvers, MA). STAT3 antibody (Cell Signaling) detection was used as a loading control. Blotted proteins were visualized using enhanced chemiluminescence (ECL; Amersham, Amersham, UK). For detection of multiple STAT family members, the membranes were stripped of bound antibody and reprobed with antisera. For the quantification and identity of the recombinant GIFT15 protein, mouse IL-15 and GM-CSF antibodies were used (R&D Systems, Minneapolis, MN) and recombinant mouse IL-15 and/or mouse GM-CSF proteins were used as standards (R&D Systems, Minneapolis, MN).