Ionomycin

Splenic CD8⁺ T cells were isolated by negative selection using the MojoSort Mouse CD8⁺ T Cell Isolation Kit (Biolegend) following the manufacturer's instructions. Isolated cells were plated at 2×10⁶ per mL in RPMI 1640 containing 2.0 mM L-glutamine and 25 mM HEPES, supplemented with 10 mM Sodium Pyruvate, nonessential amino acids, 100 U/ml penicillin/streptomycin, 55 μM β-ME, and 10% fetal calf serum (complete media). Cells were stimulated with 1 μg/mL ionomycin (Invivogen) and 1 ng/mL phorbol myristate acetate (PMA, Invivogen) for 7 days, with complete media supplemented with 200 U/mL recombinant human IL-2 added on days 3 and 6. Following stimulation, cells were resuspended at 5×10⁵ per mL in RPMI 1640 supplemented with 0.1% bovine serum albumin, and were incubated at 4°C for 60 min with or without 100 nM (±)-AMG 487 (R&D Systems). Cells were then plated in the top well of 96 well transwell plate (3 μm polycarbonate membrane pore, Corning). The bottom well of the plate contained RPMI 1640 supplemented with 0.1% BSA, either with or without recombinant mouse CXCL9 or CXCL10 (Biolegend). Cells were allowed to migrate for 1 hr at 37°C, prior to data collection with a MACSQuant VYB flow cytometer (Miltenyi Biotech) and analysis using FlowJo version 10.

We evaluated the immunological responsiveness of T-cells from different organs by quantifying the intracellular IFN-γ production in the CD8⁺ and CD4⁺ T-cells isolated from the immunized mice using a previously reported method.^{[24 (link)]} Briefly, we prepared single cell suspensions from the lung, spleen and LN harvested 7 days after the boost immunization, using the procedure described above. Cells were cultured in Iscove modified Dulbecco medium media (Thermo Fisher Scientific) supplemented with 10% HI-FBS, 2 mM L-glutamine and 1% penicillin/ streptomycin at 37 °C. Cells from spleen and LN were plated on a 6-well plate at a 7 × 10⁶ cells/well and cultured in presence of monensin (Sigma) and 1 μg mL^−1 of SIINFEKL (InvivoGen) or 100 μg mL^−1 OVA (InvivoGen) for 6 hours to re-stimulate OVA-specific CD8⁺ or CD4⁺ T-cells, respectively. In parallel, cells from the lung were plated in the same manner and re-stimulated with 1 μg mL^−1 ionomycin and 50 ng mL^−1 PMA (InvivoGen) for 4 hours. Cells from all three organs were additionally treated with 5 μg mL^−1 brefeldin A (Thermo Fisher Scientific) for 2 and 3 hours to inhibit secretion of newly produced IFN-γ from CD8⁺ and CD4⁺ T-cells, respectively, prior to the cell collection. Cells were then stained for flow cytometric analysis.