Cell purification, cell stimulation, and IAV infection

For each experiment, cryopreserved PBMCs were thawed and washed in RP10 medium (RPMI 1640 medium supplemented with 10% FBS plus 2 mM L-glutamine, 100 U/ml penicillin, and 100 U/ml streptomycin). Autologous, untouched naïve T cells and NK cells were purified by negative selection using MACS according to the manufacturer’s instructions. Briefly, T cells were purified using the human Naïve Pan T Cell Isolation Kit (Miltenyi Biotec; CAT# 130-097-095). This kit depletes PBMCs of cells other than naïve T cells using antibodies specific to CD45RO, CD14, CD15, CD16, CD19, CD25, CD34, CD36, CD57, CD123, HLA-DR, CD235a, and CD244 yielding cells that are CD3⁺CD45RA⁺CD197⁺. NK cells were purified using the human NK Cell Isolation Kit (Miltenyi Biotec; CAT#130-092-657). The viability for each cell type was determined using a micro flow cytometer with propidium iodide (Moxi Flow; ORFLO Technologies, Ketchum, ID) or a hemocytometer. MoDCs were pelleted and re-suspended in serum-free RPMI, then plated into wells of a 96-well U-bottom tissue culture treated plate (CELLTREAT, Pepperell, MA) at a seeding density of 2 X 10⁵ cells/200uL/well and 0.5 X 10⁵cells/50uL/well. Cal/09 or Vic/11 was then adsorbed on MoDCs at a multiplicity of infection (MOI) of 3 for 1 h at 37°C and 5% CO₂ in FBS-free RPMI. At 1 HPI, virus-containing supernatant was decanted, and MoDCs were either co-cultured with NK cells (1:4 or 4:1 ratios) and/or NK cells and pan naïve T cells (1:1:1, 4:1:1, or 1:4:1 ratios) in serum-containing RP10 medium–which acts to further inhibit viral entry–for the indicated time point (34). NK cells or T cells were stimulated with 162 nM Phorbol-Myristate-Acetate and 2.68 μM ionomycin (PMA/I) for 6 h (NK cells) or 12 h (T cells) for surface marker activation or 4 h for T cell intracellular cytokine staining. MoDCs were stimulated with 30 µg/mL Poly (I:C) for 24 h (In vivogen). In transwell experiments, MoDCs and NK cells were seeded in a 24-well plate and separated during culture by a semipermeable membrane of 0.4-μm pore size (Corning).