Pre coated plates

Lung MNC were assessed for responses to A/California/07/2009 (H1N1) and A/Perth/16/2009 (H3N2)^{18 (link)}. Both viruses were used at a multiplicity of infections of 0.08. Following overnight stimulation, IFN-γ expressing cells were detected using the Ferret interferon-gamma ELISpot kit with pre-coated plates (Mabtech, Nacka, Sweden). Results from duplicate tests were averaged. Data were analysed by subtracting the mean number of spots in the control wells (cells and allantoic fluid) from the mean counts of spots in wells with cells and antigen.

T cells and DCs were cultured in complete media consisting of RPMI 1640 with Glutamax (Life Technologies, Grand Island, NY, USA), supplemented with 5% heat inactivated human serum (Sigma, St. Louis, MO, USA). DC were cultured overnight with 500 U/mL each of recombinant IL-4 and GM-CSF (Peprotech, Rocky Hill, NJ, USA) and matured with 1000 U/mL recombinant IFN-γ (Peprotech) and 1 ng/mL LPS (Sigma). Prior to coculture with DC were tested for maturation status by CD83, CD86, and HLA-DR expression by flow cytometry and IL-12 production by ELISA (R&D Systems, Minneapolis, MN, USA). T cells were labeled with violet proliferation dye 450 (BD) according to the manufacturer’s instructions. T cells were cultured with DC at a 10:1 ratio, incubated at 37°C for the indicated timepoints, and analyzed for proliferation and activation by flow cytometry. Supernatants were collected and cytokines were measured by multiplex analyses (MesoScale Discovery, Rockville, MD, USA). For ELISPOT analysis, cells were collected on day 6 of MLR and analyzed for IFN-γ spot production using pre-coated plates (MabTech, Cincinnati, OH, USA). For intracellular detection of cytokines, cells were collected on day 6 of the MLR and treated with PMA (Sigma), ionomycin (Sigma), and GolgiPlug (BD, San Jose, CA, USA) for 6 h prior to addition of antibodies for flow analysis.