IFN-γ ELISpot Assay for CD8+ T Cell Response

IFN-γ ELISpot assay was performed as previously described [11 (link)], with some modifications. Briefly, multiscreen-IP plates (Millipore, Bedford, MA) were coated with anti-IFN-γ mAb (AN-18, BioLegend, San Diego, CA) at 5μg/ml in PBS. Enriched CD8⁺ T cells from infected mice were incubated with MHC II^-/- BMDCs with media alone or 5μM of Mtb peptide, in duplicate. To confirm Qa-1 restriction, BMDCs were pre-incubated with 2 μg/ml anti-Qa-1 blocking mAb (6A8.6F10.1A6, BD, Franklin Lakes, NJ) or mouse IgG1 isotype control (clone MOPC-21) (BioXCell, West Lebanon, NH) prior to adding peptide and lymphocytes. After 18h incubation at 37°C, plates were washed using PBS/0.05% Tween 20 and developed using biotinylated α-IFN-γ mAb (R4.6A2, eBioscience, San Diego, CA), followed by streptavidin-conjugated alkaline phosphatase (Jackson ImmunoResearch Laboratories, West Grove, PA) and a BCIP/NBT substrate kit (Bio-Rad, Hercules, CA) according to the manufacturer’s instructions. Spots were counted using an ImmunoSpot reader (Cellular Technology, Shaker Heights, OH).