CD1a-Mediated Lipid Antigen Presentation

Biotinylated CD1a monomers paired with β₂-microglobulin were provided by the NIH Tetramer Facility. CD1a-coated plates and lipid antigens were prepared as described previously^{11 (link)}. Briefly, streptavidin-coated plates (Thermo Scientific) were incubated with biotinylated human CD1a-β₂-microglobulin monomers (1 μg/well) and anti-CD11a (0.25 μg/ml) in PBS for 18 hours at room temperature. After washing with PBS, the plates were incubated with citrate buffer (pH 3.4) for 20 minutes, repeated twice with three washing steps in between. Lipid antigens were dissolved in PBS containing 0.05% Tween 20 (1-100 μM) by sonication for 30 minutes at room temperature in a water bath sonicator (Branson), followed by heating at 56°C for 20 minutes, prior to addition to plates coated with CD1a. After 48 hours of incubation at room temperature, the plates were washed with PBS three times. T cells suspended in complete media were added to the plates (1-2×10⁴ skin CD4 T cells, or 1×10⁵ lymph node CD4 T cells per well), and cultured for 3-5 days at 37°C in a CO₂ incubator (Thermo Scientific). Culture supernatants were used for cytokine analysis by ELISA.