MHC-I in vitro binding assay.

Peptide binding to H2-K^d or H2-L^d was measured by determination of the stabilization of MHC-I molecules on RMA-S-K^d and RMA-S-L^d cells, respectively. RMA-S cells were incubated overnight at 26°C to promote the expression of empty MHC-I molecules at the cell surface (58). For the binding assay, 2 × 10⁵ cells were incubated with peptides (50 or 100 μM) for 1 hour at 26°C followed by 2 hours at 37°C. For the stability assay, cells were incubated with 50 μM peptide at 26°C for 1 hour, then transferred at 37°C for 2, 4, or 6 hours. Cells were then washed with FACS buffer, and cell surface expression of H2-K^d or H2-L^d molecules was detected by flow cytometry using anti–H2-K^d (clone SF1-1.1, BioLegend) or anti–H2-L^d (30-5-7S, Thermo Fisher Scientific). Results are represented either as the mean fluorescence intensity (MFI) for H2-K^d or H2-L^d or as normalized binding, which was determined as MFI ratio (MFI observed in the presence of peptide/MFI observed in the absence of peptide). As a positive control for H2-L^d binding, AH1-A5 (SPSYAYHQF), an optimized peptide ligand with better affinity for H2-L^d than native AH1 (23) derived from envelope protein (gp70) of an endogenous ecotropic murine leukemia provirus, was used (59). The influenza epitope HA515 (IYSTVASSL) was used as positive control for H2-K^d binding (60).