A high-throughput immunopeptidome platform for MHC II alleles to characterize antigen-specific CD4 ⁺ T cells

CD4 ⁺ T cells play a pivotal role in adaptive immunity, recognizing peptide antigens presented by MHC II molecules during infections and tumor development. Identifying immunodominant MHC II epitopess is essential for understanding CD4 ⁺ T cell responses; however, current methods such as mass spectrometry, suffer from low sensitivity and throughput, while computational algorithms show variable accuracy. To overcome these challenges, we developed EliteMHCII, a high-throughput immunopeptidome profiling platform that identifies antigen-derived MHC II epitopes and measures peptide binding affinity across 24 globally common MHC II alleles. Using EliteMHCII, we assessed the immunodominant epitopes of the SARS-CoV-2 RBD protein. Validation in vaccinated individuals and humanized mouse models revealed a strong correlation between high-affinity peptides and robust CD4 ⁺ T cell responses, while low-affinity peptides failed to elicit responses. Therefore, our immunopeptidome profiling platform, EliteMHCII, serves as a rapid, high throughput, feasible platform for CD4 ⁺ T cell epitope discovery at a global populational level in the context of infectious diseases and cancer immunotherapy.