CD4⁺ T cells orchestrate adaptive immune responses via binding of antigens to their receptors through specific peptide/MHC-II complexes. To study these responses, it is essential to identify protein-derived MHC-II peptide ligands that constitute epitopes for T cell recognition. However, generating cells expressing single MHC-II alleles and isolating these proteins for use in peptide elution or binding studies is time consuming. Here, we express human MHC alleles (HLA-DR4 and HLA-DQ6) as native, noncovalent αβ dimers on yeast cells for direct flow cytometry-based screening of peptide ligands from selected antigens. We demonstrate rapid, accurate identification of DQ6 ligands from pre-pro-hypocretin, a narcolepsy-related immunogenic target. We also identify 20 DR4-binding SARS-CoV-2 spike peptides homologous to SARS-CoV-1 epitopes, and one spike peptide overlapping with the reported SARS-CoV-2 epitope recognized by CD4⁺ T cells from unexposed individuals carrying DR4 subtypes. Our method is optimized for immediate application upon the emergence of novel pathogens.

CD4 ⁺T 细胞通过特定的肽/MHC-II 复合物将抗原与其受体结合，从而协调适应性免疫反应。为了研究这些反应，必须确定构成 T 细胞识别表位的蛋白质衍生的 MHC-II 肽配体。然而，生成表达单个 MHC-II 等位基因的细胞并分离这些蛋白质以用于肽洗脱或结合研究是耗时的。在这里，我们将人类 MHC 等位基因（HLA-DR4 和 HLA-DQ6）表达为酵母细胞上的天然非共价 αβ 二聚体，用于直接流式细胞术筛选来自选定抗原的肽配体。我们证明了从嗜睡症相关的免疫原性靶标 pre-pro-hypocretin 中快速、准确地识别 DQ6 配体。我们还鉴定了 20 个与 SARS-CoV-1 表位同源的结合 DR4 的 SARS-CoV-2 刺突肽，⁺来自携带 DR4 亚型的未暴露个体的 T 细胞。我们的方法经过优化，可在新病原体出现时立即应用。