A funnel approach to enable analyses of epitope-specific human CD4 T cells specific for influenza and SARS-CoV-2

Protection against pathogens relies heavily on the adaptive immune response, whose key regulators are CD4 T cells. CD4 T cells, notable for their complex repertoire and functional potential, can most easily be dissected by identifying, quantifying, characterizing, and isolating epitope-specific cells. In the study reported here, we present a systematic and unbiased strategy that has enabled the identification of highly immunogenic peptide epitopes derived from influenza virus and SARS-CoV-2, presented by human HLA-DR proteins. Coupling the use of HLA-DR transgenic mice with infection and vaccination and highly sensitive epitope-specific cytokine ELISpot assays, we have narrowed the potential epitopes from 450 to 600 peptides to 5-15 peptides for each allele by an iterative process of elimination and selection, which we have termed a funnel approach. These epitopes have been validated in HLA-DR-typed human CD4 T cells directly ex vivo and enabled the derivation and implementation of HLA-DR peptide tetramers. Tetramer staining of human PBMCs enriched for CD4 T memory populations from healthy adult subjects, highlighted this approach as a sensitive and specific method for identifying novel epitopes, and subsequent CD4 T-cell responses to human viral infections.IMPORTANCETracking single epitope-specific CD4 T cells enables sophisticated analyses of the human response to infectious pathogens, vaccines, and probing the human CD4 T-cell immune memory compartment. The studies presented here provide an unbiased strategy for accomplishing this goal and provide a verified compilation of candidate HLA-DR-restricted CD4 T-cell peptide epitopes for future studies by researchers in the field of human immunology.