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

Protection from pathogenic organisms relies heavily on the adaptive immune response, for which key regulators are CD4 T cells. CD4 T cells, notable in the complexity of their repertoire and functional potential, can most easily be dissected with the ability to identify, quantify, characterize and isolate epitope-specific cells. In the study reported here, we present a systematic and unbiassed strategy that has enabled 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 with highly sensitive epitope specific cytokine ELISpot assays, we have narrowed the potential epitopes from 450-600 peptides to 5-15 peptides, 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 derivation and implementation of HLA-DR peptide tetramers. Tetramer staining of human PBMCs enriched CD4 T memory populations from healthy adult subjects highlighting this approach as a sensitive and specific method of identifying novel epitopes and subsequent CD4 T cell responses to human viral infections.