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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\u201315 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\n                      <jats:italic toggle=\"yes\">ex vivo<\/jats:italic>\n                      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.\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>Tracking 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.<\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/mbio.00449-26","type":"journal-article","created":{"date-parts":[[2026,5,19]],"date-time":"2026-05-19T13:00:20Z","timestamp":1779195620000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["A funnel approach to enable analyses of epitope-specific human CD4 T cells specific for influenza and SARS-CoV-2"],"prefix":"10.1128","volume":"17","author":[{"given":"K. A.","family":"Richards","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/022kthw22","id-type":"ROR","asserted-by":"publisher"}],"name":"David H. 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