TCR sequences can be matched against pools of cognate viral peptides. (A) CEF-reactive CD8⁺ T cells were sorted by IFNγ secretion assay from 2 healthy donor (donor 1 and donor 2) PBMCs stimulated with the CEF peptides using flow cytometry. (B) Deconvolution of the reactivities of donor 1 and 2 PBMCs against individual peptides in the CEF pool using IFNγ ELISPOT. IFNγ secreting cells were quantified, as spot-forming cell (SFC) per million of CEF-reactive T cells (right; P values computed by comparison with dimethyl sulfoxide, using 1-sided 2-sample t-test of square root of spot counts). (C) Paired TCRα and TCRβ read counts from the sequencing of CEF-reactive single T cells. (D) Multiple paired TCRαβ sequences were enriched in CEF-reactive CD8⁺ T cells compared with nonreactive CD8⁺ T cells based on single-cell TCR sequencing (Red-selected for downstream cloning and expression; solid red bars, selected on the basis of prevalence; open red bars, selected based on TRBV usage; open triangles, TCRs for which antigen specificity was identified). A TCR with sequences identical to a BMLF1-specific TCR sequence previously reported in literature is indicated. (E) Dominant TCRs were expressed on Jurkat∆αβ reporter cells by lentiviral transduction, with stabilized expression of CD3 verified by flow cytometry. (F) Cognate antigens for CEF-reactive TCRs were determined by screening against individual peptides among the CEF peptide pool, with reactivity detected by IL-2 ELISA, CD69 staining, or luciferase assay. (G) Comparison of the functional avidity of FluA M1-specific TCRs identified from donors 1 and 2. EBV, Epstein-Barr virus; FluA, influenza A; HCMV, human cytomegalovirus.