TAME increases detection of antigen-specific T cells by more than 100-fold. (A) PBMCs from healthy persons were stained with influenza A–Matrix1_58-66, CMVpp65_495-503, and MART1_26-35 HLA-A2.1 tetramers labeled with PE. Enrichment was performed and the pre-enriched, enriched, and depleted fractions were analyzed. CD3⁺ CD8⁺ lineage-negative cells were gated and the percentage and corresponding frequencies of cells stained by the respective tetramers are reported. (B) Tetramer-enriched populations were further analyzed using anti-CD45RA, anti-CD11a, and anti-CD27 antibodies, permitting accurate discrimination of naive and memory T cells. CD45RA⁺ or CD45RA⁻ cells were gated and assessed for their expression of CD11a and CD27. Dot plots shown in black represent the phenotype of tetramer-positive populations, and in gray bulk CD8⁺ T cells are shown as a reference population. All numbers and percentages refer to tetramer-positive population. (C) As the use of frozen cells would be of interest for monitoring banked samples, we confirmed that TAME is not influenced by standard freeze/thawing of PBMCs. Fresh and frozen PBMC from the same donor were stained with influenza A–Matrix1_58-66, CMVpp65_495-503, and MART1_26-35 HLA-A2.1 tetramers labeled with PE. Enrichment was performed and tetramer frequency is reported. Plots are gated on CD3⁺ cells. Background staining of non-CD8⁺ T cells was unchanged by the freezing procedure.