Tetramer-based Enrichment of Antigen-specific CD4+ T Cells

Untouched CD4⁺ T cells were isolated from PBMCs using magnetic microbeads (Miltenyi Biotec). Tetramer staining and enrichment was performed as described previously (Su et al., 2013 (link)). In brief, cells were incubated for 30 min with live/dead Aqua marker (Invitrogen), washed, and then labeled with either gE or IE63 tetramers at room temperature for 45 min (14 µg/ml). Surface markers AF700-labeled anti-CD3 (UCHT3; BD), FITC-labeled anti-CD4 (SK3; BD), Pacific blue–labeled anti-CD45RA (MHCD45RA28; BD), and PE-cyanine 7 (PECy7)–labeled anti-CD56 (B159; BD), anti-CD14 (M5E2; BD), and anti-CD8 (SK1; BD) were incubated at room temperature for 15 min. Before tetramer enrichment, 1/10th staining volume was removed and added to TruCount tubes (BD) to give an absolute count of the starting number of CD4⁺ naive and memory T cells. The remaining staining volume was enriched for tetramer-positive cells using anti-PE microbeads (Miltenyi Biotec) and added to a separate TruCount tube. Samples were acquired using an LSR Fortessa (BD), and the frequency of tetramer-positive cells determined by dividing the absolute counts of tetramer positive cells by the starting number of CD4⁺ naive/memory T cells.