Costimulation through baCD3/CD28 efficiently generates TK⁺ human lymphocytes with a preserved CD4/CD8 ratio. PBMCs (5 × 10⁶) were stimulated either with aCD3, aCD3/CD28, or baCD3/CD28. (A) After 48 hours, CD4⁺ (top row) or CD8⁺ cells (bottom row) were analyzed by flow cytometry for DNA content. The horizontal lines overlie cells in the S/M phase of the cell cycle with the relative percentages. Results with cells from one representative donor of 2 are shown. (B) At 48 and 72 hours after stimulation, cells were transduced with the RV. At day 6, TK⁺ cells were quantified by flow cytometry as LNGFR-expressing cells, along with staining for CD8. Quadrants were set according to isotype-control staining. The inserts report the percentages of cells for each quadrant. Results with cells from one representative of 5 different donors are shown. (C) At days 6, 10, and 14, cells were counted by trypan blue exclusion and analyzed by flow cytometry. Cell counts for CD4⁺ (○) or for CD8⁺ (•) TK⁺ lymphocytes are reported over time. Results with cells from one representative donor of 4 are shown.