(A) IFN-γ production. PBMCs obtained from macaque 02269 before and 14 days following infusion of a ΔCD19⁺ CMV-specific T cell clone were stimulated with medium, PMA/ionomycin, or peptide antigen and examined by cytokine flow cytometry. Data are gated on CD3⁺CD8⁺ cells and are representative of results from macaque 02258. (B) Transferred T cells that reexpress CD62L lack direct cytotoxicity but acquire cytotoxic function after stimulation. Left: PBMCs obtained 14–70 days after infusion of a T_CM-derived CD19⁺CD8⁺ clone to macaque 02258 were pooled, sorted into CD19⁺CD62L^–CD8⁺ and CD19⁺CD62L⁺CD8⁺ fractions (purity >80%), and examined for lysis of autologous unpulsed (white bars) or peptide-pulsed target cells (black bars) (E/T ratio, 5:1). The cultured T_CM-derived ΔCD19⁺CD8⁺ clone served as positive control for lysis. Right: Sorted CD19⁺CD62L^–CD8⁺ and CD19⁺CD62L⁺CD8⁺ T cells were stimulated using anti-CD3 and anti-CD28 mAbs for 14 days and then assayed for lysis of peptide-pulsed target cells (E/T ratio, 5:1). (C) Granzyme B expression. PBMCs and the transferred T_CM-derived clone were stained with mAbs to CD62L, CD8, and CD19 as well as intracellular granzyme B. Cells were analyzed by flow cytometry after gating on CD62L⁺CD8⁺, CD62L^–CD8⁺, CD19⁺CD62L⁺CD8⁺, and CD19⁺CD62L^–CD8⁺ cells. (D) Proliferation. PBMCs obtained from macaque 02269 on days 14–70 after infusion were sorted into CD19⁺CD62L⁺CD8⁺ (left panel) and CD19⁺CD62L^–CD8⁺ subsets (right panel), labeled with CFSE, and stimulated with peptide-pulsed APCs as described in Methods. After 5 days, CFSE dilution was assessed by flow cytometry after gating on CD19⁺CD3⁺CD8⁺ cells. M3 gate, proportion of cells that have undergone more than 5 divisions.