Human CD8⁺T_CM/Epersist long-term (100 days) in huIL-15 NOG mice and remain functional. T_CM/E and T_EM/E (10⁷) were injected intravenously at day 0, and irradiated NS0-IL-15 cells (1.5 × 10⁷) were administered 3 times a week starting at day 0, until mice were killed at day 100. (A) Mean percentage of human CD45⁺CD8⁺ cells (± SE) in mouse PBL, bone marrow, and spleen at day 100 was determined by flow cytometry (n = 5). (B) TCR Vβ repertoire of the input and long-term engrafted T_CM/E and T_EM/E. Bone marrow was pooled from mice, and human CD45⁺ cells were sorted and expanded by stimulation with anti-CD3. The percentage of CD3⁺ cells positive for the indicated TCR Vβ genes was determined by flow cytometry. (C) Bone marrow harvested at day 100 from mice engrafted with T_CM/E and T_EM/E was analyzed by flow cytometry for expression of human CD45, CD62L, CCR7, and CD28. Gating was based on staining with isotype control mAb, and the percentage of double-positive cells is indicated. (D) IL-2 production from CD45⁺ T cells derived from day 100 bone marrow of mice engrafted with T_CM/E and T_EM/E. Supernatants were collected after T cells were coincubated overnight with LCL-pp65, and IL-2 levels were determined using cytometric bead array. (E) Cytotoxic activity of human T cells derived from day 100 bone marrow of mice engrafted with T_CM/E and T_EM/E, and stimulated with anti-CD3 mAb. Target cells included OKT3-expressing LCLs, auto-LCLs or LCL-pp65. Mean percentage of ⁵¹Cr release (± SD) of triplicate wells. (F) Intracellular IFN-γ staining of human T cells derived from day 100 bone marrow of mice engrafted with T_CM/E and T_EM/E and coincubated overnight with LCL-pp65, LCL-OKT3, or auto-LCLs.