Functional analysis of human lymphocytes in spleen of NOD/SCID/γ_c^null mice. Four to 6 months after the transplantation of 2 × 10⁴ to 5 × 10⁴ CB CD34⁺cells, human lymphocytes in spleen were cultured and expanded for functional analyses. (A) Cell proliferation after 10-day culture; PHA (1 μg/mL) and hIL-2 (50 IU/mL) for the initial 48 hours followed by hIL-2 (50 IU/mL) only for 8 days. (B) Supernatants of the spleen cells after 48-hour stimulation with PHA and hIL-2 were taken, and the production of human cytokines was evaluated using Cytometric Bead Array Kit for human cytokine (BD PharMingen). (C) PHA-stimulated human lymphocytes were further stimulated with 10 ng/mL PMA and 1 μg/mL ionomycin for 6 hours. Brefeldin A was added during the last 2 hours. Then the cells were stained with membrane CD56 and intracellular cytokines and were analyzed using flow cytometry. (D) RNA was isolated from the spleen and BM of NOD/SCID/γ_c^null mice with or without transplanted CD34⁺cells, and the expression of mRNA for human IL-2 and IL-15 was examined by RT-PCR. Human or mouse HPRT was used as a positive control. (E-G) Anti–CD3-dependent cytotoxic T-lymphocyte activity and NK activity were evaluated by measuring the release of calcine-AM into the supernatant after cytolysis of target cells labeled with calcine. Results are expressed as the percentage of specific lysis. PB MNCs of a healthy adult were used as a positive control. Representative data with similar results of 4 independent experiments are shown.