Efficient immunologic reconstitution in mice following ex vivo bone marrow HSC gene transfer. (A) HSCs from SCID-X1 mice were transduced with either the A2UCOE-IL2RG or SFFV-IL2RG lentiviral vectors (Figure 1B) and transplanted into lethally irradiated 3KO Il2rg^−/−Rag2^−/−c5^−/− mice (see “Materials and methods, SCID mouse model and ex vivo lentiviral vector–mediated IL2RG gene transfer”). At 3 months following engraftment, spleens were analyzed for reconstitution of T-, B-, and NK-cell lineages by FACS. Cells were stained with anti-CD8, -CD4, -NK1.1, -IgM, and -B220 antibodies. Reconstitution of all cell lineages is observed in A2UCOE-IL2RG vector–transduced mice and SFFV-IL2RG vector–transduced mice (2 SCID-X1 mice transduced with the A2UCOE-IL2RG and 2 transduced with SFFV-IL2RG lentiviral vectors are shown). Mock indicates 3KO; Il2rg^−/−Rag2^−/−c5^−/− SCID-X1 mouse that received only untransduced HSCs. Percentages within quadrants represent the percentage of the total cell population analyzed that is present in that quadrant. (B) T-cell proliferation assay. Splenocytes isolated from mice transduced with A2UCOE-IL2RG were stimulated with Concanavalin A (Con A), IL-2, and Con A plus IL-2. Proliferating cells were assessed by incorporation of ³H-thymidine and expressed as a proliferation index (the ratio of the stimulated cells to unstimulated cells). All 5 mice transduced with A2UCOE-IL2RG showed an increased cell proliferation index compared with an untreated animal.