Monoclonality of certain IL-15Tg leukemic cells and polyclonal CD8 T-cell expansion was demonstrated in IL-15Rα–IL-15 double-Tg mice. (A) Smaller body weights of IL-15Rα–IL-15 double-Tg mice compared with IL-15Tg and IL-15RαTg mice. The body weights of IL-15Tg, IL-15RαTg, and IL-15Rα–IL-15 double-Tg litters (n = 5 for each group) were compared. At 12 weeks and later time points (not shown), there was a significant difference between the double- and single-Tg groups (P < .05). (B) Flow cytometric assessment of the clonality of CD8 T cells in IL-15Rα–IL-15 double-Tg mice. To assess whether the CD8 T cells in IL-15Rα–IL-15 double-Tg mice maintained polyclonality based on their usage of the TCR repertoire, cells were analyzed by flow cytometry using anti–Vβ5.1/5.2, anti–Vβ8.1/8.2, and anti–Vβ11 Abs. If polyclonal, the percentages of staining for the Vβ should be 8%-15%, whereas the disappearance of this subset indicates the loss of polyclonality in the 13-month-old IL-15Tg mouse. (C) Genomic Southern blot analysis to assess the clonality of the CD8 T cells from IL-15Tg mice. DNA was extracted from CD8 T cells purified from the spleen and lymph nodes of WT mice, IL-15Rα^−/−IL-15Tg mice, a terminal-stage IL-15Tg mouse #A, a terminal-stage IL-15Tg mouse #B, or cells from the K2 leukemia cell line or the B1 leukemic cell line and a germline control (mouse embryonic fibroblast). Ten micrograms of each DNA was digested with HindIII or AleI enzymes and loaded to each lane. A ³²P-labeled TCRβ constant-region fragment was used as a probe. The positions of constant region 1 and 2 in the germline control are indicated as C1 and C2, respectively.