Akt-inhibited MiHA-specific CD8⁺ T cells show enhanced expansion capacity in immunodeficient mice. (A) CD8⁺ T cells containing equal amounts (5500) of MiHA-specific T cells, cultured for 14 days in the absence or presence of 8 µM Akt inhibitor, were infused in NSG mice. Equal numbers of CD4⁺ T cells (3 × 10⁶) were co-infused to support engraftment, resulting in a CD4:MiHA-specific T-cell ratio of 1:490. Mice were vaccinated once per week with DCs and supported with IL-15 every 2 to 3 days. On day 21, mice were euthanized and PB and spleen were analyzed by flow cytometry for the presence of MiHA-specific CD8⁺ T cells. (B) Tetramer staining and (C) differentiation consistency of cultured cells prior to infusion. (D) Representative tetramer staining and absolute numbers of MiHA-specific CD8⁺ T cells in PB and spleen (n = 10 mice per group). (E) T-cell differentiation phenotype of control and Akt-inhibited MiHA-specific CD8⁺ T cells after in vivo rechallenge; of each group, 1 representative mouse of 3 is shown. (F) IFN-γ production of Akt-inhibited MiHA-specific CD8⁺ T cells after in vivo rechallenge and overnight peptide stimulation; 2 independent mice are shown. Percentages represent IFN-γ⁺ cells within CD137^hi cells. Statistical analysis was performed by using a one-tailed Student t test.