Enhanced expansion capacity of functional Akt-inhibited MiHA-specific CD8⁺ T cells in vitro. MiHA-specific CD8⁺ T cells cultured for 14 days in the absence or presence of 8 µM Akt inhibitor were rechallenged with peptide-loaded T2 cells and cultured for 7 days without Akt inhibitor. Medium with cytokines was refreshed every 2 to 3 days. Flow cytometry analysis was performed on day 7 to determine the number of MiHA-specific T-cells. (A) Representative tetramer staining on day 14 of priming and after 7 days of rechallenge. (B) Relative expansion to input of MiHA-specific CD8⁺ T cells during rechallenge of 3 independent donors, mean + standard error of the mean (SEM). (C) Expansion of MiHA-specific CD8⁺ T cells, calculated from an estimated precursor frequency of 1:10⁷, during priming and rechallenge of 3 independent donors, mean + SEM. (D-G) Rechallenged cells were stimulated overnight with 5 µM peptide and stained for CD107a, IFN-γ, and CD137. Representative plots of CD137 and (D) CD107a or (F) IFN-γ staining within CD8⁺ T cells. Percentages represent CD107a⁺ or IFN-γ⁺ cells within CD137^hi cells. Percentage of (E) CD107a⁺ or (G) IFN-γ⁺ within CD137^hi cells was analyzed for 2 independent cultures, mean + SEM. Statistical analysis was performed by using a 2-way ANOVA followed by a Bonferroni post hoc test. (H) Killing of CFSE-labeled target cell lines with or without endogenous ARHGDIB expression or peptide loading, by MACS-enriched ARHGDIB-specific T cells (45% ARHGDIB-specific CD8⁺ T cells) derived from a rechallenged Akt-inhibited culture (n = 3). **P < .01; ***P < .001.