Inhibiting Akt signaling during priming reserved T-cell differentiation of MiHA-specific CD8⁺ T cells. CD8⁺ T cells were cultured with peptide-loaded DCs for 7 days with or without 8 µM Akt inhibitor (inh). Medium-containing cytokines and Akt inhibitor were refreshed every 2 to 3 days. Flow cytometry analysis was performed on days 7 and 14 to determine T-cell differentiation of the MiHA-specific CD8⁺ T cells. (A) Representative tetramer staining on day 14 of culture. (B) Expansion of MiHA-specific T cells, calculated from an estimated precursor frequency of 1:10⁷ (n = 4). (C) Representative staining for T-cell differentiation based on CCR7 and CD45RO expression, gated on MiHA-specific CD8⁺ T cells. (D) Median fluorescence intensity (MFI) of CCR7 expression of MiHA-specific CD8⁺ T cells relative to control (open bars, control; solid bars, Akt-inhibitor; n = 4). (E) Differentiation determined as in (C) of MiHA-specific CD8⁺ T cells on days 7 and 14 of culture (open bars, control; solid bars, Akt-inhibitor; n = 4). (F) Additional phenotypical analysis of MiHA-specific CD8⁺ T cells on day 14 of culture in the absence (open) or presence (solid) of 8 µM Akt inhibitor. (G-H) MiHA-specific CD8⁺ T cells cultured for 14 days in the absence or presence of 8 µM Akt inhibitor were fluorescence-activated cell sorter (FACS) purified and then analyzed by PCR. Two cultures of independent donors are shown; dashed line: 3% vs 60% and solid line: 20% vs 41% CCR7⁺CD45RO⁺ cells in control and Akt-inhibited MiHA-specific CD8⁺ T cells, respectively. Statistical analysis was performed by using (E) a 2-way ANOVA followed by a Bonferroni post hoc test or (D) paired Student t test. APC, allophycocyanin; PE, phycoerythrin.