Differentiation phenotype and IL-7 responsiveness of human tk⁺ mH antigen-specific CTLs. (A) The differentiation phenotype of tk⁺ mH antigen-specific CTLs after either 2 rounds (mH^×2) or 4 rounds (mH^×4) of antigenic stimulation was evaluated by flow cytometry and is expressed as the percentage of CD45RA⁻/CCR7⁻ (left panel), of CD28⁻/CD27⁺ (middle panel), and of IL-15Rβ⁺/IL-7Rα⁺ cells (right panel). Each symbol represents the result from a single donor of a group of n = 2 HLA-A2^pos/HA-1^neg and n = 3 HLA-A2^pos/H-Y^neg donors. Results from a t test paired statistical analysis are shown (*P < .05). (B) The percentage of either mH^×2 or mH^×4 tk⁺ mH antigen-specific CTLs binding annexin V after culture in the absence of antigen and in the presence (■) or in the absence (□) of exogenous IL-7 was evaluated by flow cytometry. Means ± SD of the data from n = 2 HLA-A2^pos/HA-1^neg and n = 2 HLA-A2^pos/H-Y^neg donors and results from a paired t test statistical analysis are shown (*P < .05, **P < .01). (C) The expansion of mH^x2tk⁺ mH antigen-specific CTLs after multiple, weekly rounds of stimulation with PHA (x-axis) is expressed as fold growth (y-axis). (D) The percentage of IL-7Rα⁺ cells (y-axis) was evaluated by flow cytometry before each round of PHA stimulation (x-axis). Results from linear regression analysis are shown (exact P values).