Polyfunctionality profile and cytolytic activity of A2/Melan-A⁺ CD8⁺ T cells after UCBT. (A) Staining for IFN-γ, IL-2, and CD107a was performed on T-cell microcultures derived from graft inoculums and UCBT recipients after stimulation with cognate peptide. Background frequencies measured in the absence of cognate peptide were subtracted. Left panel represents the frequencies of cells expressing combinations of IFN-γ, IL-2, and CD107a. Right panel depicts the frequencies of cells exhibiting 1, 2, and 3 functions. n represents the number of subjects. (B) ⁵¹Cr release assays were performed with 221.A2 targets pulsed with Melan-A_26-35 A27L peptide. Samples were considered positive when ⁵¹Cr release measured in the presence of cognate peptide was ≥ 10% of total release and ≥ 2 standard deviations above ⁵¹Cr release measured in the absence of peptide. (i) Bars represent the mean and standard error of frequencies of cytolytic A2/Melan-A⁺ CD8⁺ T-cell microcultures derived from the graft inoculum (G) and from samples obtained from study subjects after UCBT. n represents the number of subjects tested at each time point. (ii) Box-and-whisker plots represent percentage of specific lysis exhibited by T-cell microcultures derived from the graft inoculums or from samples obtained from UCBT recipients. Percentage of specific lysis was computed as (⁵¹Cr release in presence of cognate peptide − ⁵¹Cr release in absence of peptide) ×100/total ⁵¹Cr release. n represents the number of T-cell microcultures tested at each time point. (iii) Squares represent the mean and standard error of lysis in the presence (squf) or absence (squo) of cognate peptide. n represents the number of T-cell microcultures tested at each time point. There were no significant differences between time points. Statistical significance was tested with ANOVA or the Mann-Whitney U test.