Conversion of CD8⁺ T_N into T_SCM can be demonstrated at the antigen-specific level. (A) Identification of WT1-specific CD8⁺ T cells restricted to the host nonshared HLA-A*0101 by dextramer staining of, from top to bottom, donor LP, day 30 peripheral blood, and day 30 BM harvested from UPN#4. For each point analyzed, left plot depicts the percentage of dextramer-positive CD8⁺ T cells (WT1-dex). Dextramers carrying the same restriction element but loaded with an irrelevant peptide were used to set the gate and distinguish rare antigen-specific cells from background. Central plot shows CD45RA and CD62L expression on dextramer-positive cells (quadrants are set on the polyclonal CD8⁺ population). Right plot depicts CD95 expression on CD45RA⁺CD62L⁺ cells when detected (CD95 gate is set on the polyclonal CD8⁺ population). (B) Histograms summarizing the quantification and phenotypic characterization of CD8⁺ T cells specific for either WT1 (UPN#4, UPN#8, UPN#11, and UPN#13) or PRAME (UPN#2) in 5 patients affected by acute leukemia and with HLA typing suitable for dextramer analysis. For all cytometric dextramer acquisitions, no fewer than 1 × 10⁶ cells were stained and no fewer than 10⁵ events on the CD3⁺ gate were recorded. n.a., sample not available; n.e., population not evaluable.