Functional verification of pHLA A*0201 tetramer staining for PR1-specific CD8⁺ T cells. BMMCs from patient 12 (Table 1) were stimulated with PR1 peptide at 10 μg/mL and stained with a panel of mAbs to assess cell surface phenotype and intracellular cytokine production as described in the Methods. (A) Gating strategy used to identify PR1-specific CD8⁺ T cells. The FSC-A versus FSC-H profile was used to exclude cell aggregates and large cells from the analysis; lymphocytes were then identified on the basis of light scatter characteristics. Live T cells were subsequently discriminated from dead cells, monocytes and B cells in a CD3 versus ViViD/CD14/CD19 bivariate plot before gating on CD8⁺ T cells. (B) Function and phenotype of PR1-specific CD8⁺ T cells. Background staining levels were assessed in control experiments processed identically in the absence of PR1 peptide (left panel). In the presence of PR1 peptide, 0.27% of memory CD8⁺ T cells expressed IFNγ (middle panel); no other cytokines were detected in this experiment (see “Methods”). Antigen-specific CD8⁺ T cells that produced IFNγ in response to PR1 peptide, depicted as colored dots superimposed on a cloud plot representing the phenotypic distribution of the total CD8⁺ T-cell population, displayed a heterogeneous memory phenotype (right panel) consistent with similar analyses conducted in conjunction with cognate pHLA A*0201 tetramer staining (Figure 4).