Gating scheme and representative flow cytometric data showing the identification of CD8⁺ T-cell populations specific for CMV pp65_495-503 and PR1 in peripheral blood and bone marrow of a patient with CML pre-HSCT. (A) The forward scatter-area (FSC-A) versus forward scatter-height (FSC-H) profile was used to exclude cell aggregates and large cells from the analysis; live T cells were discriminated from dead cells, monocytes and B cells in a CD3 versus ViViD/CD14/CD19 bivariate plot. Next, fluorochrome aggregates were gated out and small lymphocytes were identified in a FSC-A versus side scatter-area (SSC-A) plot. Antigen-specific cells were then identified in a CD8 versus pHLA A*0201 tetramer plot. Gating of antigen-specific CD8⁺ T cells was performed after background assessment, which differs according to the coreceptor binding properties of each pHLA A*0201 tetrameric form as described previously.^11,16,43  Individual plots were examined visually by 2 independent observers; only consistent interpretations were included for further analysis. (B) Representative data showing the identification of PR1-specific (left columns) and CMV-specific (right columns) CD8⁺ T-cell populations in the peripheral blood (PB) and bone marrow (BM) of a patient with CML pre-HSCT (patient 13; Table 1). Cognate D227K/T228A, WT and Q115E pHLA A*0201 tetramers were used in 3 separate and parallel analyses for each antigen specificity; drawn gates identify only distinct populations of antigen-specific CD8⁺ T cells for clarity. In the example shown, PR1-specific CD8⁺ T cells preferentially localized to the bone marrow and bound cognate antigen with high avidity; in contrast, CMV-specific CD8⁺ T cells were evenly distributed between the bone marrow and peripheral blood compartments and, atypically, did not bind cognate antigen with high avidity. Differential staining intensities between antigen specificities likely reflect avidity ranges that lie within the capture window for each pHLA A*0201 tetrameric form.