PR1/HLA-A2 visualization on normal and leukemia cells. Visualization was with fluorochrome-conjugated 8F4 in confocal microscopy (A) and flow cytometry (B-C). (A) From top to bottom: T2 cells loaded with PR1, T2 cells loaded with pp65, leukocytes from patient AML2, and leukocytes (PBMCs and granulocytes) from normal donor ND1. Cells were costained with Alexa Fluor 488 (A488)–conjugated anti–HLA-A2 (green; left panels) and Alexa Fluor 647 (A647)–conjugated 8F4 (red) and DAPI (blue; middle panels). Images were viewed with a Leica Microsystems SP2 SE confocal microscope with 10×/25 air, 63×/1.4 oil objectives and Leica Type F immersion oil. Leica LCS software (Version 2.61) was used for image analysis. Scale bar on merged images = 10 μm. (B) PR1 peptide is presented on HLA-A2⁺ PBMCs from normal donor ND2. Fresh peripheral blood was purified from red cells by lysis, stained with PE-conjugated 8F4 and the phenotype markers CD14, CD3, CD19, CD16, and Live/Dead Aqua viability indicator, and analyzed by flow cytometry (top panel). Scatter profiles and lineage markers identified the indicated cell types. CD14⁺ monocytes from HLA-A2⁺ NDs consistently expressed more PR1/HLA-A2 than lymphocytes and granulocytes. Healthy donor ND10 bone marrow cells were labeled with Live/Dead Fixable Aqua, 8F4, HLA-A2, CD34, CD33, CD13, CD14, and a lineage “dump” cocktail composed of Pacific Blue–conjugated CD3, CD7, CD10, CD19, and CD20 (middle panel). Myeloblasts were identified as viable Lin⁻CD33⁺CD34⁺; monocytes were CD14⁺. Healthy donor ND10 bone marrow cells were labeled with CD45, CD33, CD11b, CD16, and HLA-A2, and 8F4 (bottom panel). Granulocytes were identified based on scatter characteristics and then examined for expression of CD11b and CD16. Promyelocytes were identified as CD11b^lo/CD16^lo; immature granulocytes were CD11b^hi/CD16^lo; mature granulocytes stained brightly for both markers CD11b and CD16. (C) Histograms show representative labeling of AML samples (red) and fresh bone marrow cells (blue) with 8F4, mAb directed to lineage markers (the Lin cocktail was CD3, CD4, CD7, CD8, CD10, CD14, CD16, CD19, and CD20, all in Pacific Blue or V450 conjugates), CD38, CD34, and Live/Dead Fixable Aqua (Invitrogen; top panel). For each sample, filled histograms show live cells, and open histograms show Lin⁻CD34⁺CD38⁻ stem cells. Normal Lin⁻CD34⁺CD38⁻ cells show slightly higher 8F4 MFI than total Lin⁻ cells; in contrast, LSCs show lower PR1 expression compared with total blasts. Bottom panel combines 8F4 data from 3 different experiments. Each point represents one patient and is the mean value from 1 to 3 independent experiments. MFI for each sample was normalized and presented as a percentage of the MFI of the positive peak of Simply Cellular compensation beads labeled with 8F4. (B-C) The vertical line through the histograms represents background fluorescence established in the fluorescence-minus-one (FMO; gray) labeling control.