Binding of recombinant biotinylated GzmB (GzmB-Bio) to human leukocytes and cell lines analyzed by flow cytometry (FACS). (A-D) Different leukocyte subpopulations show distinct GzmB-binding patterns. (A-C) Determination of human leukocyte subpopulations and representative FACS analyses of GzmB-Bio (10 μg /mL) binding. Living cells from the chosen region (R1) were analyzed. For granulocytes and lymphocytes, background staining with SA-PE only is shown. (A) Granulocytes homogeneously bind GzmB-Bio at a low level. (B) NK cells do not bind GzmB-Bio. (C) The different lymphocyte subpopulations display strong variations in GzmB binding. Monocytes show strong and homogeneous, B cells show heterogeneous, and CD4+ and CD8+ T cells show lowest GzmB-Bio binding. Note that the cells, which are CD4^low and bind GzmB-Bio strongly, are monocytes. (D) Quantitative triplicate analysis of the experiment shown in Figure 2A-C. Bars showing GzmB-Bio/SA-PE (▪) represent the average MFI with standard deviation. As specificity control, we used biotinylated transferrin (TF-Bio; ▦). SA-PE alone is indicated by □. Note the logarithmic data presentation. (E) Titration experiment with different GzmB-Bio concentrations binding to distinct human lymphocyte subpopulations. representes monocytes; , B cells; and ▦, CD8⁺ T cells, (F) Jurkat () and K562 cells (▴) bind GzmB-Bio in a concentration-dependent manner. Mono indicates monocytes.