Use of four-colour flow cytometry to evaluate conjugate formation between human peripheral blood mononuclear cells and tumour target cells

A four-colour flow cytometry technique is described for the determination of the number and phenotype of conjugate-forming mononuclear effector cells from human blood. The discriminatory power of previously described techniques was improved by labelling the effector cells with antibodies that simultaneously identified natural killer (NK), T, and myeloid cells and by labelling the tumour target cells with fluorescein octadecyl ester (FOE), so that cell conjugates could be differentiated from nonbinding effector cells more effectively than by the use of light scatter. The simultaneous characterisation of the three classes of conjugate-forming cells within a heterogeneous human peripheral blood mononuclear cell (PBMC) population made it possible to investigate conjugate formation by each subpopulation without purification, or semipurification (e.g., by monocyte removal), of the subpopulations. The binding of PBMCs to K562 and RPMI 1788 targets was examined. Binding for all three PBMC subpopulations was optimal at 37 degrees C and was negligible at 0 degree C (except for some T cell binding to RPMI 1788 cells). At 37 degrees C, maximum binding was essentially achieved by 10 min. Sodium azide inhibited the majority of the conjugation by NK and T cells, and that inhibition could be removed by washing the cells prior to conjugation, whereas azide had a negligible effect on the binding by monocytes. It appears that effective conjugation by human peripheral blood NK and T lymphocytes requires the operation of an energy-dependent process, differentiating it from conjugation by monocytes.