Characterization of effector‐target conjugates for cloned human natural killer and human lymphokine activated killer cells by flow cytometry

The present studies demonstrate that the intracellular fluorochromes calcein and hydroethidine can be used for quantification of effector‐target conjugates involving cloned human natural killer (NK) or interleukin‐2 (IL‐2) activated human lymphokine activated killer (LAK) cells by dual color flow cytometry without potential artifacts that might result from extensive modification of effector and/or target cell membranes. Cloned NK cells and LAK cells form conjugates with cultured cell lines regardless of susceptibility to lysis. The strength of the interactions in these conjugates was investigated using a variable speed vortexer. Even relatively gentle vortexing disrupted most conjugates involving fresh human peripheral blood lymphocytes (PBL) but only about one‐fourth of conjugates between K‐562 cells and human PBL that had been cultured with or without IL‐2 by this treatment. The rate of conjugate formation for LAK cells was determined to be about 3 times faster than for cloned NK cells, and both rates are considerably faster than the reported rate of formation of cytotoxic T lymphocyte (CTL) target conjugates. The differences in the rate of conjugate formation are apparently not related to target cell specificity, since LAK cells form conjugates with susceptible and resistant cell lines at comparable rates. When effector‐target conjugates are incubated at 37°C in the absence of calcium—thereby precluding lysis—the percentage of conjugated LAK or cloned NK cells decreases logarithmically with time. These results suggest that an initial equilibrium between free and conjugated lymphocytes gradually shifts in favor of unconjugated cells.