In vitro propagation of cultured human natural killer cells expressing the HNK‐1 differentiation antigen and spontaneous cytotoxic function

Human natural killer (NK) cells expressing the HNK‐1 differentiation antigen were established in long‐term tissue culture for over 3 months. The fluorescence‐activated cell sorter‐purified HNK‐1⁺ cells required both phytohemagglutinin and exogenous interleukin 2 to propagate in long‐term culture. After 2 weeks of culture, virtually all of the growing cells exhibited the surface membrane phenotype associated with immature HNK‐1⁺ cells, since they simultaneously expressed the HNK‐1, Leu‐4 and Leu‐2a but lacked the M1, Leu‐3a and T6 antigens, and Fcγ receptors. They exhibited a lymphoblastoid appearance, contained cytoplasmic granules, and exhibited spontaneous cytotoxic function against a broader spectrum of target cells than did fresh HNK‐1⁺ cells from the same donor. Cultured HNK‐1⁺ cells lacked antibody‐dependent cell‐mediated cytotoxic (ADCC) function, while fresh HNK‐1⁺ were fully capable of ADCC function. On the other hand, cultured HNK‐1⁻ cells were lymphoblasts without cytoplasmic granules or NK cytotoxic function. The cultured HNK‐1⁺ cells gradually lost their HNK‐1 antigen expression over time, although the expression of other surface antigens (e.g., Leu‐4 and Leu‐2a) was unchanged. With prolonged culture (> 2 months), they also exhibited decreasing cytotoxic function and a diminished number of cytoplasmic granules. They were eventually indistinguishable from HNK‐1⁻ cells after 3 months of culture. These observations were not influenced by adding interferon‐γ to the cultures. The results demonstrate that the immature form of NK cells (that express T cell antigens) preferentially proliferate in long‐term cultures when incubated with phytohemagglutinin and interleukin 2.