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

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Abstract
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.