Evaluation of the Role of IgG Antibodies in Human Natural Cell-Mediated Cytotoxicity Against the Myeloid Cell Line K562

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Abstract
The possible role of anti-target cell antibodies in human natural cell-mediated cytotoxicity against the K562 myeloid leukemia cell line was investigated in a 4-hr 51chromium release assay (CRA) by using, as effector cells, peripheral blood lymphocytes from normal, healthy donors. We investigated whether anti-target cell antibodies (IgG) were involved in the mechanism of natural cytotoxicity, either 1) as “arming” antibodies bound to the Fc receptor of the natural killer (NK) cell in vivo, or 2) by synthesis during the in vitro incubation period of the CRA. NK activity was not affected by procedures that were expected to remove cytophilic IgG from effector lymphocytes, such as washing whole, unseparated blood at 37°C three times before exposure to Ficoll-Hypaque; extensive washing (up to ten times) of purified lymphocytes at 37°C; or incubation of lymphocytes at 37°C for as long as 48 hr. After exposure of effector cells to trypsin for 30 min at 37°C, NK activity was always significantly reduced, and was not spontaneously recovered, even after incubation in culture medium for up to 48 hr. NK activity was not restored to trypsinized lymphocytes by incubation with normal autologous or allogeneic sera at 4°C, 24°C, or 37°C for times ranging from 0.5 to 18 hr before the CRA. Further, K562 was not sensitized for ADCC by these same normal sera, in contrast to the strong ADCC, which resulted when the target cells were preincubated with rabbit anti-K562 sera. Only when normal sera were left in the assay for 4 hr did we very occasionally observe boosting of cytotoxicity of untreated or trypsinized lymphocytes. More frequently, sera left in the assay caused some inhibition of cytotoxicity. When effector cell suspensions were incubated before the CRA, or for the duration of a 4 hr or 18 hr CRA, with various concentrations of potent Fab or F(ab′)2 fragments of goat anti-human F(ab′)2, NK activity was not specifically inhibited. Further, protein A from Staphylococcus aureus, which binds to the Fc portion of most subclasses of human IgG, did not inhibit NK when left in the assay at concentrations up to 100 µg/ml, but significantly inhibited, in a dose-dependent manner, K cell activity to Change cells coated with human anti-HLA serum. Thus, no clear evidence in support of a role for IgG in human NK activity against K562 cells could be demonstrated.

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