Abstract
Macrophage colony-stimulating factor (M-CSF) is known to stimulate proliferation of monocyte/macrophage progenitors and enhance in vitro antitumor cytotoxicity by murine macrophages. In this paper we have shown that recombinant human M-CSF causes human peripheral blood monocytes to differentiate in culture into metabolically active macrophage-like cells. These cells mediate very efficient antibody-dependent cellular cytotoxicity (ADCC) against human melanoma and neuroblastoma cell lines in the presence of two murine IgG3 mAbs (3F8 and R24). They also mediate antibody-independent cytotoxicity (or cytostasis) to a lesser extent. Human serum had an inconsistent effect on ADCC, but often induced similar high levels of ADCC. Cytotoxicity was measured using a novel ELISA to detect surviving tumor cells after ADCC. Two conventional isotope-release assays (51Cr and [3H]TdR) underestimated or entirely failed to detect ADCC by M-CSF-activated monocytes. Optimal activation occurred with 100-300 U/ml of M-CSF, and required 9-11 d for completion. Most of the M-CSF cultured monocytes expressed the low-affinity Fc receptor (CD16). ADCC by cells of the monocyte/macrophage lineage using murine IgG3 mAbs may have significance for the immunotherapy of human malignancies.
Highlights
The technique used to detect surviving target cells was a variation of the conventional ELISA, with surviving tumor cells used as the adsorbed antigen, and tumor-specific anti-GD2 or anti-GD3 mAbs used as the primary antibody
Due to the fact that cultured monocytes showed high nonspecific binding of most antibody subclasses, we found that only IgG3 antibodies worked satisfactorily in our assay
Since no modulation of these antigens was observable by flow cytometry, and no population of antigen-negative cells emerged after exposure to antibody, we considered this approach acceptable . (Further proof that our ELISA method reflected target cell death rather than antigen modulation was provided by the flow cytometry and tumor outgrowth experiments described below.)
Summary
The interface layer contained >95% of the starting monocyte population, and was typically 40% monocytes by size and granularity on flow cytometry These cells were suspended in BS-RPMI and allowed to adhere for 1-2 h in 96-well flat-bottomed tissue culture plates (Falcon Labware, Oxnard, CA) at a concentration of 1 .6 x 105 cells per well. Adherent cells were cultured for 9-12 d in 150 ul of medium : either 10% BS-RPMI supplemented with growth factors, or 20% human serum in RPMI. Control wells receiving antibody but no monocytes typically had 5-20% fewer cells at the end of 72 h, when compared with targets incubated in medium alone. To adjust for any antiproliferative effects of antibody, all cytotoxicity data were calculated based on parallel controls containing target cells cultured under identical conditions of antibody, medium, and cytokines. Determinations were performed in duplicate and the mean was reported
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