Fc receptors are important effector molecules of neutrophilic granulocytes (polymorphonuclear neutrophils [PMN]), connecting phagocytic cells and the specific immune response. Neutrophils from healthy donors express the low-affinity receptors for IgG Fc gamma RII (CD32) and Fc gamma RIII (CD16), but not the high-affinity receptor Fc gamma RI (CD64). The latter has been found on neutrophils from patients with certain bacterial infections and can be induced in vitro after incubation with interferon-gamma. We show here that neutrophils strongly express Fc gamma RI after in vivo application of recombinant human granulocyte colony-stimulating factor (rhG-CSF). PMN from patients receiving rhG-CSF displayed higher cytotoxicity against Daudi lymphoma cells in vitro compared with control patients and with healthy donors. Fab fragments against Fc gamma RII (monoclonal antibody [MoAb] IV.3) inhibited neutrophil-mediated cytotoxicity of healthy donors but not of patients during rhG-CSF therapy. Therefore, expression of Fc receptors by PMN was investigated by flow cytometry and the mean fluorescence intensity (MFI) was compared. After staining with MoAb 32.2 against Fc gamma RL, the median MFI of neutrophils from G-CSF patients (median, 4.78; range, 2.40 to 8.50; n = 5) was significantly higher (P = .002 and P = .001, respectively) than the median MFI of patients not receiving G-CSF (median, 1.23; range, 1.01 to 1.58; n = 6) and the median MFI of healthy donors (median, 1.04; range, 0.67 to 1.12; n = 6). Fc gamma RI disappeared after the discontinuing of the G- CSF injections, but was reinduced during the next treatment cycle with rhG-CSF. The high expression of Fc gamma RI during rhG-CSF therapy correlated with enhanced cytotoxicity. In vitro incubation with rhG-CSF also enhances cytotoxicity, but only minor increments in Fc gamma RI expression were observed. Thus, during in vivo application of rhG-CSF neutrophils acquire an additional potent receptor for mediating tumor cell killing in vitro by induction of the high-affinity receptor for IgG (Fc gamma RI, CD64).

We have investigated the capacity of polymorphonuclear phagocytes (PMN) to lyse malignant B-cell lines using antibodies and antibody derivates to a range of different B-cell antigens. PMN were found to mediate lysis of all tested B-cell lines in the presence of HLA class II antibodies L227, L243, F3.3, and CR3/43. Target cell lysis was significantly enhanced when PMN isolated during granulocyte colony- stimulating factor (G-CSF) treatment were compared with PMN from healthy donors. Only G-CSF primed PMN, expressing Fc gamma RI (CD64), lysed B cells in the presence of monoclonal antibody (MoAb) 1D10 or Lym- 1 to HLA class II related epitopes. Remarkably, PMN were consistently unable to kill malignant B cells with antibodies to the B-cell related antigens CD19, CD20, CD21, CD37, and CD38. These target antigen restriction was not observed with mononuclear effector cells, which mediated cytotoxicity with antibodies to HLA class II, but also with mouse/human chimeric constructs to CD19, CD37, and CD38. Blocking studies with Fc gamma RI antibodies and reverse antibody-dependent cellular cytotoxicity (ADCC) experiments against Fc gamma R antibody expressing hybridoma targets confirmed the pivotal role of Fc gamma RI in enhanced killing by G-CSF primed neutrophils. Bispecific antibodies (BsAb) with one specificity for Fc gamma RI, and another for a tumor associated antigen, offer an interesting approach to improve effector cell recruitment for immunotherapy. In our studies, very effective lysis was observed with G-CSF primed PMN and an [HLA class II x Fc gamma RI] BsAb. The therapeutic implications of these findings and the possible use of BsAb in combination with G-CSF are discussed.

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