title: Rituximab reduces the number of peripheral blood B-cells in vitro mainly by effector cell-mediated mechanisms.
abstract: OBJECTIVE
  The humanized CD20 mono- clonal antibody, rituximab, has significant anti-tumor activity in patients with B-cell non-Hodgkin's lymphoma and induces depletion of B-cells in vivo. It was the objective of this study to define the contribution of the different mechanisms of action of rituximab on primary normal and malignant B-cells.
  
  
  METHODS
  Primary human B-lymphocytes and effector cell fractions were isolated from peripheral blood of normal donors using an immunomagnetic separation technique. Blood samples from 20 patients with chronic lymphocytic leukemia (CLL) were studied and the B-lymphoblastoid Daudi cell line was used as a control. B-cells were cultured in the presence or absence of rituximab adding a secondary hyper-crosslinking antibody, serum as source of complement or different effector cell fractions. The cells were analyzed by immunofluorescence staining and flow cytometry.
  
  
  RESULTS
  In contrast to the B-lymphoblastoid Daudi cell line, the number of highly purified normal peripheral blood CD19+ cells was only minimally affected by rituximab in the presence of autologous serum. A significant reduction in the number of B-cells was observed when mononuclear cells from peripheral blood were added back. To identify the cell type which mediates this effect, CD3+ T-cells, CD56+ cells, and CD14+ monocytes were added to selected CD22+ B-cells. A marked B-cell decrease was only observed in the presence of CD56+ and CD14+ cells in an effector to target ratio of 10:1. The experiments with mononuclear cells from patients with CLL showed a B-cell reduction by rituximab, which was significantly enhanced following addition of granulocyte-macrophage colony-stimulating factor (GM-CSF).
  
  
  CONCLUSIONS
  These data support the important role of cell-mediated mechanisms in the B-cell-depleting action of rituximab and suggest that pre-treatment with GM-CSF could improve the response to rituximab in patients with CLL.
authors.affiliation: German Cancer Research Center, Heidelberg, Germany. mtvoso@rm.unicatt.it
authors.forename: Maria Teresa
authors.initials: MT
authors.lastname: Voso
authors.forename: Gitta
authors.initials: G
authors.lastname: Pantel
authors.forename: Sergio
authors.initials: S
authors.lastname: Rutella
authors.forename: Mirjam
authors.initials: M
authors.lastname: Weis
authors.forename: Francesco
authors.initials: F
authors.lastname: D'Alò
authors.forename: Raffaella
authors.initials: R
authors.lastname: Urbano
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Leone
authors.forename: Rainer
authors.initials: R
authors.lastname: Haas
authors.forename: Stefan
authors.initials: S
authors.lastname: Hohaus
mesh_terms: D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D058846:Antibodies, Monoclonal, Murine-Derived; D001402:B-Lymphocytes / Q000166:cytology* / Q000187:drug effects* / Q000473:pathology; D017252:CD3 Complex / Q000378:metabolism; D019002:CD56 Antigen / Q000378:metabolism; D002452:Cell Count; D002455:Cell Division / Q000187:drug effects; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D006801:Humans; D015448:Leukemia, B-Cell / Q000473:pathology; D018950:Lipopolysaccharide Receptors / Q000378:metabolism; D000069283:Rituximab
chemical_list: D000911:Antibodies, Monoclonal; D058846:Antibodies, Monoclonal, Murine-Derived; D017252:CD3 Complex; D019002:CD56 Antigen; D018950:Lipopolysaccharide Receptors; D000069283:Rituximab