title: Enhanced outgrowth of EBV-transformed chronic lymphocytic leukemia B cells mediated by coculture with macrophage feeder cells.
abstract: B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the clonal expansion of CD5-expressing B lymphocytes that produce mAbs often reactive with microbial or autoantigens. Long-term culture of B-CLL clones would permit the collection and characterization of B-CLL mAbs to study antigen specificity and of B-CLL DNA to investigate molecular mechanisms promoting the disease. However, the derivation of long-term cell lines (eg, by EBV), has not been efficient. We have improved the efficiency of EBV B-CLL transformation of CpG oligonucleotide-stimulated cells by incubating patient peripheral blood mononuclear cells in the presence of an irradiated mouse macrophage cell line, J774A.1. Using this approach, peripheral blood mononuclear cells isolated from 13 of 21 B-CLL patients were transformed as documented by IGHV-D-J sequencing. Four clones grew and retained CD5 expression in culture for 2 to 4 months. However, despite documentation of EBV infection by expression of EBNA2 and LMP1, B-CLL cells died after removal of macrophage feeder cells. Nevertheless, using electrofusion technology, we generated 6 stable hetero-hybridoma cell lines from EBV-transformed B-CLL cells, and these hetero-hybridomas produced immunoglobulin. Thus, we have established enhanced methods of B-CLL culture that will enable broader interrogation of B-CLL cells at the genetic and protein levels.
authors.affiliation: Duke Human Vaccine Institute, Duke University Medical Center, Durham, NC 27710, USA.
authors.forename: Kwan-Ki
authors.initials: KK
authors.lastname: Hwang
authors.forename: Xi
authors.initials: X
authors.lastname: Chen
authors.forename: Daniel M
authors.initials: DM
authors.lastname: Kozink
authors.forename: Marietta
authors.initials: M
authors.lastname: Gustilo
authors.forename: Dawn J
authors.initials: DJ
authors.lastname: Marshall
authors.forename: John F
authors.initials: JF
authors.lastname: Whitesides
authors.forename: Hua-Xin
authors.initials: HX
authors.lastname: Liao
authors.forename: Rosa
authors.initials: R
authors.lastname: Catera
authors.forename: Charles C
authors.initials: CC
authors.lastname: Chu
authors.forename: Xiao-Jie
authors.initials: XJ
authors.lastname: Yan
authors.forename: Micah A
authors.initials: MA
authors.lastname: Luftig
authors.forename: Barton F
authors.initials: BF
authors.lastname: Haynes
authors.forename: Nicholas
authors.initials: N
authors.lastname: Chiorazzi
mesh_terms: D000818:Animals; D048708:Cell Growth Processes / Q000502:physiology*; D002461:Cell Line, Transformed; D002472:Cell Transformation, Viral / Q000235:genetics / Q000502:physiology; D002478:Cells, Cultured; D018920:Coculture Techniques; D019309:Epstein-Barr Virus Nuclear Antigens / Q000235:genetics; D061252:Feeder Cells / Q000166:cytology* / Q000502:physiology; D004854:Herpesvirus 4, Human / Q000235:genetics / Q000502:physiology*; D006801:Humans; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000235:genetics / Q000473:pathology*; D008264:Macrophages / Q000166:cytology* / Q000502:physiology; D051379:Mice; D015854:Up-Regulation; D014764:Viral Proteins / Q000235:genetics
chemical_list: C402641:EBNA-2 protein, Human herpesvirus 4; D019309:Epstein-Barr Virus Nuclear Antigens; D014764:Viral Proteins