title: Cytotoxic CD4+ T cells in patients with B cell chronic lymphocytic leukemia kill via a perforin-mediated pathway.
abstract: OBJECTIVE
  B-cell chronic lymphocytic leukemia (B-CLL) is a clonal expansion of CD5+B cells that accumulate due to their uncontrolled growth and resistance to apoptosis. We have previously shown that up to 50% of blood CD4+ T cells in B-CLL patients have a cytotoxicity-related CD28- CD57+ phenotype and high content of both granzyme B and perforin (PF). In this study we investigate the cytotoxic potential of these cells against autologous B-CLL cells.
  
  
  METHODS
  Blood CD4+ or CD8+ T cells were positively isolated from B-CLL patients and cultured under a range of conditions with autologous purified B-CLL cells and with bispecific [anti-CD3 x anti-CD19] antibodies. Apoptosis of labeled B-CLL cells was assessed using the change of mitochondrial membrane potential with the fluorescent dye DiOC6 and confirmed by annexin V binding.
  
  
  RESULTS
  There was time- and dose-dependent killing of B-CLL cells by both CD8+ and CD4+ T cells and this ranged from 6.6 - 68.0% for CD4+ cells and 6.4 - 57.8% for CD8+ cells. Almost complete inhibition by concanamycin A suggests that CD4+ T cells like CD8+ T cells induced apoptosis through a perforin-mediated pathway, but not via Fas/FasL (as indicated by lack of blocking with brefeldin A), tumor necrosis factor alpha or TRAIL.
  
  
  CONCLUSIONS
  This study shows that blood CD4+PF+ T cells enriched in B-CLL patients, are able to kill autologous B-CLL cells ex vivo, through bispecific antibodies via a perforin mediated mechanism.
authors.affiliation: Department of Immunology,Tbilisi State University, Georgia. n_porakishvili@hotmail.com
authors.forename: Nino
authors.initials: N
authors.lastname: Porakishvili
authors.forename: Lela
authors.initials: L
authors.lastname: Kardava
authors.forename: Andrew P
authors.initials: AP
authors.lastname: Jewell
authors.forename: Kwee
authors.initials: K
authors.lastname: Yong
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Glennie
authors.forename: Arne
authors.initials: A
authors.lastname: Akbar
authors.forename: Peter M
authors.initials: PM
authors.lastname: Lydyard
mesh_terms: D017209:Apoptosis; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D018920:Coculture Techniques; D003602:Cytotoxicity, Immunologic; D005260:Female; D006801:Humans; D007700:Kinetics; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000276:immunology* / Q000473:pathology; D008297:Male; D008562:Membrane Glycoproteins / Q000378:metabolism*; D054353:Perforin; D052899:Pore Forming Cytotoxic Proteins
chemical_list: D008562:Membrane Glycoproteins; D052899:Pore Forming Cytotoxic Proteins; D054353:Perforin