title: Perforin-mediated lysis of tumor cells by Mycobacterium bovis Bacillus Calmette-Guérin-activated killer cells.
abstract: Immunotherapy with Bacillus Calmette-Guérin (BCG) is clinically established in the treatment of superficial bladder cancer. In our attempt to clarify the underlying immunological mechanism, we could previously show that stimulation of PBMC with BCG leads to the generation of cytotoxic BCG-activated killer (BAK) cells. Among others, these BAK cells as well as lymphokine-activated killer (LAK) cells have been suggested as possible effector cells during BCG therapy. To understand BCG-induced activation of effector lymphocytes more precisely, we investigated the lytic pathways of human BAK cells and compared BAK cell cytotoxicity with LAK cell cytotoxicity. Perforin and Fas ligand (FasL) are the major cytolytic molecules of cytotoxic lymphocytes. Our results demonstrate that BAK and LAK cells showed an increased expression of perforin and FasL as compared with unstimulated controls. Killing of T-24 bladder tumor as well as Jurkat cells by BAK and LAK cells was predominantly mediated via perforin as demonstrated by a drastically reduced lysis in the presence of concanamycin A and EGTA/MgCl2, respectively. In contrast, lysis (radioactive release assay) and membrane disintegration (Annexin V binding) of both targets by BAK and LAK cells could not be blocked with an inhibitory anti-FasL monoclonal antibody (NOK-1). Nevertheless, T-24 and Jurkat were susceptible to killing by recombinant soluble FasL and by Chinese hamster ovary cells expressing membrane-bound FasL. We conclude that cellular mediators of BCG effector mechanisms, such as BAK and LAK cells, kill their targets via perforin and independent of the FasL pathway. Because we also found increased numbers of perforin-expressing lymphocytes in patients after BCG therapy, our findings have potential clinical relevance because BCG therapy would not be impaired by FasL resistance of target cells, which recently has been described for some tumors.
authors.affiliation: Department of Immunology and Cell Biology, Research Center Borstel, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Brandau
authors.forename: H
authors.initials: H
authors.lastname: Suttmann
authors.forename: J
authors.initials: J
authors.lastname: Riemensberger
authors.forename: U
authors.initials: U
authors.lastname: Seitzer
authors.forename: J
authors.initials: J
authors.lastname: Arnold
authors.forename: C
authors.initials: C
authors.lastname: Durek
authors.forename: D
authors.initials: D
authors.lastname: Jocham
authors.forename: H D
authors.initials: HD
authors.lastname: Flad
authors.forename: A
authors.initials: A
authors.lastname: Böhle
mesh_terms: D000276:Adjuvants, Immunologic / Q000494:pharmacology; D000818:Animals; D001500:BCG Vaccine / Q000276:immunology* / Q000494:pharmacology; D016466:CHO Cells / Q000378:metabolism; D006224:Cricetinae; D003602:Cytotoxicity, Immunologic / Q000276:immunology; D053222:Fas Ligand Protein; D006801:Humans; D007376:Interleukin-2 / Q000494:pharmacology; D015979:Killer Cells, Lymphokine-Activated / Q000276:immunology; D007694:Killer Cells, Natural / Q000276:immunology*; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D008213:Lymphocyte Activation / Q000187:drug effects / Q000276:immunology; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000276:immunology*; D009163:Mycobacterium bovis / Q000276:immunology*; D054353:Perforin; D052899:Pore Forming Cytotoxic Proteins; D014407:Tumor Cells, Cultured; D001749:Urinary Bladder Neoplasms / Q000276:immunology* / Q000378:metabolism / Q000628:therapy; D019459:Urothelium / Q000276:immunology / Q000378:metabolism; D019014:fas Receptor / Q000096:biosynthesis / Q000276:immunology
chemical_list: D000276:Adjuvants, Immunologic; D001500:BCG Vaccine; C505661:FASLG protein, human; D053222:Fas Ligand Protein; D007376:Interleukin-2; D008562:Membrane Glycoproteins; D052899:Pore Forming Cytotoxic Proteins; D019014:fas Receptor; D054353:Perforin