title: Stably transfected antisense granzyme B and perforin constructs inhibit human granule-mediated lytic ability.
abstract: In human NK cells and CTL it has been shown that release of lytic molecules is, at least in part, responsible for the lysis of target cells (TC). Of the various types of molecules thought to be involved in cell-mediated cytotoxicity (CMC), perforin and the serine proteases (granzymes A and B) are the best described. Using mammalian expression vectors (pRSV-neo and pSV2-neo), antisense constructs for perforin and granzyme B were independently electroporated into YT-INDY, a human non-MHC-restricted, IL-2-independent, cytotoxic lymphocyte. Transfected YT-INDY was then selected for expression of the plasmid by antibiotic G418 resistance. The presence of plasmid was confirmed by detection of the integrated plasmid G418 resistance gene using PCR. The presence of antisense perforin in YT-INDY (YT-xPFP) inhibited lytic ability by > 95% compared to YT-INDY transfected with plasmid alone or plasmid with unrelated antisense (YT-neo, YT-ctrl, respectively). Likewise, the presence of antisense GrB (YT-xGrB) inhibited the lytic ability of YT-INDY by > 95%. Western analysis revealed a 30% decrease in the level of perforin and a 55% decrease in granzyme B protein levels compared to YT-neo. Northern analysis using oligo probes complementary to perforin and granzyme B mRNA showed a decrease in their respective message levels. In conclusion, stably transfected antisense constructs for perforin and granzyme B essentially eliminated the lytic ability of YT-INDY. These results strongly indicate that both perforin and granzyme B are required by this human cytotoxic lymphocyte for effective TC lysis.
authors.affiliation: Department of Microbiology, Indiana University School of Medicine, Indianapolis 46202-5200, USA.
authors.forename: M R
authors.initials: MR
authors.lastname: Bochan
authors.forename: W S
authors.initials: WS
authors.lastname: Goebel
authors.forename: Z
authors.initials: Z
authors.lastname: Brahmi
mesh_terms: D001483:Base Sequence; D015550:Cell Degranulation; D003602:Cytotoxicity, Immunologic; D016373:DNA, Antisense / Q000737:chemistry; D053804:Granzymes; D006801:Humans; D007111:Immunity, Cellular; D007694:Killer Cells, Natural / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000502:physiology*; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000737:chemistry; D054353:Perforin; D052899:Pore Forming Cytotoxic Proteins; D012697:Serine Endopeptidases / Q000502:physiology*; D013602:T-Lymphocytes, Cytotoxic / Q000378:metabolism*; D014162:Transfection
chemical_list: D016373:DNA, Antisense; D008562:Membrane Glycoproteins; D015345:Oligonucleotide Probes; D052899:Pore Forming Cytotoxic Proteins; D054353:Perforin; C509213:GZMB protein, human; D053804:Granzymes; D012697:Serine Endopeptidases