title: Granzyme B and the downstream granzymes C and/or F are important for cytotoxic lymphocyte functions.
abstract: Although the functions of granzyme A (GzmA) and GzmB are well-defined, a number of orphan granzymes of unknown function are also expressed in cytotoxic lymphocytes. Previously, we showed that a targeted loss-of-function mutation for GzmB was associated with reduced expression of several downstream orphan granzyme genes in the lymphokine-activated killer cell compartment. To determine whether this was caused by the retained phosphoglycerate kinase I gene promoter (PGK-neo) cassette in the GzmB gene, we retargeted the GzmB gene with a LoxP-flanked PGK-neo cassette, then removed the cassette in embryonic stem cells by transiently expressing Cre recombinase. Mice homozygous for the GzmB null mutation containing the PGK-neo cassette (GzmB-/-/+PGK-neo) displayed reduced expression of the closely linked GzmC and F genes in their MLR-derived CTLs and lymphokine-activated killer cells; removal of the PGK-neo cassette (GzmB-/-/DeltaPGK-neo) restored the expression of both genes. Cytotoxic lymphocytes derived from mice with the retained PGK-neo cassette (GzmB-/-/+PGK-neo) had a more severe cytotoxic defect than those deficient for GzmB only (GzmB-/-/DeltaPGK-neo). Similarly, GzmB-/-/+PGK-neo mice displayed a defect in the allogeneic clearance of P815 tumor cells, whereas GzmB-/-/DeltaPGK-neo mice did not. These results suggest that the retained PGK-neo cassette in the GzmB gene causes a knockdown of GzmC and F expression, and also suggest that these granzymes are relevant for the function of cytotoxic lymphocytes in vitro and in vivo.
authors.affiliation: Division of Oncology, Department of Internal Medicine, Siteman Cancer Center, Washington University School of Medicine, St. Louis Children's Hospital, St. Louis, MO 63110, USA.
authors.forename: Paula A
authors.initials: PA
authors.lastname: Revell
authors.forename: William J
authors.initials: WJ
authors.lastname: Grossman
authors.forename: Dori A
authors.initials: DA
authors.lastname: Thomas
authors.forename: Xuefang
authors.initials: X
authors.lastname: Cao
authors.forename: Rajesh
authors.initials: R
authors.lastname: Behl
authors.forename: Jane A
authors.initials: JA
authors.lastname: Ratner
authors.forename: Zhi Hong
authors.initials: ZH
authors.lastname: Lu
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Ley
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D003602:Cytotoxicity, Immunologic / Q000235:genetics; D053222:Fas Ligand Protein; D005260:Female; D018390:Gene Targeting / Q000379:methods; D053804:Granzymes; D016131:Lymphocyte Subsets / Q000201:enzymology / Q000276:immunology; D008297:Male; D008562:Membrane Glycoproteins / Q000172:deficiency / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D016254:Mutagenesis, Insertional; D009374:Neoplasms, Experimental / Q000201:enzymology / Q000276:immunology / Q000473:pathology / Q000517:prevention & control; D054353:Perforin; D010735:Phosphoglycerate Kinase / Q000235:genetics; D052899:Pore Forming Cytotoxic Proteins; D011401:Promoter Regions, Genetic; D012697:Serine Endopeptidases / Q000096:biosynthesis / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D013602:T-Lymphocytes, Cytotoxic / Q000201:enzymology* / Q000276:immunology*
chemical_list: D053222:Fas Ligand Protein; C505658:Fasl protein, mouse; D008562:Membrane Glycoproteins; D052899:Pore Forming Cytotoxic Proteins; D054353:Perforin; D010735:Phosphoglycerate Kinase; D053804:Granzymes; C509214:Gzmb protein, mouse; C509216:Gzmc protein, mouse; C056485:Gzmf protein, mouse; D012697:Serine Endopeptidases