title: A method for detecting intracellular perforin in mouse lymphocytes.
abstract: Cytotoxic lymphocytes destroy pathogen-infected and transformed cells through the cytotoxic granule exocytosis death pathway, which is dependent on the delivery of proapoptotic granzymes into the target cell cytosol by the pore-forming protein, perforin. Despite the importance of mouse models in understanding the role of cytotoxic lymphocytes in immune-mediated disease and their role in cancer immune surveillance, no reliable intracellular detection method exists for mouse perforin. Consequently, rapid, flow-based assessment of cytotoxic potential has been problematic, and complex assays of function are generally required. In this study, we have developed a novel method for detecting perforin in primary mouse cytotoxic T lymphocytes by immunofluorescence and flow cytometry. We used this new technique to validate perforin colocalization with granzyme B in cytotoxic granules polarized to the immunological synapse, and to assess the expression of perforin in cytotoxic T lymphocytes at various stages of activation. The sensitivity of this technique also allowed us to distinguish perforin levels in Prf1(+/+) and Prf1(+/-) mice. This new methodology will have broad applications and contribute to advances within the fields of lymphocyte biology, infectious disease, and cancer.
authors.affiliation: Cancer Immunology Program, Peter MacCallum Cancer Centre, East Melbourne, Victoria 3002, Australia; ilia.voskoboinik@petermac.org amelia.brennan@petermac.org.
authors.forename: Amelia J
authors.initials: AJ
authors.lastname: Brennan
authors.affiliation: Cancer Immunology Program, Peter MacCallum Cancer Centre, East Melbourne, Victoria 3002, Australia; Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, Victoria 3010, Australia;
authors.forename: Imran G
authors.initials: IG
authors.lastname: House
authors.affiliation: Cancer Immunology Program, Peter MacCallum Cancer Centre, East Melbourne, Victoria 3002, Australia; Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, Victoria 3010, Australia;
authors.forename: Jane
authors.initials: J
authors.lastname: Oliaro
authors.affiliation: Cancer Immunology Program, Peter MacCallum Cancer Centre, East Melbourne, Victoria 3002, Australia;
authors.forename: Kelly M
authors.initials: KM
authors.lastname: Ramsbottom
authors.affiliation: Cancer Immunology Program, Peter MacCallum Cancer Centre, East Melbourne, Victoria 3002, Australia;
authors.forename: Magdalena
authors.initials: M
authors.lastname: Hagn
authors.affiliation: Department of Immunology, Juntendo University School of Medicine, Tokyo 113-8421, Japan;
authors.forename: Hideo
authors.initials: H
authors.lastname: Yagita
authors.affiliation: Cancer Immunology Program, Peter MacCallum Cancer Centre, East Melbourne, Victoria 3002, Australia; Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, Victoria 3010, Australia; Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria 3010, Australia;
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Trapani
authors.affiliation: Cancer Immunology Program, Peter MacCallum Cancer Centre, East Melbourne, Victoria 3002, Australia; Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, Victoria 3010, Australia; Department of Pathology, University of Melbourne, Parkville, Victoria 3010, Australia; and Department of Genetics, University of Melbourne, Parkville, Victoria 3010, Australia ilia.voskoboinik@petermac.org amelia.brennan@petermac.org.
authors.forename: Ilia
authors.initials: I
authors.lastname: Voskoboinik
mesh_terms: D000818:Animals; D002469:Cell Separation; D002478:Cells, Cultured; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D053804:Granzymes / Q000378:metabolism*; D006801:Humans; D054992:Immunological Synapses / Q000378:metabolism*; D042541:Intracellular Space / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D054353:Perforin / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*
chemical_list: D054353:Perforin; D053804:Granzymes