title: Visualization of cytolytic T cell differentiation and granule exocytosis with T cells from mice expressing active fluorescent granzyme B.
abstract: To evaluate acquisition and activation of cytolytic functions during immune responses we generated knock in (KI) mice expressing Granzyme B (GZMB) as a fusion protein with red fluorescent tdTomato (GZMB-Tom). As for GZMB in wild type (WT) lymphocytes, GZMB-Tom was absent from naïve CD8 and CD4 T cells in GZMB-Tom-KI mice. It was rapidly induced in most CD8 T cells and in a subpopulation of CD4 T cells in response to stimulation with antibodies to CD3/CD28. A fraction of splenic NK cells expressed GZMB-Tom ex vivo with most becoming positive upon culture in IL-2. GZMB-Tom was present in CTL granules and active as a protease when these degranulated into cognate target cells, as shown with target cells expressing a specific FRET reporter construct. Using T cells from mice expressing GZMB-Tom but lacking perforin, we show that the transfer of fluorescent GZMB-Tom into target cells was dependent on perforin, favoring a role for perforin in delivery of GZMB at the target cells' plasma membranes. Time-lapse video microscopy showed Ca++ signaling in CTL upon interaction with cognate targets, followed by relocalization of GZMB-Tom-containing granules to the synaptic contact zone. A perforin-dependent step was next visualized by the fluorescence signal from the non-permeant dye TO-PRO-3 at the synaptic cleft, minutes before the labeling of the target cell nucleus, characterizing a previously undescribed synaptic event in CTL cytolysis. Transferred OVA-specific GZMB-Tom-expressing CD8 T cells acquired GZMB-Tom expression in Listeria monocytogenes-OVA infected mice as soon as 48h after infection. These GZMB-Tom positive CD8 T cells localized in the splenic T-zone where they interacted with CD11c positive dendritic cells (DC), as shown by GZMB-Tom granule redistribution to the T/DC contact zone. GZMB-Tom-KI mice thus also provide tools to visualize acquisition and activation of cytolytic function in vivo.
authors.affiliation: Centre d'Immunologie de Marseille-Luminy (CIML), Aix-Marseille University, UM2, Marseille, France.
authors.forename: Pierre
authors.initials: P
authors.lastname: Mouchacca
authors.forename: Anne-Marie
authors.initials: AM
authors.lastname: Schmitt-Verhulst
authors.forename: Claude
authors.initials: C
authors.lastname: Boyer
mesh_terms: D000818:Animals; D015550:Cell Degranulation; D002454:Cell Differentiation; D002478:Cells, Cultured; D015870:Gene Expression; D055879:Gene Knock-In Techniques; D053804:Granzymes / Q000096:biosynthesis* / Q000235:genetics; D007694:Killer Cells, Natural / Q000378:metabolism; D007700:Kinetics; D008088:Listeriosis / Q000276:immunology; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D008198:Lymph Nodes / Q000276:immunology; D008213:Lymphocyte Activation; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D022142:Secretory Vesicles / Q000201:enzymology / Q000378:metabolism*; D059010:Single-Cell Analysis; D013602:T-Lymphocytes, Cytotoxic / Q000502:physiology*; D000097573:Red Fluorescent Protein
chemical_list: D053804:Granzymes; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D000097573:Red Fluorescent Protein; C509214:Gzmb protein, mouse