title: Visualization of granzyme B-expressing CD8 T cells during primary and secondary immune responses to Listeria monocytogenes.
abstract: CD8 T cells contribute to long-term protection against Listeria monocytogenes infection by differentiating into memory T cells. These rapidly respond to antigen or inflammation upon secondary infection. In this study we used CD8 T cells from OT1 mice and CD4 T cells from OT2 mice expressing a fluorescent chimeric granzyme (GZMB-Tom) protein to monitor the primary response to infection with ovalbumin-expressing L. monocytogenes (Lm-OVA). We show that, unlike poorly responding CD4 T cells, CD8 T cells readily proliferated and expressed high levels of GZMB-Tom as early as 2 days after infection. FACS analysis showed GZMB-Tom expression in undivided CD8 T cells, with its level increasing over one to four divisions. OT1 T cells were visualized in the T-cell zone by confocal microscopy. This showed GZMB-Tom-containing granules oriented towards MHCII-positive cells. Twenty hours later, most OT1 T cells had divided but their level of GZMB-Tom expression was reduced. Recently divided OT1 cells failed to express GZMB-Tom. Fourteen hours after secondary infection, GZMB-Tom was re-expressed in memory OT1 T cells responding either to Lm-OVA or L. monocytogenes. Differences in the activation phenotype and in the splenic distribution of OT1 T cells were observed, depending on the challenge. Notably, OTI T cells with polarized granules were only observed after challenge with cognate antigen. This work showed that the GZMB-Tom knock-in mice in which GZMB-Tom faithfully reproduced GZMB expression, provide useful tools to dissect mechanisms leading to the development of anti-bacterial effector and memory CD8 T cells and reactivation of the memory response to cognate antigen or inflammatory signals.
authors.affiliation: Centre d'Immunologie de Marseille-Luminy, UM2 Aix-Marseille Université, Marseille Cedex 9, France; INSERM U1104, Marseille, France; CNRS UMR7280, Marseille, France.
authors.forename: Pierre
authors.initials: P
authors.lastname: Mouchacca
authors.forename: Lionel
authors.initials: L
authors.lastname: Chasson
authors.forename: Melissa
authors.initials: M
authors.lastname: Frick
authors.forename: Chloé
authors.initials: C
authors.lastname: Foray
authors.forename: Anne-Marie
authors.initials: AM
authors.lastname: Schmitt-Verhulst
authors.forename: Claude
authors.initials: C
authors.lastname: Boyer
mesh_terms: D000818:Animals; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology / Q000473:pathology; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology* / Q000473:pathology; D049109:Cell Proliferation; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics / Q000276:immunology*; D053804:Granzymes / Q000235:genetics / Q000276:immunology*; D000949:Histocompatibility Antigens Class II / Q000235:genetics / Q000276:immunology; D007249:Inflammation / Q000235:genetics / Q000276:immunology / Q000473:pathology; D008089:Listeria monocytogenes / Q000276:immunology*; D008088:Listeriosis / Q000235:genetics / Q000276:immunology* / Q000473:pathology; D051379:Mice; D008822:Mice, Transgenic; D015398:Signal Transduction / Q000235:genetics / Q000276:immunology; D013997:Time Factors
chemical_list: D000949:Histocompatibility Antigens Class II; D053804:Granzymes; C509214:Gzmb protein, mouse
keywords: cytolytic T lymphocyte; fluorescent granzyme B; immune response to bacteria; memory T cells