Granzyme B‐tdTomato, a new probe to visualize cytolytic effector cell activation

Mouchacca, Pierre; Guyot, Vincent; Grégoire, Claude; Schmitt-Verhulst, Anne-Marie; Boyer, Catherine

doi:10.1002/eji.201141671

Cited by 4 publications

(14 citation statements)

References 10 publications

(10 reference statements)

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“…GZMB-Tom was analyzed at the G610 channel as described [39] and this fluorescence is designated as tdTom.…”

Section: Methodsmentioning

confidence: 99%

“…Immunoblots to define the MW of the GZMB-Tom fusion protein expressed in GZMB-Tom-KI/KI CTL were performed as described [39]. A protein of around 80kDa was recognized by the anti-RFP Ab that cross-reacts with tdTom (Fig.S2).…”

Section: Methodsmentioning

confidence: 99%

“…As previously described [39], CTL and target cells (RMA-S cells loaded with either the relevant OVA SIINKEFKL or irrelevant pKB1 peptide or EG7-DEVD cells) were mixed at different effector to target ratios (1/1 or 3/1) in the presence of anti-Lamp-1/CD107 mAb [34] for 1–2h at 37°C. Labeling with anti-CD8 mAb was then performed before FACS analysis at the LSRII-561.…”

Section: Methodsmentioning

confidence: 99%

“…Fixed or fixed and permeabilized cells were prepared for confocal analysis as described [39] and analyzed using a Leica SPX5 or Zeiss780 confocal microscope.…”

Section: Methodsmentioning

confidence: 99%

“…With that aim and based on our previous experience [39], we have constructed by homologous recombination a KI mouse genetically modified to express a fusion protein between GZMB and tdTomato (GZMB-Tom), a powerful red fluorescent tandem dimer protein [40], in place of GZMB, maintaining the gene expression control elements of the endogenous gmzb gene. This mouse, called GZMB-Tom-KI, has normal lymphocyte development and distribution.…”

Section: Introductionmentioning

confidence: 99%

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Visualization of Cytolytic T Cell Differentiation and Granule Exocytosis with T Cells from Mice Expressing Active Fluorescent Granzyme B

2013

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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.

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“…GZMB-Tom was analyzed at the G610 channel as described [39] and this fluorescence is designated as tdTom.…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…Fixed or fixed and permeabilized cells were prepared for confocal analysis as described [39] and analyzed using a Leica SPX5 or Zeiss780 confocal microscope.…”

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Visualization of Cytolytic T Cell Differentiation and Granule Exocytosis with T Cells from Mice Expressing Active Fluorescent Granzyme B

2013

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A fluorescent reporter model for the visualization and characterization of T_DC

Fiore,

Sala,

Laura

et al. 2023

Eur J Immunol

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TDC are hematopoietic cells that combine dendritic cell (DC) and conventional T cell markers and functional properties. They were identified in secondary lymphoid organs (SLOs) of naïve mice as cells expressing CD11c, major histocompatibility molecules (MHC)‐II, and the T cell receptor (TCR). Despite thorough characterization, a physiological role for TDC remains to be determined. Unfortunately, using CD11c as a marker for TDC has the caveat of its upregulation on different cells, including T cells, upon activation. Here we took advantage of Zbtb46‐GFP reporter mice to explore the frequency and localization of TDC in different tissues at steady state and upon viral infection. RNA sequencing analysis confirmed that TDC sorted from Zbtb46‐GFP mice have a gene signature that is distinct from conventional T cells and DC. In addition, this reporter model allowed for identification of TDC in situ not only in SLOs but also in the liver and lung of naïve mice. Interestingly, we found that TDC numbers in the SLOs increased upon viral infection, suggesting that TDC might play a role during viral infections. In conclusion, we propose a visualization strategy that might shed light on the physiological role of TDC in several pathological contexts, including infection and cancer.This article is protected by copyright. All rights reserved

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Murine CD8+T cell cytotoxicity against schistosomula induced by inoculation of schistosomal 22.6/26GST coupled Sepharose 4B beads

Zhou

Zhang

Sun

et al. 2012

Vaccine

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Granzyme B‐tdTomato, a new probe to visualize cytolytic effector cell activation

Cited by 4 publications

References 10 publications

Visualization of Cytolytic T Cell Differentiation and Granule Exocytosis with T Cells from Mice Expressing Active Fluorescent Granzyme B

Visualization of Cytolytic T Cell Differentiation and Granule Exocytosis with T Cells from Mice Expressing Active Fluorescent Granzyme B

A fluorescent reporter model for the visualization and characterization of T_DC

Murine CD8+T cell cytotoxicity against schistosomula induced by inoculation of schistosomal 22.6/26GST coupled Sepharose 4B beads

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Granzyme B‐tdTomato, a new probe to visualize cytolytic effector cell activation

Cited by 4 publications

References 10 publications

Visualization of Cytolytic T Cell Differentiation and Granule Exocytosis with T Cells from Mice Expressing Active Fluorescent Granzyme B

Visualization of Cytolytic T Cell Differentiation and Granule Exocytosis with T Cells from Mice Expressing Active Fluorescent Granzyme B

A fluorescent reporter model for the visualization and characterization of TDC

Murine CD8+T cell cytotoxicity against schistosomula induced by inoculation of schistosomal 22.6/26GST coupled Sepharose 4B beads

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A fluorescent reporter model for the visualization and characterization of T_DC