Studying the biology of cytotoxic T lymphocytes in vivo with a fluorescent granzyme B-mTFP knock-in mouse
- Cellular Neurophysiology, Center for Integrative Physiology and Molecular Medicine (CIPMM), Saarland University, Germany
- Department of Molecular Neurobiology, Max-Planck-Institute of Experimental Medicine, Germany
- Diabetes Research Institute and Cell Transplant Center, University of Miami Miller School of Medicine, United States
- Department of Surgery, University of Miami Miller School of Medicine, United States
- Department of Microbiology and Immunology, University of Miami Miller School of Medicine, United States
- Department of Ophthalmology, University of Miami Miller School of Medicine, United States
- Diabetes Research Institute Federation, United States
- The Rolf Luft Research Center for Diabetes and Endocrinology, Karolinska Institutet, Karolinska University Hospital, Sweden
- Sensory and Neuroendocrine Physiology, Center for Integrative Physiology and Molecular Medicine (CIPMM), Saarland University, Germany
Figures
Figure 1 with 1 supplement
Generation of GzmB-mTFP knock-in mice.
(A) CRISPR-Cas9 strategy to generate the GzmB-mTFP-KI. wt, wild-type; KI, GzmB-mTFP-KI; numbered black boxes, Gzmb exons; red bar, Stop codon; yellow bar, GGSGGSGGS-linker; green box, mTFP coding …
Figure 1—figure supplement 1
Design of the HDR fragment to generate the GzmB-mTFP-KI The HDR fragment was designed to replace the endogenous Stop codon of the Gzmb gene by sequences encoding a GGSGGSGGS-linker, mTFP and a Stop codon.
Some sites were mutated (green bold) to protect the HDR fragment against cleavage in the presence of the sgRNA used, and of sgRNAs that had initially been envisioned for this project but were …
Figure 2
Endogenous GzmB-mTFP fluorescence co-localizes with CG markers and migrates to the IS.
(A) SIM images of primary CTLs from GzmB-mTFP-KI mice on day five after activation. CTLs were fixed and stained with Alexa647 conjugated anti-GzmB antibody (upper panel) or Alexa647 conjugated …
Figure 3
GzmB-mTFP containing cytotoxic granules fuse at the IS.
(A) Selected live-cell TIRF microscopy images of GzmB-mTFP in a CTL in contact with an anti-CD3 coated coverslip. Fusion events are indicated with open arrows (seven consecutive frames per fusion …
Figure 4
CTLs from GzmB-mTFP-KI mice kill target cells as efficient as wild-type CTLs.
(A) Live cell killing assay showing a wild-type (left) and GzmB-mTFP-KI (right) CTL in contact with P815 target cells stably expressing Casper3-GR (FRET construct containing Tag-GFP and Tag-RFP with …
Figure 5 with 3 supplements
In vivo imaging of allorejection in the anterior chamber of the eye (ACE) visualized by CTLs from GzmB-TFP-KI mice.
(A) Binocular image of a mouse eye with islets of Langerhans from DBA/2 mice transplanted into the anterior chamber of the eye (ACE) 4 days prior to the image. Islets can be identified as small …
Figure 5—figure supplement 1
Longitudinal and repetitive observations of transplanted islets inside the ACE.
(A) Two-photon image at POD14 of the capillary network of a transplanted islet. Rhodamine-dextran was injected into the tail vein of the mouse 15 min before acquisition to label blood vessels. …
Figure 5—video 1
In vivo imaging of migrating T cells during infiltration of a transplanted pancreatic islet.
Same islet as in Figure 5D. A maximum intensity projection of 11 planes covering a 40 µm thick stack over 32 min is shown. The movie was acquired with a confocal laser scanning microscope. Faint …
Figure 5—video 2
In vivo imaging of migrating T cells in the cornea by two-photon microscopy Same experiment as in Figure 5E.
A maximum intensity projection of 11 planes covering a 40 µm thick stack with 2 µm interval distance over 40 min is shown. The first part of the video shows the overall migration pattern of …
Author response image 1
Islets of Langerhans were incubated with CTLs derived from GzmB-mTFP KI (left) and GzmB-mTFP KI/Perforin KO mice (middle) and imaged by confocal microscopy.
CTLs started to infiltrate the islets and kill them, which resulted in a detachment of islets from the culture dish. Quantification of the detachment at day 1 and day 2 of co-culture (right panel) …
Videos
Video 1
In vivo imaging of the capillary network of a transplanted pancreatic islet.
The pancreatic islet was transplanted into the ACE 14 days prior the experiment. The movie is a maximum intensity projection of 11 planes covering a 40 µm thick stack over 37 min and was acquired …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | anti-GAPDH (rabbit polyclonal) | Cell Signaling Technology | Cat# 2118L; Clone-14C10 | WB (1:5000) |
| Antibody | anti-tRFP (rabbit polyclonal) | Evrogen | Cat# ab233 | WB (1:2000) |
| Antibody | anti-Granzyme B (rabbit polyclonal) | Cell Signaling Technology | Cat# 4275S | WB (1:2000) |
| Antibody | Alexa647-coupled anti-Granzyme B (mouse IgG1, κ) | BioLegend | Cat# 515405; clone-GB11 | IF (1:200) |
| Antibody | Alexa647-coupled anti-perforin (mouse, IgG2b, κ) | BioLegend | Cat# 308109; clone-dG9 | IF (1:200) |
| Antibody | Hamster anti-mouse (CD3ε) | BD Pharmingen | Cat# 553058; clone-145–2 C11 | 30 µg/ml |
| Antibody | Rat Brilliant Violet421 anti-mouse CD8a (rat, IgG2a, κ) | BioLegend | Cat# 100737; clone-53–6.7 | IHC (1:200) |
| Antibody | Rat APC anti-mouse CD4 (rat, IgG2b, κ) | BioLegend | Cat# 100412; clone-GK1.5 | IHC (1:200) |
| Antibody | Rat anti-mouse CD107a-PE (rat, IgG2a, κ) | BioLegend | Cat# 121611; clone-ID4B | FACS (1:200) |
| Recombinant DNA reagent | Casper3-GR (plasmid) | Evrogen | Cat# FP971 | |
| Recombinant DNA reagent | pMAX-Granzyme B-mCherry (mouse) | This paper | Figure 3, Available from the authors upon request | |
| Recombinant DNA reagent | pMAX-Casper3-GR (plasmid) | This paper | Figure 4A; Available from the authors upon request | |
| Biological sample (Mus musculus) | Primary CD8+ T lymphocytes | This paper | Freshly isolated from spleen of Granzyme B-mTFP knock-in mouse(8–20 weeks old); Mouse available from EMMA mouse depository |
Additional files
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Source data 1
Chitiralaetal_Source Data.xlsx.
- https://cdn.elifesciences.org/articles/58065/elife-58065-data1-v1.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/58065/elife-58065-transrepform-v1.docx
