Source: Lirussi, D. et al., Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development. J. Vis. Exp. (2018)
The video demonstrates an immunological method to monitor in vivo adjuvant-induced cytotoxic T lymphocyte or CTL production. A mouse is pre-injected with genetically engineered, proliferative dye-stained donor CD8+ T lymphocytes. Subcutaneous injection of ovalbumin with adjuvants generates cytotoxic CD8+ T lymphocytes that are identified post-harvesting through immunostaining and flow cytometry.
All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
All mice used in this study were from the C57BL/6 background. All the animals were kept under pathogen-free conditions.
1. Carboxyfluorescein succinimidyl ester (CFSE) Staining of OT-I T Cells and Adoptive Transfer
NOTE: OT-I mice are transgenically generated animals that express a T cell receptor (TCR) with fixed α and β chains that together recognize the immuno-dominant peptide of ovalbumin, OVA, SIINFEKL. As a result, these mice have a considerably high number of SIINFEKL-specific CD8+ T cells (97%) when compared to normal or OVA-vaccinated mice (≤ 1%).
2. Immunization (Endo-free OVA +/- Adjuvant)
3. Isolation of Lymphocytes and Staining for Flow Cytometry Analysis
4. Flow Cytometry
Table 1: Antibody stainings for flow cytometry. Fluorophore-conjugated antibody clones used and recommended staining concentrations (2x).
Dyes – Antibodies | Clone | Fluorophore/channel-filter | Concentration 2X |
Thy1.1 (CD90.1) | HIS51 | PE-Cy7 – 780/60 YG | 1:750 |
CD8 | 53-6.7 | APC – 670/14 R | 1:280 |
CD4 | RM4-5 | BV 421 – 450/50 V | 1:100 |
CFSE | – | FITC – 530/30 YG | (according to CFSE-staining protocol) |
DCM (Dead Cell Marker) | – | -/ U.V. – 450/50 UV | 1:500 |
Figure 1: The assay timeline. The assay timeline represents the initial OT-I T cell transfer at day 0, the s.c. vaccination at day 1, and the sampling spleen and draining of lymph nodes 2 days later.
Figure 2: Flow diagram of the gating strategy followed to measure the proliferation of CD8+ T cells (OT-I, Thy1.1+) by flow cytometry. Two samples are represented in different colors (red and light blue) for better visualization. A-B. Single cells are discriminated from doublets successively in the first two gates by plotting forward-scatter-height vs. forward-scatter-area and side-scatter-width vs. side-scatter-area. C. Cells gated in B are displayed by their fluorescence intensities of the BV 650 channel (autofluorescence) plotted against their CFSE intensity (where diming indicates cells' divisions/proliferation) in the 530/30 YG channel. This gate allows the selection of true CFSE-positive cells by discriminating those that have high autofluorescence. D. CFSE positive cells were gated with their high fluorescence intensity of Pe-Cy7 (Thy1.1, marker for OT-I cells) vs. their APC intensity (CD8). E. BV 450 (CD4) plotted against APC (CD8) for previously gated Thy1.1 positive cells to accurately select CD8+ T cells. F. Previously gated CD8+ cells are plotted in a histogram against the CFSE intensity to finally gate the proliferated population.
The authors have nothing to disclose.
BD LSR Fortessa Cell Analyzer | BD | Special Order | Flow Cytometer |
CFSE | Molecular Probes | C34554 | Proliferation Dye |
MojoSort Mouse CD8 T Cell Isolation Kit | Biolegend | 480007 | Magnetic Isolation Beads and antibodies for negative selection of untouched CD8 T cells. |
LIVE/DEAD Fixable Blue Dead Cell Stain Kit, for UV excitation | Molecular Probes | L23105 | Dead Cell Marker |
CD90.1 (Thy-1.1) Monoclonal Antibody (HIS51), PE-Cyanine7 | eBioscience | 25-0900-82 | antibody |
APC anti-mouse CD8a Antibody | BioLegend | 100712 | antibody |
BV421 Rat Anti-Mouse CD4 | BD | 740007 | antibody |
Z2 coulter Particle count and Size Analyzer | Beckman Coulter | 9914591DA | Cell counter. Z2 Automated particle/cell counter |
EndoGrade Ovalbumin (10 mg) | Hyglos(Germany) | 321000 | Ovalbumin endotoxin free tested. |
Cell Strainer 100µm nylon | Corning | 352360 | Cell strainer (100 µm pore mesh cups). |
Sample Vials | Beckman Coulter | 899366014 | Sample vials for Z2 automated counter |
C57BL/6 mice (CD90.2) | Harlan (Rossdorf, Germany) | Company is now Envigo | |
OT-I (C57BL/6 background, CD90.1) | Harlan (Rossdorf, Germany) | Inbreed at our animal facility. Company from where adquired is now Envigo | |
FACS tubes | Fischer (Corning) | 14-959-5 | Corning Falcon Round-Bottom Polystyrene Tubes |
Falcon 15 mL tubes | Fischer (Corning) | 05-527-90 | Falcon 15mL Conical Centrifuge Tubes |
PBS (500 mL) | Fischer (Gibco) | 20-012-027 | Gibco PBS (Phosphate Buffered Saline), pH 7.2 |
Red lamp (heating lamp) | Dirk Rossmann GmbH (Germany) | 405096 | Heating infrred lamp (100 wats) |
IsoFlo (Isoflurane) | Abbott Laboratories (USA) | 5260.04-05. | Isoflurane anesthesic (250 mL flask). |
Tabletop Anesthesia Machine/Mobile Anesthesia Machine with CO2 Absorber | Parkland Scientific | V3000PK | Isoflurane anesthesia machine. |
RPMI 1640 medium | Gibco (distributed by ThermoFischer) | 11-875-093 | Base medium with Glutamine (500 mL) |
Pen-Strept antibiotic solution (Gibco) | Gibco (distributed by ThermoFischer) | 15-140-148 | Gibco Penicillin-Streptomycin (10,000 U/mL) |
Fetal Bobine Serum (Gibco) | Gibco (distributed by ThermoFischer) | 10082147 | Fetal Bovine Serum, certified, heat inactivated, US origin |
ACK Lysing Buffer (100 ml) | Gibco (distributed by ThermoFischer) | A1049201 | Amonium Chloride Potasium (ACK) Whole Blood Lysis Buffer, suitable for erytrocyte lysis in spleen suspensions also |
Plastic Petri Dishes | Nunc (distributed by ThermoFischer) | 150340 | 60 x 15mm Plastic Petri Dish, Non-treated |
Cell Clump Filter | CellTrics (Sysmex) | 04-004-2317 | CellTrics® 50 μm, sterile |