An Immunological Technique to Monitor Adjuvant-Mediated Cytotoxic T Lymphocyte Generation

Abstract

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.

Protocol

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 anima…

Representative Results

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 fo…

Materials

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