A Technique for the Generation of Antigen-Specific CAR T Cells

Abstract

Source: Davidson, H. W., et al. High-Efficiency Generation of Antigen-Specific Primary Mouse Cytotoxic T Cells for Functional Testing in an Autoimmune Diabetes Model. J. Vis. Exp. (2019).

This video demonstrates a technique for generating antigen-specific chimeric antigen receptor (CAR) T cells through retroviral transduction. Activated murine CD8+ T cells are added to a microwell plate coated with recombinant fibronectin fragments. Retroviral vectors containing transgenic RNA encoding an antigen-specific chimeric antigen receptor (CAR) are then introduced, and the plate is centrifuged to facilitate transduction. After incubation, the transduced cells express antigen-specific CARs on their surface.

Protocol

1. Generation and validation of single chain Fab antibody (scFab)-CARs. NOTE: CARs typically contain 3 critical domains—an antigen-targeting domain, a spacer/transmembrane domain, and a cytoplasmic signaling domain. The precise design of each CAR depends on the intended target, and so, apart from the key features of the construct relevant to the generation of the retrovirus, will not be described in detail in this protocol. The overall design of the CARs u…

Representative Results

Figure 1: Schematic of the CAR retroviral construct. The CAR comprises a targeting domain derived from the Fab fragment of a suitable mouse monoclonal antibody, and a spacer/ membrane anchor/ signaling domain from mouse CD28, CD137 and CD247. The synthetic cDNA is inserted into the pMIG-II retroviral expression vector. Restriction endonuclease sites used for generating the mAb287-CAR are shown.</…

Materials

2-Mercaptoethanol (50mM)	Gibco	21985-023	50 uM
5' RACE PCR	Clontech	634859
anti-mouse CD28 antibodies	eBioscience	14-0281-86	final concentration at 1µg/ml
anti-mouse CD3e antibody	eBioscience	145-2C11	final concentration at 1µg/ml
Biotin Rat Anti-Mouse IFN-γ	BD Biosciences	554410	Working concentration at 0.5 µg/ml
BSA	Sigma	A7030
Endo-free Maxi-Prep kit	Qiagen	12362
Gentamicin	Gibco	15750-060	Final 50 µg/ml.
Heat inactivated FCS	Hyclone	SH30087.03	Final 10% FCS
HEPES (100X)	Gibco	15630-080	1X
Insulin-Transferrin-Selenium-Ethanolamine (ITS 100x)	ThermoFisher	51500056	Final concentraion is 1x
Lipofectamine 2000	Invitrogen	11668019
LS Columns	Miltenyi Biotec	130-042-401
MACS Separation Buffer	Miltenyi Biotec	130-091-221
Mouse CD8a+ T Cell Isolation Kit	Miletenyi Biotec Inc	130-104-075
Mouse CD8a+ T Cell Isolation Kit	Miltenyi Biotec	130-104-075
Opti-MEM medium	ThermoFisher	31985070
Penicillin-Streptomycin (5000U/ml)	ThermoFisher	15070063	50 U/ml
Phoenix-ECO cells	ATCC	CRL-3214
Phosphate-buffered saline (PBS)	Gibco	10010-023
pMIG II	Addgene	52107
pMSCV-IRES-GFP II	Addgene	52107
Red cell lysis buffer	Sigma	R7767
RetroNectin	Takara	T100A	Working concentration at 50 µg/ml in PBS
rhIL-2 (stock concentration 105 IU/ul)	Peprotech	200-02	Final concentration at 200 IU/ml
rmIL-7 ( stock concentration 50ng/ul)	R&D	407-ML-005	Final concentration at 0.5ng/ml
RPMI-1640	Gibco	11875-093
Sterile Cell Strainers	Fisher Scientific	22-363-548
Tryple	Gibco	12605-028