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

Published: May 31, 2024

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.</…

開示

The authors have nothing to disclose.

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

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記事を引用
A Technique for the Generation of Antigen-Specific CAR T Cells. J. Vis. Exp. (Pending Publication), e22263, doi: (2024).

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