High-Efficiency Generation of Antigen-Specific Primary Mouse Cytotoxic T Cells for Functional Testing in an Autoimmune Diabetes Model

Summary

This article describes a protocol for the generation of antigen-specific CD8 T cells, and their expansion in vitro, with the aim of yielding high numbers of functional T cells for use in vitro and in vivo.

Abstract

Type 1 Diabetes (T1D) is characterized by islet-specific autoimmunity leading to beta cell destruction and absolute loss of insulin production. In the spontaneous non-obese diabetes (NOD) mouse model, insulin is the primary target, and genetic manipulation of these animals to remove a single key insulin epitope prevents disease. Thus, selective elimination of professional antigen presenting cells (APCs) bearing this pathogenic epitope is an approach to inhibit the unwanted insulin-specific autoimmune responses, and likely has greater translational potential.

Chimeric antigen receptors (CARs) can redirect T cells to selectively target disease-causing antigens. This technique is fundamental to recent attempts to use cellular engineering for adoptive cell therapy to treat multiple cancers. In this protocol, we describe an optimized T-cell retrovirus (RV) transduction and in vitro expansion protocol that generates high numbers of functional antigen-specific CD8 CAR-T cells starting from a low number of naive cells. Previously multiple CAR-T cell protocols have been described, but typically with relatively low transduction efficiency and cell viability following transduction. In contrast, our protocol provides up to 90% transduction efficiency, and the cells generated can survive more than two weeks in vivo and significantly delay disease onset following a single infusion. We provide a detailed description of the cell maintenance and transduction protocol, so that the critical steps can be easily followed. The whole procedure from primary cell isolation to CAR expression can be performed within 14 days. The general method may be applied to any mouse disease model in which the target is known. Similarly, the specific application (targeting a pathogenic peptide/MHC class II complex) is applicable to any other autoimmune disease model for which a key complex has been identified.

Introduction

Given the likely reduced risk of unwanted off-target effects, antigen-specific immune therapies (ASI) are promising treatments for autoimmune diseases such as T1D. Accumulating evidence suggests that immune responses to (prepro)insulin may be particularly important in T1D¹. In the past decade, studies from multiple groups, including our own, strongly suggest that presentation of an epitope containing insulin B chain amino acids 9 to 23 by specific MHC class II molecules (B:9-23/MHCII), plays an important role in the development of T1D in mice and humans^2,3,4....

Protocol

Mice were maintained under specific pathogen-free conditions at a Transgenic Mouse Facility, and all animal experiments were performed in accordance with protocols approved by the Baylor College of Medicine animal care and use committee.

NOTE: The experiment requires preparing the virus and the T cells in parallel. Table 1 summarizes the protocol. The key reagents and buffers are listed in the Table of Materials. We focus on the generation and expansion of CAR-T cells targeting specific populations of APCs in this protocol.

1. Generation and validation of single chain Fab antibody ....

Results

Typically, the transduction efficiency using this protocol is ~60-90%. In the experiment shown in Figure 3, prior to sorting approximately, 70% of the CD8 T cells co-expressed GFP. They also co-expressed CD28 and CD3 (Figure 3C). Importantly, all of the “test” GFP⁺ cells also co-stained with IA^g7-B:R3 tetramers, but not with the control tetramer (Figure 4). Similarly, the sorted.......

Discussion

This protocol describes an efficient method for producing antigen-specific CD8 CAR-T cells by retroviral transduction. The transduction efficiency of our protocol is typically high, and robust expression of the CAR is generally observed. The expanded CAR T cells retain the essential features of the parent-activated T cells, and antibody specificity, and are suitable for both in vitro and in vivo use. We have applied Ab-CAR CD8 T cells in reprograming Type 1 Diabetes in NOD mice⁷.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2-Mercaptoethanol (50mM)	Gibco	21985-023	50 μM
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
IAg7-CLIP tetramer-BV421	NIH tetramer Facility at Emory	per approval	Working concentration at 6 µg/ml
IAg7-insulin P8E tetramer-BV421	NIH tetramer Facility at Emory	per approval	Working concentration at 6 µg/ml
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
Purified Rat Anti-Mouse IFN-γ	BD Biosciences	551216	Working concentration at 3 µg/ml
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