Generation of Human Chimeric Antigen Receptor Regulatory T Cells

Summary

This protocol provides a streamlined workflow to generate and test human chimeric antigen receptor regulatory T cells (CAR Tregs).

Abstract

Chimeric antigen receptor (CAR) T-cell therapy has reshaped the face of cancer treatment, leading to record remission rates in previously incurable hematological cancers. These successes have spurred interest in adapting the CAR platform to a small yet pivotal subset of CD4⁺ T cells primarily responsible for regulating and inhibiting the immune response, regulatory T cells (Tregs). The ability to redirect Tregs' immunosuppressive activity to any extracellular target has enormous implications for creating cell therapies for autoimmune disease, organ transplant rejection, and graft-versus-host disease. Here, we describe in detail methodologies for bona fide Treg isolation from human peripheral blood, genetic modification of human Tregs utilizing either lentivirus or CRISPR/Cas9-aided knock-in using adeno-associated virus-mediated homologous directed repair (HDR) template delivery, and ex vivo expansion of stable human CAR Tregs. Lastly, we describe the assessment of human CAR Treg phenotypic stability and in vitro suppressive function, which provides insights into how the human CAR Tregs will behave in preclinical and clinical applications.

Introduction

Chimeric antigen receptor (CAR) T-cell therapies have revolutionized the treatment of hematological malignancies, achieving remarkably high remission rates in previously untreatable cancers^1,2. Encouraging early results using CAR T cells to treat glioblastoma^3,4,5 highlight CAR technology's versatility and future potential to target a wide range of malignancies. As the field explores further applications of CARs, regulatory T cells (Tregs) have emerged as a promising cell type. Tregs play a crucial role in maintai....

Protocol

1. Human Treg isolation

1. Leukopak processing
   1. Transfer the contents of the leukopak into a 50 mL conical tube. Add an equal volume of Dulbecco's phosphate-buffered saline (DPBS) +2% fetal bovine serum (FBS) by mixing gently with a pipette.
   2. Spin at 300 × g for 10 min at room temperature (RT). Carefully aspirate the supernatant. Reconstitute the cell pellet in 2 mL of DPBS + 2% FBS. Add 8 mL of ammonium chloride solution by pipette to the cell suspension a.......

Discussion

This protocol provides a streamlined and comprehensive methodology for generating and evaluating human chimeric antigen receptor regulatory T cells (CAR Tregs). The success of CAR technology in treating hematological cancers has inspired its application to the immunosuppressive subset of T cells, Tregs. Unlike conventional T cells, Tregs inhibit immune responses, offering potential treatments for conditions resulting from unwanted immunity, such as autoimmune disease, organ transplant rejection, graft-versus-host disease.......

Materials

Name	Company	Catalog Number	Comments
Adeno-associated virus (AAV)	Charles River Laboratories
CAR target-expressing K562 cells			e.g., CD19-K562
Cesium-137 irradiator
Anti-human CD8 PerCP (clone SK1)	Biolegend	344708
Anti-human CD4 PE/Cy7 (clone SK3)	Biolegend	344612
DynaMag-15 magnet	ThermoFisher	12301D
Ghost BV510 viability dye	TONBO	13-0870-T100
K562 cells	American Type Culture Collection	CCL-243
0.5 M EDTA, pH 8.0	Gibco	15575020
1 M HEPES	Gibco	15630080
Ammonium chloride solution	STEMCELL Technologies	7850
Anti-human CD127 PE (clone hIL-7R-M21)	BD Biosciences	557938
Anti-human CD25 APC (clone BC96)	Biolegend	302610
Anti-human CD4 FITC (clone SK3)	Biolegend	344604
Anti-human CD71 PE (clone SK1)	Biolegend	334106
Anti-human CD8 PerCP (clone SK1)	Biolegend	344707
Anti-human CTLA-4 PerCP-e710	ThermoFisher	46-1529-42
Anti-human EGFR APC (clone AY13)	Biolegend	352905
Anti-human FOXP3 eFluor 450	ThermoFisher	48-4776-42
Anti-human HELIOS PE	Biolegend	137216
Ca2+ and Mg2+ free Dulbecco’s Phosphate Buffered Saline (DPBS)	Gibco	14190144
Cell counter (TC20 Automated Cell Counter)	Bio-Rad	1450102
Cell Counting Slides	Bio-Rad	1450016
CellTrace Violet Cell Proliferation Kit	ThermoFisher	C34571
DNA LoBind Tubes	Eppendorf	22431021
Easy 50 EasySep magnet	STEMCELL Technologies	18002
EasySep Human CD4+ T cell Enrichment Kit	STEMCELL Technologies	19052
EasySep Human CD8+ T cell Enrichment Kit	STEMCELL Technologies	19053
EasySep magnet	STEMCELL Technologies	18000
eBioscience Foxp3 transcription factor staining buffer set	ThermoFisher	00-5523-00
Falcon Round-Bottom Polystyrene Test Tubes with Cell Strainer Snap Cap, 5 mL	Fisher Scientific	08-771-23	40μm
Fetal Bovine Serum (FBS)	Gibco	26140079
Flow cytometer	Beckman Coulter		CytoFLEX LX U3-V5-B3-Y5-R3-I2
Fluorescence-activated cell sorter	BD Biosciences		FACS Aria III Cell Sorter
GlutaMAX	Gibco	35050061
Human CD3/28 T Cell Expansion and Activation Dynabeads	Gibco	11131D
Invitrogen Neon Transfection System	ThermoFisher	10431915
Invitrogen Neon Transfection System 100 μL Kit	ThermoFisher	10114334
Lentivirus	VectorBuilder
MEM Non-Essential Amino Acids Solution	Gibco	11140050
Myc Tag antibody A647 (clone 9B11)	Cell Signaling Technologies	2233S
Opti-MEM I Reduced Serum Medium	ThermoFisher	31985062
Penicilin-Streptomycin solution	Gibco	15140122
Recombinant human interleukin-2 (rhIL-2)	Peprotech	200-02
RPMI 1640 medium, no glutamine	Gibco	11875093
Sodium pyruvate	Gibco	11360070
Spectral Flow Cytometer	Cytek		Northern Lights
TRAC gRNA	Synthego		Sequence (CAGGGTTCTGGATATCTGT)
TrueCut Cas9 Protein v2	ThermoFisher	A36496
Trypan Blue solution	Sigma	T8154-100ML
1/10 Leukopak	STEMCELL Technologies	200-0092	1-2 billion PBMC