Enrich and Expand Rare Antigen-specific T Cells with Magnetic Nanoparticles

Summary

Antigen-specific T cells are difficult to characterize or utilize in therapies due to their extremely low frequency. Herein, we provide a protocol to develop a magnetic particle which can bind to antigen-specific T cells to enrich these cells and then to expand them several hundred-fold for both characterization and therapy.

Abstract

We have developed a tool to both enrich and expand antigen-specific T cells. This can be helpful in cases such as to A) detect the existence of antigen-specific T cells, B) probe the dynamics of antigen-specific responses, C) understand how antigen-specific responses affect disease state such as autoimmunity, D) demystify heterogeneous responses for antigen-specific T cells, or E) utilize antigen-specific cells for therapy. The tool is based on a magnetic particle that we conjugate antigen-specific and T cell co-stimulatory signals, and that we term as artificial antigen presenting cells (aAPCs). Consequently, since the technology is simple to produce, it can easily be adopted by other laboratories; thus, our purpose here is to describe in detail the fabrication and subsequent use of the aAPCs. We explain how to attach antigen-specific and co-stimulatory signals to the aAPCs, how to utilize them to enrich for antigen-specific T cells, and how to expand antigen-specific T cells. Furthermore, we will highlight engineering design considerations based on experimental and biological information of our experience with characterizing antigen-specific T cells.

Introduction

With the rise of many immunotherapies, there is a need to be able to characterize and control immune responses. In particular, the adaptive immune response is of interest because of the specificity and durability of the cells. Recently, chimeric-antigen-receptor T cell therapies have been approved for cancer therapy; however, the antigen-receptors are based off the common cell surface antigen CD19, instead of the antigens specific to the cancer¹. Beyond the specificity, immunotherapies can also suffer from the lack of control, and limited understanding the dynamic immune response within cancer or autoimmunity.

One of....

Protocol

All mice were maintained per guidelines approved by the Johns Hopkins University's Institutional Review Board.

1. Load Dimeric Major Histocompatibility Complex Immunoglobulin Fusion Protein (MHC-Ig) with Desired Antigen Peptide Sequence.

NOTE: If using H-2Kb:Ig, then follow the protocol detailed in Step 1.1; if using H-2Db:Ig, then follow the protocol detailed in Step 1.2.

1. Active loading of peptide sequence into H-2Kb:Ig.
   1. Prepare necessary buffers. Prepare the denaturation buffer by making a solution of 150 mM NaCl and 15mM Na₂CO₃ in deionized water and then adjus....

Discussion

We have created a novel antigen-specific T cell isolation technology based on nanoparticle artificial antigen presenting cells (aAPCs). Nanoparticle aAPCs have peptide-loaded MHC on the surface that allows antigen-specific T cell binding and activation alongside co-stimulatory activation. aAPCs are also paramagnetic, and thus can be used to enrich rare antigen-specific T cells using a magnetic field. We have optimized and studied key nanoparticle properties of size, ligand density, and ligand choice and their influence o.......

Materials

Name	Company	Catalog Number	Comments
DimerX I: Recombinant Soluble Dimeric Human HLA-A2:Ig Fusion Protein	BD Biosciences	551263
DimerX I: Recombinant Soluble Dimeric Mouse H-2D[b]:Ig	BD Biosciences	551323
DimerX I: Recombinant Soluble Dimeric Mouse H-2K[b]:Ig Fusion Protein	BD Biosciences	550750
Vivaspin 20 MWCO 50 000	GE Life Sciences	28932362
Vivaspin 2 MWCO 50 000	GE Life Sciences	28932257
Purified Human Beta 2 Microglobulin	Bio-Rad	PHP135
nanomag-D-spio, NH2, 100 nm nanoparticles	Micromod	79-01-102
Super Mag NHS Activated Beads, 0.2 µm	Ocean Nanotech	SN0200
Anti-Biotin MicroBeads UltraPure	Miltenyi	130-105-637
EZ-Link NHS-Biotin	ThermoFisher	20217
Sulfo-SMCC Crosslinker	ProteoChem	c1109-100mg
2-Iminothiolane hydrochloride	Sigma-Aldrich	I6256 Sigma
96 Well Half-Area Microplate, black polystyrene	Corning	3875
FITC Rat Anti-Mouse Ig, λ1, λ2, & λ3 Light Chain  Clone  R26-46	BD Biosciences	553434
FITC Mouse Anti-Armenian and Syrian Hamster IgG  Clone  G192-1	BD Biosciences	554026
B6.Cg-Thy1a/Cy Tg(TcraTcrb)8Rest/J (transgenic PMEL) mice	Jackson Laboratory	005023
C57BL/6J (B6 wildtype) mice	Jackson Laboratory	000664
CD8a+ T Cell Isolation Kit, Mouse	Miltenyi	130-104-075
MS Columns	Miltenyi	130-042-201
LS Columns	Miltenyi	130-042-401
Streptavidin-Phycoerythrin, SAv-PE	Biolegend	405203
N52 disk magnets of 0.75 inches	K&J Magnetics	DX8C-N52
APC anti-mouse CD8a Antibody, clone 53-6.7	Biolegend	100711
LIVE/DEAD Fixable Green Dead Cell Stain Kit, for 488 nm excitation	ThermoFisher	L-34969