Nanoparticle-Based Enrichment and Expansion of Antigen-Specific CD8+ T Cells

Abstract

Source: Hickey, J. W., et al. Enrich and Expand Rare Antigen-specific T Cells with Magnetic Nanoparticles. J. Vis. Exp. (2018).

This video describes a protocol for isolating rare-antigen-specific CD8+ T cells using magnetic nanoparticle-based artificial antigen-presenting cells (aAPCs) functionalized with antigenic peptide-loaded MHC-Igs and anti-CD28 antibodies. Interaction of the aAPCs with CD8+ T cells specifically expressing the antigenic peptide-targeting T cell receptors, followed by their expansion in vitro, results in the enrichment and expansion of this CD8+ T cell population.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Enrich Antigen-specific CD8+ T Cells with Prepared Nanoparticle Artificial Antigen-Presenting Cells.

1. Isolate CD8+ T cells.
   1. Euthanize the animals by exposure to isoflurane followed by cervical dislocation.
   2. Remove the spleen and lymph nodes from wild-type C57BL/6j mice and place them in a solution of PBS. Macerate the organs and elute the cells through a sterile 70 µm cell strainer with frequent washes of PBS.
   3. To ....

Materials

List of materials used in this article

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
nanomag-D-spio, NH2, 100 nm nanoparticles	Micromod	79-01-102
96-Well Half-Area Microplate, black polystyrene	Corning	3875
Super Mag NHS-Activated Beads, 0.2 µm	Ocean Nanotech	SN0200
Anti-Biotin MicroBeads UltraPure	Milteny	130-105-637
EZ-Link NHS-Biotin	ThermoFisher	20217
C57BL/6J (B6 wildtype) mice	Jackson Laboratory	664
CD8a+ T Cell Isolation Kit, Mouse	Miltenyi	130-104-075
MS Columns	Milteny	130-042-201
LS Columns	Milteny	130-042-401
Streptavidin-Phycoerythrin, SAvPE	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