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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.
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.
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....
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.
To complete a successful enrichment and expansion of antigen-specific T cells, the peptide-loaded MHC-Ig and co-stimulatory molecules should be successfully attached to the aAPC particle. Based on the 3 methods of particle attachment, we provide some representative data for a successful conjugation procedure outcome (Figure 5a). Indeed, if the ligand density is too low, then there will not be effective stimulation of antigen-specific CD8+ T cells where this o.......
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.......
J.W.H. thanks the NIH Cancer Nanotechnology Training Center at the Johns Hopkins Institute for NanoBioTechnology, the National Science Foundation Graduate Research Fellowship (DGE-1232825), and the ARCS foundation for fellowship support. This work was funded by support from the National Institutes of Health (P01-AI072677, R01-CA108835, R21-CA185819), TEDCO/Maryland Innovation Initiative, and the Coulter Foundation (JPS).
....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 |
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