Enrichment and Quantification of Epitope-specific CD4+ T Lymphocytes using Ferromagnetic Iron-gold and Nickel Nanowires

Daniel E Shore,Bethanie J H Stadler,Marc K Jenkins,Jaime F Modiano,Thamotharampillai Dileepan

doi:10.1038/s41598-018-33910-0

Daniel E Shore, Bethanie J H Stadler + Show 3 more

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https://doi.org/10.1038/s41598-018-33910-0

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Abstract

Epitope-specific CD4+ T lymphocytes were magnetically enriched using ferromagnetic Ni and Fe-Au nanowires coated with a monomer containing a major histocompatibility complex class II-bound peptide epitope (pMHCII). The enriched lymphocytes were subsequently quantified using fluorescence-activated cell sorting (FACS). This was the first use of magnetic nanowires for cell sorting using FACS, and improvements in both specificity and fluorescent signal strength were predicted due to higher particle moments and lengths than conventional paramagnetic beads. Three different types of nanowires (Ni, Fe with Au tip and Fe-Au multilayers) were made by electrodeposition. Ni nanowires separated fewer T cells than Au tipped Fe nanowires, likely because Ni has a lower magnetic moment than Fe. Fe-Au multilayer nanowires separated more T cells than Au-tipped Fe nanowires because there was more monomer per nanowire. Also, increasing the amount of monomer increased the number of CD4+ cells separated. Compared to conventional paramagnetic beads, the nanowires had lower specificity for CD4+ T cells, but had stronger fluorescent signals due to more fluorophores per particle. This results in broader FACS baseline separation between the positive and negative cells, which is useful to detect T cells, even those with lower binding affinity for pMHCII ligands.

Highlights

IntroductionTo use the tetramers for magnetic cell separation and T cell quantification, commercial paramagnetic beads (embedded in a plastic, spherical particle) are attached to the fluorescent tetramer using an antibody that binds to the fluorescent streptavidin molecules
To use the tetramers for magnetic cell separation and T cell quantification, commercial paramagnetic beads are attached to the fluorescent tetramer using an antibody that binds to the fluorescent streptavidin molecules
Multiple groups have shown that electrodeposited Ni nanowires have minimal cytotoxicity[5,6] especially if the surfaces are coated with polyethylene glycol (PEG) and/or RGD11,14

Summary

Introduction

To use the tetramers for magnetic cell separation and T cell quantification, commercial paramagnetic beads (embedded in a plastic, spherical particle) are attached to the fluorescent tetramer using an antibody that binds to the fluorescent streptavidin molecules. Kim et al used antibody functionalized Ni-silicide (NiSi) nanowires (60–100 nm diameter, 5–10 μm long) to tag and separate CD4+ T lymphocyte cells from a mixture of lymphocyte cells They found ~95% capture efficiency for both the nanowires and the commercial paramagnetic beads[13]. Having more fluorophores per particle should increase the strength of the fluorescent signal detected by FACS to improve detection of T cells even when TCRs have a low affinity for the pMHCII ligand[2,3] All these factors make electrodeposited nanowires an attractive alternative to paramagnetic beads for magnetic cell enrichment

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