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Abstract:The advent of major histocompatibility complex (MHC) tetramer technology has been a major contribution to T cell immunology, because tetramer reagents permit detection of antigen-specific T cells at the single-cell level in heterogeneous populations by flow cytometry. However, unlike MHC class I tetramers, the utility of MHC class II tetramers has been less frequently reported. MHC class II tetramers can be used successfully to enumerate the frequencies of antigen-specific CD4 T cells in cells activated in vit… Show more
“…Dextramers of class II pMHC molecules improve detection of antigen-specific CD4 + T cells (16), which are emerging as important components of anti-tumor immunity (17,18) and are generally lower-affinity than CD8 + T cells(19). Moreover, TCRs derived from tumor-reactive CD8 + T cells are constrained by thymic selection to a low-affinity range (1,4) and are generally assisted in pMHC recognition by CD8:MHC I interaction (20).…”
Peptide-major histocompatibility complex (pMHC) multimers enable the detection, characterization, and isolation of antigen-specific T-cell subsets at the single-cell level via flow cytometry and fluorescence microscopy. These labeling reagents exploit a multivalent scaffold to increase the avidity of individually weak T-cell receptor (TCR)-pMHC interactions. Dextramers are an improvement over the original streptavidin-based tetramer technology because they are more multivalent, improving sensitivity for rare, low-avidity T cells, including self/tumor-reactive clones. However, commercial pMHC dextramers are expensive, and in-house production is very involved for a typical biology research laboratory. Here, we present a simple, inexpensive protocol for preparing pMHC dextramers by doping in biotinylated dextran during conventional tetramer preparation. We use these pMHC dextramers to identify patient-derived, tumor-reactive T cells. We apply the same dextran doping technique to prepare TCR dextramers and use these novel reagents to yield new insight into MHC I-mediated antigen presentation.
“…Dextramers of class II pMHC molecules improve detection of antigen-specific CD4 + T cells (16), which are emerging as important components of anti-tumor immunity (17,18) and are generally lower-affinity than CD8 + T cells(19). Moreover, TCRs derived from tumor-reactive CD8 + T cells are constrained by thymic selection to a low-affinity range (1,4) and are generally assisted in pMHC recognition by CD8:MHC I interaction (20).…”
Peptide-major histocompatibility complex (pMHC) multimers enable the detection, characterization, and isolation of antigen-specific T-cell subsets at the single-cell level via flow cytometry and fluorescence microscopy. These labeling reagents exploit a multivalent scaffold to increase the avidity of individually weak T-cell receptor (TCR)-pMHC interactions. Dextramers are an improvement over the original streptavidin-based tetramer technology because they are more multivalent, improving sensitivity for rare, low-avidity T cells, including self/tumor-reactive clones. However, commercial pMHC dextramers are expensive, and in-house production is very involved for a typical biology research laboratory. Here, we present a simple, inexpensive protocol for preparing pMHC dextramers by doping in biotinylated dextran during conventional tetramer preparation. We use these pMHC dextramers to identify patient-derived, tumor-reactive T cells. We apply the same dextran doping technique to prepare TCR dextramers and use these novel reagents to yield new insight into MHC I-mediated antigen presentation.
“…Third, interactions between some partners may be too weak to be detected by the ECIA, and detecting these may require the use of higher-order multimers. For example, analysis of binding of low-affinity T cell receptors to peptide-bound MHC molecules can require the use of large clusters of MHC-peptide complexes assembled on dextran polymers (dextramers) [40]. …”
The immunoglobulin superfamily (IgSF) encompasses hundreds of cell surface proteins containing multiple immunoglobulin-like (Ig) domains. Among these are neural IgCAMs, which are cell adhesion molecules that mediate interactions between cells in the nervous system. IgCAMs in some vertebrate IgSF subfamilies bind to each other homophilically and heterophilically, forming small interaction networks. In Drosophila, a global ‘interactome’ screen identified two larger networks in which proteins in one IgSF subfamily selectively interact with proteins in a different subfamily. One of these networks, the ‘Dpr-ome’, includes 30 IgSF proteins, each of which is expressed in a unique subset of neurons. Recent evidence shows that one interacting protein pair within the Dpr-ome network is required for development of the brain and neuromuscular system.
“…These fluorescent tetramers can be used to tag, track, and quantify populations of epitope-specific T cells using fluorescence-activated cell sorting (FACS). Some groups have attached biotinylated pHMC monomers to fluorescent streptavidin molecules using a sub-stoichiometric ratio 2 , 3 . Biotinylated dextran was then used to create multimers with more than four monomers to tag and quantify T cells using FACS 2 , 3 .…”
Section: Introductionmentioning
confidence: 99%
“…Some groups have attached biotinylated pHMC monomers to fluorescent streptavidin molecules using a sub-stoichiometric ratio 2 , 3 . Biotinylated dextran was then used to create multimers with more than four monomers to tag and quantify T cells using FACS 2 , 3 . Figure 1 ( a ) Cartoon of monomer structure and fluorescently-tagged tetramer with paramagnetic bead (PB) attached to the tetramer using an antibody.…”
Section: Introductionmentioning
confidence: 99%
“…1c ). 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.…”
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.
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