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Comparison of peptide-major histocompatibility complex tetramers and dextramers for the identification of antigen-specific T cells.

ABSTRACT: Fluorochrome-conjugated peptide-major histocompatibility complex (pMHC) multimers are widely used for flow cytometric visualization of antigen-specific T cells. The most common multimers, streptavidin-biotin-based 'tetramers', can be manufactured readily in the laboratory. Unfortunately, there are large differences between the threshold of T cell receptor (TCR) affinity required to capture pMHC tetramers from solution and that which is required for T cell activation. This disparity means that tetramers sometimes fail to stain antigen-specific T cells within a sample, an issue that is particularly problematic when staining tumour-specific, autoimmune or MHC class II-restricted T cells, which often display TCRs of low affinity for pMHC. Here, we compared optimized staining with tetramers and dextramers (dextran-based multimers), with the latter carrying greater numbers of both pMHC and fluorochrome per molecule. Most notably, we find that: (i) dextramers stain more brightly than tetramers; (ii) dextramers outperform tetramers when TCR-pMHC affinity is low; (iii) dextramers outperform tetramers with pMHC class II reagents where there is an absence of co-receptor stabilization; and (iv) dextramer sensitivity is enhanced further by specific protein kinase inhibition. Dextramers are compatible with current state-of-the-art flow cytometry platforms and will probably find particular utility in the fields of autoimmunity and cancer immunology.

SUBMITTER: Dolton G

PROVIDER: S-EPMC4089154 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature

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Comparison of peptide-major histocompatibility complex tetramers and dextramers for the identification of antigen-specific T cells.

Dolton G G Lissina A A Skowera A A Ladell K K Tungatt K K Jones E E Kronenberg-Versteeg D D Akpovwa H H Pentier J M JM Holland C J CJ Godkin A J AJ Cole D K DK Neller M A MA Miles J J JJ Price D A DA Peakman M M Sewell A K AK

Clinical and experimental immunology 20140701 1

Fluorochrome-conjugated peptide-major histocompatibility complex (pMHC) multimers are widely used for flow cytometric visualization of antigen-specific T cells. The most common multimers, streptavidin-biotin-based 'tetramers', can be manufactured readily in the laboratory. Unfortunately, there are large differences between the threshold of T cell receptor (TCR) affinity required to capture pMHC tetramers from solution and that which is required for T cell activation. This disparity means that te ...[more]

PMID: 24673376

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Project description:AimTo establish a simplified method for generating peptide-major histocompatibility complex (MHC) class I tetramers.MethodscDNAs encoding the extracellular domain of human lymphocyte antigen (HLA)-A*0201 heavy chain (A2) and beta(2)-microglobulin (beta(2)m) from total RNA extracted from leukocytes of HLA-A2(+) donors were cloned into separate expression vectors by reverse transcription-polymerase chain reaction. The recombinant A2 and beta(2)m proteins were expressed in Escherichia coli strain BL21(DE3) and recovered from the inclusion body fraction. Soluble A2 proteins loaded with specific antigen peptides were refolded by dilution from the heavy chain in the presence of light chain beta(2)m and HLA-A2-restricted peptide antigens. The refolded A2 monomers were biotinylated with a commercial biotinylation enzyme (BirA) and purified by low pressure anion exchange chromatography on a Q-Sepharose (fast flow) column. The tetramers were then formed by mixing A2 monomers with streptavidin-PE in a molar ratio of 4:1. Flow cytometry was used to confirm the expected tetramer staining of CD8(+) T cells.ResultsRecombinant genes for HLA-A*0201 heavy chain (A2) fused to a BirA substrate peptide (A2-BSP) and mature beta(2)m from HLA-A2(+) donor leukocytes were successfully cloned and highly expressed in E. coli. Two soluble monomeric A2-peptide complexes were reconstituted from A2-BSP in the presence of beta(2)m and peptides loaded with either human cytomegalovirus pp65(495-503) peptide (NLVPMVATV, NLV; designated as A2-NLV) or influenza virus matrix protein Mp58-66 peptide (GILGFVFTL, GIL; designated as A2-GIL). Refolded A2-NLV or A2-GIL monomers were biotinylated and highly purified by single step anion exchange column chromatography. The tetramers were then formed by mixing the biotinylated A2-NLV or A2-GIL monomers with streptavidin-PE, leading to more than 80% multiplication as revealed by SDS-PAGE under non-reducing, unboiled conditions. Flow cytometry revealed that these tetramers could specifically bind to CD8(+) T cells from a HLA-A2(+) donor, but failed to bind to those from a HLA-A2- donor.ConclusionThe procedure is simple and efficient for generating peptide-MHC tetramers.

Project description:Minor Histocompatibility (H) antigens are major histocompatibility complex (MHC)/Human Leukocyte Antigen (HLA)-bound peptides that differ between allogeneic hematopoietic stem cell transplantation (HCT) recipients and their donors as a result of genetic polymorphisms. Some minor H antigens can be used as therapeutic T cell targets to augment the graft-vs.-leukemia (GVL) effect in order to prevent or manage leukemia relapse after HCT. Graft engineering and post-HCT immunotherapies are being developed to optimize delivery of T cells specific for selected minor H antigens. These strategies have the potential to reduce relapse risk and thereby permit implementation of HCT approaches that are associated with less toxicity and fewer late effects, which is particularly important in the growing and developing pediatric patient. Most minor H antigens are expressed ubiquitously, including on epithelial tissues, and can be recognized by donor T cells following HCT, leading to graft-vs.-host disease (GVHD) as well as GVL. However, those minor H antigens that are expressed predominantly on hematopoietic cells can be targeted for selective GVL. Once full donor hematopoietic chimerism is achieved after HCT, hematopoietic-restricted minor H antigens are present only on residual recipient malignant hematopoietic cells, and these minor H antigens serve as tumor-specific antigens for donor T cells. Minor H antigen-specific T cells that are delivered as part of the donor hematopoietic stem cell graft at the time of HCT contribute to relapse prevention. However, in some cases the minor H antigen-specific T cells delivered with the graft may be quantitatively insufficient or become functionally impaired over time, leading to leukemia relapse. Following HCT, adoptive T cell immunotherapy can be used to treat or prevent relapse by delivering large numbers of donor T cells targeting hematopoietic-restricted minor H antigens. In this review, we discuss minor H antigens as T cell targets for augmenting the GVL effect in engineered HCT grafts and for post-HCT immunotherapy. We will highlight the importance of these developments for pediatric HCT.

Dataset Information

Comparison of peptide-major histocompatibility complex tetramers and dextramers for the identification of antigen-specific T cells.

Publications

Comparison of peptide-major histocompatibility complex tetramers and dextramers for the identification of antigen-specific T cells.

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