MHC–peptide tetramers for the analysis of antigen-specific T cells

Sims, Stuart; Willberg, Christian; Klenerman, Paul

doi:10.1586/erv.10.66

Cited by 53 publications

(41 citation statements)

References 70 publications

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“…Such assemblies can be centered on a single streptavidin, for example, streptavidin:MHC (major histocompatibility complex) tetramers, to analyze immune function [7]. Assemblies can also depend on multiple streptavidin molecules holding together networks, such as for amplification in histochemistry [8] or to bridge biomolecules to facilitate structure determination by electron microscopy, atomic force microscopy, and X-ray crystallography [9–11].…”

Section: Introductionmentioning

confidence: 99%

Plug-and-Play Pairing via Defined Divalent Streptavidins

Fairhead

Krndija

Lowe

et al. 2014

Journal of Molecular Biology

134

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Streptavidin is one of the most important hubs for molecular biology, either multimerizing biomolecules, bridging one molecule to another, or anchoring to a biotinylated surface/nanoparticle. Streptavidin has the advantage of rapid ultra-stable binding to biotin. However, the ability of streptavidin to bind four biotinylated molecules in a heterogeneous manner is often limiting. Here, we present an efficient approach to isolate streptavidin tetramers with two biotin-binding sites in a precise arrangement, cis or trans. We genetically modified specific subunits with negatively charged tags, refolded a mixture of monomers, and used ion-exchange chromatography to resolve tetramers according to the number and orientation of tags. We solved the crystal structures of cis-divalent streptavidin to 1.4 Å resolution and trans-divalent streptavidin to 1.6 Å resolution, validating the isolation strategy and explaining the behavior of the Dead streptavidin variant. cis- and trans-divalent streptavidins retained tetravalent streptavidin's high thermostability and low off-rate. These defined divalent streptavidins enabled us to uncover how streptavidin binding depends on the nature of the biotin ligand. Biotinylated DNA showed strong negative cooperativity of binding to cis-divalent but not trans-divalent streptavidin. A small biotinylated protein bound readily to cis and trans binding sites. We also solved the structure of trans-divalent streptavidin bound to biotin-4-fluorescein, showing how one ligand obstructs binding to an adjacent biotin-binding site. Using a hexaglutamate tag proved a more powerful way to isolate monovalent streptavidin, for ultra-stable labeling without undesired clustering. These forms of streptavidin allow this key hub to be used with a new level of precision, for homogeneous molecular assembly.

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Section: Introductionmentioning

confidence: 99%

Plug-and-Play Pairing via Defined Divalent Streptavidins

Fairhead

Krndija

Lowe

et al. 2014

Journal of Molecular Biology

134

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“…Several techniques for immune monitoring of specific T-cell responses are now available including assays which provide information about the specific T cell recognition of cancer antigens, irrespective of their functional activity, such as those based on the use of tetramers [26], assays aimed at detecting T-cell precursors by amplifying cells that proliferate in response to the antigenic stimulation [27], as well as assays that measure the secretion of a particular cytokine [28] All these test do not provide information about the anti-tumour lytic activity of the immune cells [9,28]. On the other hand, the assessment of cytotoxicity, is generally measured on the basis of the Chromium or Europium release assay, Such cytotoxicity assays measure the percentage of targets lysed by a bulk population of effectors, but they do not provide any information about the frequency of cyotoxic T cells.…”

Section: Discussionmentioning

confidence: 99%

Evaluation of antigen specific recognition and cell mediated cytotoxicity by a modified lysispot assay in a rat colon carcinoma model

Bordignon

Cordiali‐Fei

Rinaldi

et al. 2012

J Exp Clin Cancer Res

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BackgroundAntigen-specific CD8+ cytotoxic T lymphocytes represent potent effector cells of the adaptive immune response against viruses as well as tumours. Therefore assays capable at exploring the generation and function of cytotoxic T lymphocytes represent an important objective for both clinical and experimental settings.MethodsHere we show a simple and reproducible assay for the evaluation of antigen-specific CD8+ cytotoxic T lymphocytes based on a LysiSpot technique for the simultaneous determination of antigen-specific IFN-γ production and assessment of tumor cytolysis. The assay was developed within an experimental model of colorectal carcinoma, induced by the colorectal tumor cell line DHD-K12 that induces tumors in BDIX rats and, in turn, elicits a tumor- specific immune response.ResultsUsing DHD-K12 cells transfected to express Escherichia coli β-galactosidase as target cells, and by the fine setting of spot colours detection, we have developed an in vitro assay that allows the recognition of cytotoxic T lymphocytes induced in BDIX rats as well as the assessment of anti-tumour cytotoxicity. The method highlighted that in the present experimental model the tumour antigen-specific immune response was bound to killing target cells in the proportion of 55%, while 45% of activated cells were not cytotoxic but released IFN-γ. Moreover in this model by an ELISPOT assay we demonstrated the specific recognition of a nonapeptide epitope called CSH-275 constitutionally express in DHD-K12 cells.ConclusionsThe assay proved to be highly sensitive and specific, detecting even low frequencies of cytotoxic/activated cells and providing the evaluation of cytokine-expressing T cells as well as the extent of cytotoxicity against the target cells as independent functions. This assay may represent an important tool to be adopted in experimental settings including the development of vaccines or immune therapeutic strategies

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“…The tetramer molecule is made up of a fluorescently labeled, centric streptavidin molecule bound to biotin-labeled MHC molecules, which are loaded with a peptide known to be a T cell epitope to form the peptide-MHC complex ( Figure 1A) [38]. The resulting tetramer can then be used as a reagent to bind T cells that are specific for both, the MHC type and peptide used in the tetramer (Figure 1B).…”

Section: Mhc Tetramer Assaymentioning

confidence: 99%

Strategies to Study T Cells and T Cell Targets in Allergic Disease

Schulten¹

2017

Allergen

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Type I allergy is an immunoglobulin E (IgE)-mediated chronic disease. As such, disease diagnosis and identification of targeted allergens are primarily based on specific IgE reactivity. Over the past decades, the contribution of T cells in allergy pathogenesis has been extensively studied. T cells are not only significant for the onset and maintenance of allergic disease but likely also play a key role for the induction of tolerance by allergen-specific immunotherapy (AIT). Due to the complexity of allergic T cell responses, epitopes have only been thoroughly mapped for the most dominant and prevalent allergens. Recently developed laboratory approaches enable us to perform thorough peptide screens, identifying T cell epitopes in known and novel allergenic targets, irrespective of their IgE reactivity. Monitoring allergen-specific T cells and their phenotype will provide insights into disease manifestation and progression on a molecular level.However, performing such experiments in the clinic is not feasible. The definition of dominant T cell epitopes will allow us to create a tool to assess allergen-specific T cells in the context of different disease severities, such as rhinitis, asthma, and/or immunotherapy which will likely hold the key for improved diagnostic, biomarkers, and even novel therapeutic approaches.

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MHC–peptide tetramers for the analysis of antigen-specific T cells

Cited by 53 publications

References 70 publications

Plug-and-Play Pairing via Defined Divalent Streptavidins

Plug-and-Play Pairing via Defined Divalent Streptavidins

Evaluation of antigen specific recognition and cell mediated cytotoxicity by a modified lysispot assay in a rat colon carcinoma model

Strategies to Study T Cells and T Cell Targets in Allergic Disease

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