Functional recombinant MHC class II molecules and high-throughput peptide-binding assays

Justesen, Sune; Harndahl, Mikkel; Lamberth, Kasper; Nielsen, Lise-Lotte B.; Buus, Søren

doi:10.1186/1745-7580-5-2

Cited by 62 publications

(103 citation statements)

References 90 publications

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“…The construction of HLA class II genes has been described previously [19]. Briefly, HLA-DR class II α chains were truncated at position 191 and C-terminally fused to a FOS leucine zipper segment.…”

Section: Methodsmentioning

confidence: 99%

“…Over the past decades we have developed versatile, high-yield, recombinant E. coli expression systems generating highly active, biotinylated MHC class I [18] and II [19] molecules and accompanying high throughput peptide-binding assays [19], [20]. For MHC class I, we have also developed a “one-pot, mix-and-read” tetramer approach where the consecutive admixture of MHC class I, peptide and SA (i.e.…”

Section: Introductionmentioning

confidence: 99%

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MHC Class II Tetramers Made from Isolated Recombinant α and β Chains Refolded with Affinity-Tagged Peptides

et al. 2013

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Targeting CD4+ T cells through their unique antigen-specific, MHC class II-restricted T cell receptor makes MHC class II tetramers an attractive strategy to identify, validate and manipulate these cells at the single cell level. Currently, generating class II tetramers is a specialized undertaking effectively limiting their use and emphasizing the need for improved methods of production. Using class II chains expressed individually in E. coli as versatile recombinant reagents, we have previously generated peptide-MHC class II monomers, but failed to generate functional class II tetramers. Adding a monomer purification principle based upon affinity-tagged peptides, we here provide a robust method to produce class II tetramers and demonstrate staining of antigen-specific CD4+ T cells. We also provide evidence that both MHC class II and T cell receptor molecules largely accept affinity-tagged peptides. As a general approach to class II tetramer generation, this method should support rational CD4+ T cell epitope discovery as well as enable specific monitoring and manipulation of CD4+ T cell responses.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

MHC Class II Tetramers Made from Isolated Recombinant α and β Chains Refolded with Affinity-Tagged Peptides

et al. 2013

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“…60 Alternatively, using insect cell expression systems, soluble "empty" MHC II molecules can be produced and peptide binding assessed by means of biotin or fluorochrome labeled peptides. 61,62 MHC II-peptide complex stability can be measured via the disappearance of the tagged peptide over time.…”

Section: Mhc II Peptide Binding Validationmentioning

confidence: 99%

Current tools for predicting cancer-specific T cell immunity

Gfeller¹,

Bassani-Sternberg²,

Schmidt³

et al. 2016

OncoImmunology

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Tumor exome and RNA sequencing data provide a systematic and unbiased view on cancer-specific expression, over-expression, and mutations of genes, which can be mined for personalized cancer vaccines and other immunotherapies. Of key interest are tumor-specific mutations, because T cells recognizing neoepitopes have the potential to be highly tumoricidal. Here, we review recent developments and technical advances in identifying MHC class I and class II-restricted tumor antigens, especially neoantigen derived MHC ligands, including in silico predictions, immune-peptidome analysis by mass spectrometry, and MHC ligand validation by biochemical methods on T cells.

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“…A number of studies have assessed peptide-binding to MHC-II on the surface of intact APCs (12,13), whereas a routinely used in vitro approach entails purifying soluble recombinant MHC-II molecules from different expressing systems such as B cell lines (14), insect cells (15,16), yeast (17), or Escherichia coli (18)(19)(20) and then characterizing binding of these molecules to different peptides generated either chemically by solid phase synthesis or genetically by phage display (4,21). The labor-intensive preparation of soluble proteins, lengthy binding assays, or nonquantitative data generated (e.g., peptide abundance) limits the efficiency and throughput of these methods for mapping MHC-II binding specificities across the large number of existing alleles.…”

mentioning

confidence: 99%

High-throughput engineering and analysis of peptide binding to class II MHC

Jiang

Boder

2010

Proc. Natl. Acad. Sci. U.S.A.

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Class II major histocompatibility complex (MHC-II) proteins govern stimulation of adaptive immunity by presenting antigenic peptides to CD4þ T lymphocytes. Many allelic variants of MHC-II exist with implications in peptide presentation and immunity; thus, highthroughput experimental tools for rapid and quantitative analysis of peptide binding to MHC-II are needed. Here, we present an expression system wherein peptide and MHC-II are codisplayed on the surface of yeast in an intracellular association-dependent manner and assayed by flow cytometry. Accordingly, the relative binding of different peptides and/or MHC-II variants can be assayed by genetically manipulating either partner, enabling the application of directed evolution approaches for high-throughput characterization or engineering. We demonstrate the application of this tool to map the side-chain preference for peptides binding to HLA-DR1 and to evolve novel HLA-DR1 mutants with altered peptide-binding specificity.yeast display | MHC peptide-binding interactions | MHC engineering | anchor specificity | directed evolution C lass II major histocompatibility complex (MHC-II)-restricted T cell responses are related to a great number of diseases including autoimmunity, graft rejection, and atypical immune response. MHC-II proteins are heterodimeric transmembrane proteins consisting of α and β chains containing two domains each (1), and these proteins capture antigenic peptides processed inside professional antigen-presenting cells (APCs) and present them on the APC surface for recognition by CD4þ T cells to initiate adaptive immunity (2, 3). The peptide-binding sites of MHC-II formed by the α1 and β1 domains contain several pockets that prefer to accommodate specific side chains of "anchor" residues on peptide antigens (4, 5). Thus, MHC-II are semipromiscuous binders capable of presenting numerous different peptides, but anchor pockets constrain the milieu of peptides presented by a given MHC-II. In depth characterization of peptide binding by MHC-II is therefore critical to understanding issues in vaccine design (6), autoimmune disease (7), infectious disease progression (8), and transplantation rejection (9, 10), but the lack of a rapid, efficient, robust, and quantitative methodology for characterizing the peptide-binding specificity and promiscuity of MHC alleles remains a bottleneck.MHCs are the most polymorphic glycoproteins known in nature (11), and many polymorphisms impact peptide recognition; thus, investigation of peptide-binding properties of MHC-II is a challenging problem that requires high-throughput approaches. A number of studies have assessed peptide-binding to MHC-II on the surface of intact APCs (12, 13), whereas a routinely used in vitro approach entails purifying soluble recombinant MHC-II molecules from different expressing systems such as B cell lines (14), insect cells (15, 16), yeast (17), or Escherichia coli (18-20) and then characterizing binding of these molecules to different peptides generated either chemically by solid phase sy...

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Functional recombinant MHC class II molecules and high-throughput peptide-binding assays

Cited by 62 publications

References 90 publications

MHC Class II Tetramers Made from Isolated Recombinant α and β Chains Refolded with Affinity-Tagged Peptides

MHC Class II Tetramers Made from Isolated Recombinant α and β Chains Refolded with Affinity-Tagged Peptides

Current tools for predicting cancer-specific T cell immunity

High-throughput engineering and analysis of peptide binding to class II MHC

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