Generation of Peptide MHC Class I Monomers and Multimers Through Ligand Exchange

Toebes, Mireille; Rodenko, Boris; Ovaa, Huib; Schumacher, Ton N.

doi:10.1002/0471142735.im1816s87

Cited by 49 publications

(48 citation statements)

References 11 publications

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“…For VACV/ECTV-and SIINFEKLspecific CD8 ϩ T cells, H-2 K b :Ig recombinant fusion protein (Dimer-X; BD) was incubated with synthetic TSYKFESV, ITYRFYLI, SIFRFLNI, KS YNYMLL, and SIINFEKL peptides and used as recommended by the manufacturer. On some occasions, instead of K b -peptide dimers, we used K b -TSYKFESV, -SIFRFLNI, -KSYNYMLL, and -STLNFNNL tetramers prepared by the NIAID Tetramer Facility or K b -TSYKFESV or -SIINFEKL tetramers prepared in our laboratory according to published methods (47). Stained cells were analyzed by flow cytometry at the Fox Chase Cell Sorting Facility using an LSR II system (BD).…”

Section: Methodsmentioning

confidence: 99%

Memory CD8⁺T Cells Specific for a Single Immunodominant or Subdominant Determinant Induced by Peptide-Dendritic Cell Immunization Protect from an Acute Lethal Viral Disease

Remakus

Rubio

et al. 2012

J Virol

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The antigens recognized by individual CD8+T cells are small peptides bound to major histocompatibility complex (MHC) class I molecules. The CD8+T cell response to a virus is restricted to several peptides, and the magnitudes of the effector as well as memory phases of the response to the individual peptides are generally hierarchical. The peptide eliciting a stronger response is called immunodominant (ID), and those with smaller-magnitude responses are termed subdominant (SD). The relative importance of ID and SD determinants in protective immunity remains to be fully elucidated. We previously showed that multispecific memory CD8+T cells can protect susceptible mice from mousepox, an acute lethal viral disease. It remained unknown, however, whether CD8+T cells specific for single ID or SD peptides could be protective. Here, we demonstrate that immunization with dendritic cells pulsed with ID and some but not all SD peptides induces memory CD8+T cells that are fully capable of protecting susceptible mice from mousepox. Additionally, while natural killer (NK) cells are essential for the natural resistance of nonimmune C57BL/6 (B6) to mousepox, we show that memory CD8+T cells of single specificity also protect B6 mice depleted of NK cells. This suggests it is feasible to produce effective antiviral CD8+T cell vaccines using single CD8+T cell determinants and that NK cells are no longer essential when memory CD8+T cells are present.

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Section: Methodsmentioning

confidence: 99%

Memory CD8⁺T Cells Specific for a Single Immunodominant or Subdominant Determinant Induced by Peptide-Dendritic Cell Immunization Protect from an Acute Lethal Viral Disease

Remakus

Rubio

et al. 2012

J Virol

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“…HLA molecules were expressed and refolded as described (40) in the presence of peptide ([am-phg][NVA]AGIGILT [PRG], in which am-phg is D-alpha-methyl-phenylglycine, NVA is norvaline and PRG is propargylglycine). Subsequently, pMHC complexes were loaded on a Mono-Q anion exchange column and eluted with NaCl gradient in 20 mM Tris-HCl (pH 7.0).…”

Section: Crystallizationmentioning

confidence: 99%

“…T cell staining with exchange pMHC multimers was performed essentially as described (40). Enhanced and control peptides in DMSO were added to biotinylated MHC monomers (25 mg/ml in PBS) to a final concentration of 50 mM and ultraviolet irradiated for 30 min.…”

Section: T Cell Staining and Flow Cytometrymentioning

confidence: 99%

Altered Peptide Ligands Revisited: Vaccine Design through Chemically Modified HLA-A2–Restricted T Cell Epitopes

Hoppes

Oostvogels

Luimstra

et al. 2014

The Journal of Immunology

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Virus or tumor Ag–derived peptides that are displayed by MHC class I molecules are attractive starting points for vaccine development because they induce strong protective and therapeutic cytotoxic T cell responses. In thus study, we show that the MHC binding and consequent T cell reactivity against several HLA-A*02 restricted epitopes can be further improved through the incorporation of nonproteogenic amino acids at primary and secondary anchor positions. We screened more than 90 nonproteogenic, synthetic amino acids through a range of epitopes and tested more than 3000 chemically enhanced altered peptide ligands (CPLs) for binding affinity to HLA-A*0201. With this approach, we designed CPLs of viral epitopes, of melanoma-associated Ags, and of the minor histocompatibility Ag UTA2-1, which is currently being evaluated for its antileukemic activity in clinical dendritic cell vaccination trials. The crystal structure of one of the CPLs in complex with HLA-A*0201 revealed the molecular interactions likely responsible for improved binding. The best CPLs displayed enhanced affinity for MHC, increasing MHC stability and prolonging recognition by Ag-specific T cells and, most importantly, they induced accelerated expansion of antitumor T cell frequencies in vitro and in vivo as compared with the native epitope. Eventually, we were able to construct a toolbox of preferred nonproteogenic residues with which practically any given HLA-A*02 restricted epitope can be readily optimized. These CPLs could improve the therapeutic outcome of vaccination strategies or can be used for ex vivo enrichment and faster expansion of Ag-specific T cells for transfer into patients.

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“…Indicated virus-derived, melanoma-associated, and UV-cleavable peptides were synthesized in-house as described previously (13). Recombinant HLA-A*02 subtype-specific H chains and human b 2 -microglobulin L chain were produced in Escherichia coli and isolated from inclusion bodies.…”

Section: Generation Of Pmhc Complexesmentioning

confidence: 99%

“…MHC complexes loaded with the indicated peptides of interest were generated by UV-induced ligand exchange. In brief, pMHC complexes with UV-sensitive peptide (100 mg/ml) were subjected to 366 nM UV light (Camag) for 1 h at 4˚C in the presence of rescue peptide (200 mM) (13,15).…”

Section: Generation Of Pmhc Complexesmentioning

confidence: 99%

HLA Micropolymorphisms Strongly Affect Peptide–MHC Multimer–Based Monitoring of Antigen-Specific CD8+ T Cell Responses

Buuren

Dijkgraaf

Linnemann

et al. 2014

The Journal of Immunology

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Peptide–MHC (pMHC) multimers have become one of the most widely used tools to measure Ag-specific T cell responses in humans. With the aim of understanding the requirements for pMHC-based personalized immunomonitoring, in which individuals expressing subtypes of the commonly studied HLA alleles are encountered, we assessed how the ability to detect Ag-specific T cells for a given peptide is affected by micropolymorphic differences between HLA subtypes. First, analysis of a set of 10 HLA-A*02:01–restricted T cell clones demonstrated that staining with pMHC multimers of seven distinct subtypes of the HLA-A*02 allele group was highly variable and not predicted by sequence homology. Second, to analyze the effect of minor sequence variation in a clinical setting, we screened tumor-infiltrating lymphocytes of an HLA-A*02:06 melanoma patient with either subtype-matched or HLA-A*02:01 multimers loaded with 145 different melanoma-associated Ags. This revealed that of the four HLA-A*02:06–restricted melanoma-associated T cell responses observed in this patient, two responses were underestimated and one was overlooked when using subtype-mismatched pMHC multimer collections. To our knowledge, these data provide the first demonstration of the strong effect of minor sequence variation on pMHC-based personalized immunomonitoring, and they provide tools to prevent this issue for common variants within the HLA-A*02 allele group.

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Generation of Peptide MHC Class I Monomers and Multimers Through Ligand Exchange

Cited by 49 publications

References 11 publications

Memory CD8⁺T Cells Specific for a Single Immunodominant or Subdominant Determinant Induced by Peptide-Dendritic Cell Immunization Protect from an Acute Lethal Viral Disease

Memory CD8⁺T Cells Specific for a Single Immunodominant or Subdominant Determinant Induced by Peptide-Dendritic Cell Immunization Protect from an Acute Lethal Viral Disease

Altered Peptide Ligands Revisited: Vaccine Design through Chemically Modified HLA-A2–Restricted T Cell Epitopes

HLA Micropolymorphisms Strongly Affect Peptide–MHC Multimer–Based Monitoring of Antigen-Specific CD8+ T Cell Responses

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Generation of Peptide MHC Class I Monomers and Multimers Through Ligand Exchange

Cited by 49 publications

References 11 publications

Memory CD8+T Cells Specific for a Single Immunodominant or Subdominant Determinant Induced by Peptide-Dendritic Cell Immunization Protect from an Acute Lethal Viral Disease

Memory CD8+T Cells Specific for a Single Immunodominant or Subdominant Determinant Induced by Peptide-Dendritic Cell Immunization Protect from an Acute Lethal Viral Disease

Altered Peptide Ligands Revisited: Vaccine Design through Chemically Modified HLA-A2–Restricted T Cell Epitopes

HLA Micropolymorphisms Strongly Affect Peptide–MHC Multimer–Based Monitoring of Antigen-Specific CD8+ T Cell Responses

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Product

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Memory CD8⁺T Cells Specific for a Single Immunodominant or Subdominant Determinant Induced by Peptide-Dendritic Cell Immunization Protect from an Acute Lethal Viral Disease

Memory CD8⁺T Cells Specific for a Single Immunodominant or Subdominant Determinant Induced by Peptide-Dendritic Cell Immunization Protect from an Acute Lethal Viral Disease