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Increased mobility of major histocompatibility complex I-peptide complexes decreases the sensitivity of antigen recognition.

ABSTRACT: CD8(+) cytotoxic T lymphocytes (CTL) can recognize and kill target cells expressing only a few cognate major histocompatibility complex (MHC) I-peptide complexes. This high sensitivity requires efficient scanning of a vast number of highly diverse MHC I-peptide complexes by the T cell receptor in the contact site of transient conjugates formed mainly by nonspecific interactions of ICAM-1 and LFA-1. Tracking of single H-2K(d) molecules loaded with fluorescent peptides on target cells and nascent conjugates with CTL showed dynamic transitions between states of free diffusion and immobility. The immobilizations were explained by association of MHC I-peptide complexes with ICAM-1 and strongly increased their local concentration in cell adhesion sites and hence their scanning by T cell receptor. In nascent immunological synapses cognate complexes became immobile, whereas noncognate ones diffused out again. Interfering with this mobility modulation-based concentration and sorting of MHC I-peptide complexes strongly impaired the sensitivity of antigen recognition by CTL, demonstrating that it constitutes a new basic aspect of antigen presentation by MHC I molecules.

SUBMITTER: Segura JM

PROVIDER: S-EPMC3259769 | biostudies-literature | 2008 Aug

REPOSITORIES: biostudies-literature

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Increased mobility of major histocompatibility complex I-peptide complexes decreases the sensitivity of antigen recognition.

Segura Jean-Manuel JM Guillaume Philippe P Mark Silke S Dojcinovic Danijel D Johannsen Alexandre A Bosshard Giovanna G Angelov Georgi G Legler Daniel F DF Vogel Horst H Luescher Immanuel F IF

The Journal of biological chemistry 20080625 35

CD8(+) cytotoxic T lymphocytes (CTL) can recognize and kill target cells expressing only a few cognate major histocompatibility complex (MHC) I-peptide complexes. This high sensitivity requires efficient scanning of a vast number of highly diverse MHC I-peptide complexes by the T cell receptor in the contact site of transient conjugates formed mainly by nonspecific interactions of ICAM-1 and LFA-1. Tracking of single H-2K(d) molecules loaded with fluorescent peptides on target cells and nascent ...[more]

PMID: 18579518

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Project description:BACKGROUND:Identification of antigenic peptide epitopes is an essential prerequisite in T cell-based molecular vaccine design. Computational (sequence-based and structure-based) methods are inexpensive and efficient compared to experimental approaches in screening numerous peptides against their cognate MHC alleles. In structure-based protocols, suited to alleles with limited epitope data, the first step is to identify high-binding peptides using docking techniques, which need improvement in speed and efficiency to be useful in large-scale screening studies. We present pDOCK: a new computational technique for rapid and accurate docking of flexible peptides to MHC receptors and primarily apply it on a non-redundant dataset of 186 pMHC (MHC-I and MHC-II) complexes with X-ray crystal structures. RESULTS:We have compared our docked structures with experimental crystallographic structures for the immunologically relevant nonameric core of the bound peptide for MHC-I and MHC-II complexes. Primary testing for re-docking of peptides into their respective MHC grooves generated 159 out of 186 peptides with C? RMSD of less than 1.00 Å, with a mean of 0.56 Å. Amongst the 25 peptides used for single and variant template docking, the C? RMSD values were below 1.00 A for 23 peptides. Compared to our earlier docking methodology, pDOCK shows upto 2.5 fold improvement in the accuracy and is ~60% faster. Results of validation against previously published studies represent a seven-fold increase in pDOCK accuracy. CONCLUSIONS:The limitations of our previous methodology have been addressed in the new docking protocol making it a rapid and accurate method to evaluate pMHC binding. pDOCK is a generic method and although benchmarks against experimental structures, it can be applied to alleles with no structural data using sequence information. Our outcomes establish the efficacy of our procedure to predict highly accurate peptide structures permitting conformational sampling of the peptide in MHC binding groove. Our results also support the applicability of pDOCK for in silico identification of promiscuous peptide epitopes that are relevant to higher proportions of human population with greater propensity to activate T cells making them key targets for the design of vaccines and immunotherapies.

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.

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Increased mobility of major histocompatibility complex I-peptide complexes decreases the sensitivity of antigen recognition.

Publications

Increased mobility of major histocompatibility complex I-peptide complexes decreases the sensitivity of antigen recognition.

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