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Two-stage cooperative T cell receptor-peptide major histocompatibility complex-CD8 trimolecular interactions amplify antigen discrimination.

ABSTRACT: The T cell receptor (TCR) and CD8 bind peptide-major histocompatibility complex (pMHC) glycoproteins to initiate adaptive immune responses, yet the trimolecular binding kinetics at the T cell membrane is unknown. By using a micropipette adhesion frequency assay, we show that this kinetics has two stages. The first consists of TCR-dominant binding to agonist pMHC. This triggers a second stage consisting of a step increase in adhesion after a one second delay. The second-stage binding requires Src family kinase activity to initiate CD8 binding to the same pMHC engaged by the TCR. This induced trimeric-cooperative interaction enhances adhesion synergistically to favor potent ligands, which further amplifies discrimination. Our data reveal a TCR-CD8 positive-feedback loop involved in initial signaling steps that is sensitive to a single pMHC is rapid, reversible, synergistic, and peptide discriminative.

SUBMITTER: Jiang N

PROVIDER: S-EPMC3381515 | biostudies-literature | 2011 Jan

REPOSITORIES: biostudies-literature

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Two-stage cooperative T cell receptor-peptide major histocompatibility complex-CD8 trimolecular interactions amplify antigen discrimination.

Jiang Ning N Huang Jun J Edwards Lindsay J LJ Liu Baoyu B Zhang Yan Y Beal Carrie D CD Evavold Brian D BD Zhu Cheng C

Immunity 20110120 1

The T cell receptor (TCR) and CD8 bind peptide-major histocompatibility complex (pMHC) glycoproteins to initiate adaptive immune responses, yet the trimolecular binding kinetics at the T cell membrane is unknown. By using a micropipette adhesion frequency assay, we show that this kinetics has two stages. The first consists of TCR-dominant binding to agonist pMHC. This triggers a second stage consisting of a step increase in adhesion after a one second delay. The second-stage binding requires Src ...[more]

PMID: 21256056

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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.

Dataset Information

Two-stage cooperative T cell receptor-peptide major histocompatibility complex-CD8 trimolecular interactions amplify antigen discrimination.

Publications

Two-stage cooperative T cell receptor-peptide major histocompatibility complex-CD8 trimolecular interactions amplify antigen discrimination.

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