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Programmed self-assembly of peptide-major histocompatibility complex for antigen-specific immune modulation.

ABSTRACT: A technology to prime desired populations of T cells in the body-particularly those that possess low avidity against target antigen-would pave the way for the design of new types of vaccination for intractable infectious diseases or cancer. Here, we report such a technology based on positive feedback-driven, programmed self-assembly of peptide-major histocompatibility complex (pMHC) directly on the membrane of cognate T cells. Our design capitalizes on the unique features of the protein annexin V (ANXA5), which-in a concerted and synergistic manner-couples the early onset of TCR signaling by cognate pMHC with a surge in pMHC-TCR affinity, with repeated pMHC encounters, and with widespread TCR cross-linking. In our system, ANXA5 is linked to pMHC and firmly engages the plasma membrane of cognate T cells upon (and only upon) the early onset of TCR signaling. ANXA5, in turn, exerts a mechanical force that stabilizes interactions at the TCR-pMHC interface and facilitates repeated, serial pMHC encounters. Furthermore, ANXA5 quickly arranges into uniform 2D matrices, thereby prompting TCR cross-linking. Fusion of ANXA5 to pMHC augments lymphocyte activation by several orders of magnitude (>1,000-fold), bypasses the need for costimulation, and breaks tolerance against a model self-antigen in vivo. Our study opens the door to the application of synthetic, feedback-driven self-assembly platforms in immune modulation.

SUBMITTER: Mao CP

PROVIDER: S-EPMC5924900 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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Programmed self-assembly of peptide-major histocompatibility complex for antigen-specific immune modulation.

Mao Chih-Ping CP Peng Shiwen S Yang Andrew A He Liangmei L Tsai Ya-Chea YC Hung Chien-Fu CF Wu T-C TC

Proceedings of the National Academy of Sciences of the United States of America 20180409 17

A technology to prime desired populations of T cells in the body-particularly those that possess low avidity against target antigen-would pave the way for the design of new types of vaccination for intractable infectious diseases or cancer. Here, we report such a technology based on positive feedback-driven, programmed self-assembly of peptide-major histocompatibility complex (pMHC) directly on the membrane of cognate T cells. Our design capitalizes on the unique features of the protein annexin ...[more]

PMID: 29632186

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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

Programmed self-assembly of peptide-major histocompatibility complex for antigen-specific immune modulation.

Publications

Programmed self-assembly of peptide-major histocompatibility complex for antigen-specific immune modulation.

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