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A flexible MHC class I multimer loading system for large-scale detection of antigen-specific T cells.

ABSTRACT: Adaptive immunity is initiated by T cell recognition of specific antigens presented by major histocompatibility complexes (MHCs). MHC multimer technology has been developed for the detection, isolation, and characterization of T cells in infection, autoimmunity, and cancer. Here, we present a simple, fast, flexible, and efficient method to generate many different MHC class I (MHC I) multimers in parallel using temperature-mediated peptide exchange. We designed conditional peptides for HLA-A*02:01 and H-2Kb that form stable peptide-MHC I complexes at low temperatures, but dissociate when exposed to a defined elevated temperature. The resulting conditional MHC I complexes, either alone or prepared as ready-to-use multimers, can swiftly be loaded with peptides of choice without additional handling and within a short time frame. We demonstrate the ease and flexibility of this approach by monitoring the antiviral immune constitution in an allogeneic stem cell transplant recipient and by analyzing CD8+ T cell responses to viral epitopes in mice infected with lymphocytic choriomeningitis virus or cytomegalovirus.

SUBMITTER: Luimstra JJ

PROVIDER: S-EPMC5940271 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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A flexible MHC class I multimer loading system for large-scale detection of antigen-specific T cells.

Luimstra Jolien J JJ Garstka Malgorzata A MA Roex Marthe C J MCJ Redeker Anke A Janssen George M C GMC van Veelen Peter A PA Arens Ramon R Falkenburg J H Frederik JHF Neefjes Jacques J Ovaa Huib H

The Journal of experimental medicine 20180417 5

Adaptive immunity is initiated by T cell recognition of specific antigens presented by major histocompatibility complexes (MHCs). MHC multimer technology has been developed for the detection, isolation, and characterization of T cells in infection, autoimmunity, and cancer. Here, we present a simple, fast, flexible, and efficient method to generate many different MHC class I (MHC I) multimers in parallel using temperature-mediated peptide exchange. We designed conditional peptides for HLA-A*02:0 ...[more]

PMID: 29666167

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Project description:Peptide-MHC (pMHC) multimers have become the "gold standard" for the detection and isolation of antigen-specific T-cells but recent evidence shows that normal use of these reagents can miss fully functional T-cells that bear T-cell receptors (TCRs) with low affinity for cognate antigen. This issue is particularly pronounced for anticancer and autoimmune T-cells as self-reactive T-cell populations are enriched for low-affinity TCRs due to the removal of cells with higher affinity receptors by immune tolerance mechanisms. Here, we stained a wide variety of self-reactive human T-cells using regular pMHC staining and an optimized technique that included: (i) protein kinase inhibitor (PKI), to prevent TCR triggering and internalization, and (ii) anti-fluorochrome antibody, to reduce reagent dissociation during washing steps. Lymphocytes derived from the peripheral blood of type 1 diabetes patients were stained with pMHC multimers made with epitopes from preproinsulin (PPI), insulin-? chain, glutamic acid decarboxylase 65 (GAD65), or glucose-6-phospate catalytic subunit-related protein (IGRP) presented by disease-risk allelles HLA A*02:01 or HLA*24:02. Samples from ankylosing spondylitis patients were stained with a multimerized epitope from vasoactive intestinal polypeptide receptor 1 (VIPR1) presented by HLA B*27:05. Optimized procedures stained an average of 40.5-fold (p?=?0.01, range between 1.4 and 198) more cells than could be detected without the inclusion of PKI and cross-linking anti-fluorochrome antibody. Higher order pMHC dextramers recovered more cells than pMHC tetramers in parallel assays, and standard staining protocols with pMHC tetramers routinely recovered less cells than functional assays. HLA A*02:01-restricted PPI-specific and HLA B*27:05-restricted VIPR1-specific T-cell clones generated using the optimized procedure could not be stained by standard pMHC tetramer staining. However, these clones responded well to exogenously supplied peptide and endogenously processed and presented epitopes. We also showed that anti-fluorochrome antibody-conjugated magnetic beads enhanced staining of self-reactive T-cells that could not be stained using standard protocols, thus enabling rapid ex vivo isolation of autoimmune T-cells. We, therefore, conclude that regular pMHC tetramer staining is generally unsuitable for recovering self-reactive T-cells from clinical samples and recommend the use of the optimized protocols described herein.

Project description:BACKGROUND:The total number of known three-dimensional protein structures is rapidly increasing. Consequently, the need for fast structural search against complete databases without a significant loss of accuracy is increasingly demanding. Recently, TopSearch, an ultra-fast method for finding rigid structural relationships between a query structure and the complete Protein Data Bank (PDB), at the multi-chain level, has been released. However, comparable accurate flexible structural aligners to perform efficient whole database searches of multi-domain proteins are not yet available. The availability of such a tool is critical for a sustainable boosting of biological discovery. RESULTS:Here we report on the development of a new method for the fast and flexible comparison of protein structure chains. The method relies on the calculation of 2D matrices containing a description of the three-dimensional arrangement of secondary structure elements (angles and distances). The comparison involves the matching of an ensemble of substructures through a nested-two-steps dynamic programming algorithm. The unique features of this new approach are the integration and trade-off balancing of the following: 1) speed, 2) accuracy and 3) global and semiglobal flexible structure alignment by integration of local substructure matching. The comparison, and matching with competitive accuracy, of one medium sized (250-aa) query structure against the complete PDB database (216,322 protein chains) takes about 8 min using an average desktop computer. The method is at least 2-3 orders of magnitude faster than other tested tools with similar accuracy. We validate the performance of the method for fold and superfamily assignment in a large benchmark set of protein structures. We finally provide a series of examples to illustrate the usefulness of this method and its application in biological discovery. CONCLUSIONS:The method is able to detect partial structure matching, rigid body shifts, conformational changes and tolerates substantial structural variation arising from insertions, deletions and sequence divergence, as well as structural convergence of unrelated proteins.

Dataset Information

A flexible MHC class I multimer loading system for large-scale detection of antigen-specific T cells.

Publications

A flexible MHC class I multimer loading system for large-scale detection of antigen-specific T cells.

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