Design and validation of conditional ligands for HLA‐B*08:01, HLA‐B*15:01, HLA‐B*35:01, and HLA‐B*44:05

Abstract

We designed conditional ligands restricted to HLA-B*08:01, 2B*35:01, and 2B*44:05 and proved the use of a conditional ligand previously designed for HLA-B*15:02 together with HLA-B*15:01. Furthermore, we compared the detection capabilities of specific HLA-B*15:01-restricted T cells using the HLA-B*15:01 and HLA-B*15:02 major histocompatibility complex (MHC) multimers and found remarkable differences in the staining patterns detected by flow cytometry. These new conditional ligands greatly add to the application of MHC-based technologies in the analyses of T-cell recognition as they represent frequently expressed HLA-B molecules. This expansion of conditional ligands is important to allow T-cell detection over a wide range of HLA restrictions, and provide comprehensive understanding of the T-cell recognition in a given context. V C 2015 International Society for Advancement of Cytometry Key terms CD8 T cells; MHC multimer; HLA-B molecules; flow cytometry; conditional ligands THE tracking of specific T cells by use of fluorochrome-conjugated major histocompatibility complex (MHC) multimers for flow cytometry has increased our understanding in all areas of T-cell immunology (1-3). Staining of cells for binding to MHC multimers, combined with a set of antibodies to identify the CD8 T-cell population, represents an attractive way to quantitatively and phenotypically measure the CD8 T cells with certain specificities in a complex cell sample. It is thus possible to directly investigate the frequency and phenotype of these peptide-MHC (pMHC) multimer-specific T cells. The design and use of conditional ligands for the generation of MHC monomers was introduced in 2006 and revolutionized our ability to generate MHC reagents in a high-throughput fashion (4). These are HLA-binding peptides containing an UV-labile bond next to a 2-nitrophenylglycine or 3-amino-3-(2-nitrophenyl)-propionic acid residue (denoted "J") in their amino acid sequence, and can be integrated into the standard refolding procedure. Upon short-term UV light (366 nm) exposure, the amide bond next to the "J" residue is cleaved, and the fragmented peptide can be substituted with another ligand. The rescue of the MHC Class I complex after UV light exposure is dependent on the binding affinity between the ligand and the MHC molecule. Thus, this technique allows high-throughput affinity estimation using an MHC ELISA (5,6) and the generation of large libraries of pMHC complexes for the determinations of antigen-specific T cells using MHC Class I multimers in flow (7,8) and mass cytometry (9). The UV exchange technology has been extended to several MHC Class I variants (2,6,10), but conditional ligands has previously not been defined for some of the most frequently expressed HLA-B alleles.

A Recurrent Mutation in Anaplastic Lymphoma Kinase with Distinct Neoepitope Conformations

“…In addition, the percentage of reactive T cells against our two neoepitopes is comparable to values observed for the immunodominant SARS epitope (Figure S13C in Supplementary Material). While the nominal frequency of T cells specific for most p/MHC molecules ranges from 0.00005 to 0.01% ( 52 , 53 ), our observed values for HLA-B*15 tetramers are within the range of specific T cells identified in previous reports of PBMC staining of healthy donors using HLA-B*15 tetramers ( 54 ). Our staining results support the recognition of our putative nonamer and decamer neoepitopes by CD8+ T cells, potentiating the ability for the epitopes to drive specific immune responses.…”

A new synthetic toll-like receptor 1/2 ligand is an efficient adjuvant for peptide vaccination in a human volunteer

“…Lyophilized peptides (see Tables 1 and 2) were diluted in DMSO or water/ DMSO for T-cell assays and monomer refolding, respectively. The former was performed by conventional refolding as described before [17, 18], whereas ADV-Hex HLA-A*01-peptide and FLU-NCAP HLA-B*08-peptide monomers were generated by exchange of an HLA-B*08 UV labile monomer [19]. Multimers were generated by incubating monomers with streptavidin-PE/ streptavidin-APC (Biolegend, San Diego, CA) together with glycerol and human serum albumin [20].…”

Human endogenous retroviruses form a reservoir of T cell targets in hematological cancers

“…T cell staining using DNA barcode tagged peptide-MHC multimers. HERV peptide library specific monomers, restricted to HLA-A*01:01, A*02:01, B*07:02, and B*08:01, were generated by a UV mediated peptide exchange process 65,66,69,70 . These peptide-specific monomers were then attached to their corresponding DNA barcode dextrans by incubating at 4°C for 30 min, thus providing a DNA barcodelabeled dextran for each peptide-MHC (pMHC multimer) specifically to detect the respective T cell population.…”