title: Reversible major histocompatibility complex I-peptide multimers containing Ni(2+)-nitrilotriacetic acid peptides and histidine tags improve analysis and sorting of CD8(+) T cells.
abstract: MHC-peptide multimers containing biotinylated MHC-peptide complexes bound to phycoerythrin (PE) streptavidin (SA) are widely used for analyzing and sorting antigen-specific T cells. Here we describe alternative T cell-staining reagents that are superior to conventional reagents. They are built on reversible chelate complexes of Ni(2+)-nitrilotriacetic acid (NTA) with oligohistidines. We synthesized biotinylated linear mono-, di-, and tetra-NTA compounds using conventional solid phase peptide chemistry and studied their interaction with HLA-A*0201-peptide complexes containing a His(6), His(12), or 2×His(6) tag by surface plasmon resonance on SA-coated sensor chips and equilibrium dialysis. The binding avidity increased in the order His(6) < His(12) < 2×His(6) and NTA(1) < NTA(2) < NTA(4), respectively, depending on the configuration of the NTA moieties and increased to picomolar K(D) for the combination of a 2×His(6) tag and a 2×Ni(2+)-NTA(2). We demonstrate that HLA-A2-2×His(6)-peptide multimers containing either Ni(2+)-NTA(4)-biotin and PE-SA- or PE-NTA(4)-stained influenza and Melan A-specific CD8+ T cells equal or better than conventional multimers. Although these complexes were highly stable, they very rapidly dissociated in the presence of imidazole, which allowed sorting of bona fide antigen-specific CD8+ T cells without inducing T cell death as well as assessment of HLA-A2-peptide monomer dissociation kinetics on CD8+ T cells.
authors: Ludwig Center of the University of Lausanne, 1066 Epalinges, Switzerland.
authors: Julien
authors: J
authors: Schmidt
authors: Ludwig Center of the University of Lausanne, 1066 Epalinges, Switzerland.
authors: Philippe
authors: P
authors: Guillaume
authors: Swiss Institute of Bioinformatics, Batiment Genopode, 1015 Lausanne Switzerland.
authors: Melita
authors: M
authors: Irving
authors: Division of Clinical Onco-Immunology, Ludwig Center of the University of Lausanne, 1011 Lausanne, Switzerland.
authors: Petra
authors: P
authors: Baumgaertner
authors: Division of Clinical Onco-Immunology, Ludwig Center of the University of Lausanne, 1011 Lausanne, Switzerland.
authors: Daniel
authors: D
authors: Speiser
authors: Ludwig Center of the University of Lausanne, 1066 Epalinges, Switzerland. Electronic address: immanuel.luescher@unil.ch.
authors: Immanuel F
authors: IF
authors: Luescher
mesh_terms: D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D015789:HLA-A2 Antigen / Q000737:chemistry / Q000276:immunology; D006639:Histidine / Q000737:chemistry / Q000276:immunology; D006801:Humans; D058965:MART-1 Antigen / Q000737:chemistry / Q000276:immunology; D009532:Nickel / Q000737:chemistry*; D009571:Nitrilotriacetic Acid / Q000737:chemistry* / Q000276:immunology; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000276:immunology; D013194:Staining and Labeling / Q000379:methods
chemical_list: C435939:HLA-A*02:01 antigen; D015789:HLA-A2 Antigen; D058965:MART-1 Antigen; C548667:MLANA protein, human; D010455:Peptides; C033223:polyhistidine; D006639:Histidine; D009532:Nickel; D009571:Nitrilotriacetic Acid