title: Flow cytometry-based TCR-ligand Koff -rate assay for fast avidity screening of even very small antigen-specific T cell populations ex vivo.
abstract: High epitope-specific sensitivity of CD8(+) T cells is required for optimal immune protection against intracellular pathogens as well as certain malignancies. The quality of antigen recognition of CD8(+) T cells is usually described as "avidity" to its cognate peptide MHCI complex. T cell avidity is mainly dependent on the structural qualities of the T cell receptor (TCR), as convincingly demonstrated by recombinant TCR re-expression experiments. Based on reversible MHCI multimer staining and koff -rate measurements of monomeric peptide MHCI complexes, we recently established a microscopic assay for determining the structural avidity of individual CD8(+) T cells. Here we demonstrate that this assay can be adapted for rapid flow-cytometric avidity screening of epitope-specific T cell populations. Furthermore, we show that-in combination with conventional nonreversible MHCI multimer staining-even very small epitope-specific CD8(+) T cell populations can be analyzed directly ex vivo without the need for previous TCR cloning or T cell sorting. This simplified approach provides highly accurate mean TCR-ligand koff -rate values for poly- or oligoclonal T cell populations and is ideally suited for high-throughput applications in basic research as well as clinical settings. © 2016 International Society for Advancement of Cytometry.
authors: Institute for Medical Microbiology, Immunology and Hygiene, Technische Universität München, Munich, Germany.
authors: Magdalena
authors: M
authors: Nauerth
authors: Institute for Medical Microbiology, Immunology and Hygiene, Technische Universität München, Munich, Germany.|Juno Therapeutics, Munich, Germany.
authors: Christian
authors: C
authors: Stemberger
authors: Institute for Medical Microbiology, Immunology and Hygiene, Technische Universität München, Munich, Germany.
authors: Fabian
authors: F
authors: Mohr
authors: Institute for Medical Microbiology, Immunology and Hygiene, Technische Universität München, Munich, Germany.
authors: Bianca
authors: B
authors: Weißbrich
authors: Institute for Medical Microbiology, Immunology and Hygiene, Technische Universität München, Munich, Germany.
authors: Matthias
authors: M
authors: Schiemann
authors: Juno Therapeutics, Munich, Germany.
authors: Lothar
authors: L
authors: Germeroth
authors: Institute for Medical Microbiology, Immunology and Hygiene, Technische Universität München, Munich, Germany. dirk.busch@tum.de.|DZIF - National Centre for Infection Research, Munich, Germany. dirk.busch@tum.de.|Focus Group ''Clinical Cell Processing and Purification", Institute for Advanced Study, Technische Universität München, Munich, Germany. dirk.busch@tum.de.
authors: Dirk H
authors: DH
authors: Busch
mesh_terms: D000818:Animals; D000941:Antigens / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D018984:Epitopes, T-Lymphocyte / Q000276:immunology*; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D008024:Ligands; D008213:Lymphocyte Activation / Q000276:immunology; D051379:Mice; D011948:Receptors, Antigen, T-Cell / Q000276:immunology; D013601:T-Lymphocytes / Q000276:immunology*
chemical_list: D000941:Antigens; D018984:Epitopes, T-Lymphocyte; D008024:Ligands; D011948:Receptors, Antigen, T-Cell
keywords: MHC multimer double staining; T cell functionality; T cell therapy; TCR-ligand-koff-rate; direct avidity screening; dissociation kinetics; online injection; structural TCR avidity; temperature controlled analysis; uninterrupted acquisition