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Flow cytometry‐based TCR‐ligand K off ‐rate assay for fast avidity screening of even very small antigen‐specific T cell populations ex vivo
Author(s) -
Nauerth Magdalena,
Stemberger Christian,
Mohr Fabian,
Weißbrich Bianca,
Schiemann Matthias,
Germeroth Lothar,
Busch Dirk H.
Publication year - 2016
Publication title -
cytometry part a
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.316
H-Index - 90
eISSN - 1552-4930
pISSN - 1552-4922
DOI - 10.1002/cyto.a.22933
Subject(s) - avidity , t cell receptor , flow cytometry , epitope , t cell , ex vivo , biology , streptamer , cd8 , antigen , microbiology and biotechnology , cytotoxic t cell , immune system , immunology , biochemistry , in vitro
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 k off ‐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 k off ‐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