title: Multiplex and functional detection of antigen-specific human T cells by ITRA--indirect T cell recognition assay.
abstract: The identification and functional characterization of pathogen-specific T cells plays a critical role in immunological research and diagnostics. In addition to the present standard technologies such as intracellular cytokine staining (ICS), enzyme-linked immunospot (ELISPOT) and peptide-major-histocompatibility-complex (MHC) multimer staining, we aimed to develop a multiplex detection assay, which provides fast in vitro functional data for both human CD4 and CD8 T cells with different antigen specificities in one sample. In this study, we have exploited the expression of CD83 on B cells to develop the cell array-based indirect T cell recognition assay (ITRA). In detail, B cells are pulsed with different pathogen peptide pools and fluorescently barcoded. Thereafter the B cells are pooled and co-cultured with autologous T cells. Subsequently each B cell population is analyzed via flow cytometry for CD83 expression, which indicates antigen-specific interaction with CD4 T cells. Moreover, we revealed donor dependent variations of cytotoxic activity of pathogen-specific CD4 T cells and CD8 T cells, evidenced by specific lysis of peptide-pulsed B cells. Taken together, ITRA is a novel antigen presenting cell (APC) array based method to analyze the presence and function of various antigen-specific T cells in one sample. It has the potential to be used in the future for epitope/antigen screening in research and for analysis of anti-tumor, anti-pathogen or autoimmune T cell responses in patient samples.
authors: Department of Human Medicine, Institute of Immunology, University Witten/Herdecke, 58453 Witten, Germany.
authors: Lan
authors: L
authors: Tong
authors: Department of Research and Development, Miltenyi Biotec GmbH, Bergisch Gladbach 51429, Germany.
authors: Carolin
authors: C
authors: Schuhmacher
authors: Department of Research and Development, Miltenyi Biotec GmbH, Bergisch Gladbach 51429, Germany.
authors: Mario
authors: M
authors: Assenmacher
authors: Department of Human Medicine, Institute of Immunology, University Witten/Herdecke, 58453 Witten, Germany.
authors: Kurt
authors: K
authors: Zänker
authors: Department of Research and Development, Miltenyi Biotec GmbH, Bergisch Gladbach 51429, Germany.
authors: Peter
authors: P
authors: Jähn
mesh_terms: D000256:Adenoviridae / Q000737:chemistry / Q000276:immunology; D015703:Antigens, CD / Q000235:genetics / Q000276:immunology; D000956:Antigens, Viral / Q000276:immunology / Q000494:pharmacology; D001402:B-Lymphocytes / Q000276:immunology / Q000473:pathology / Q000821:virology; D015496:CD4-Positive T-Lymphocytes / Q000187:drug effects / Q000276:immunology / Q000473:pathology*; D018414:CD8-Positive T-Lymphocytes / Q000187:drug effects / Q000276:immunology / Q000473:pathology*; D018920:Coculture Techniques; D003587:Cytomegalovirus / Q000737:chemistry / Q000276:immunology; D018984:Epitopes, T-Lymphocyte / Q000276:immunology*; D015870:Gene Expression; D004854:Herpesvirus 4, Human / Q000737:chemistry / Q000276:immunology; D006801:Humans; D007118:Immunoassay; D007136:Immunoglobulins / Q000235:genetics / Q000276:immunology; D008213:Lymphocyte Activation; D008562:Membrane Glycoproteins / Q000235:genetics / Q000276:immunology; D010455:Peptides / Q000276:immunology / Q000494:pharmacology; D061251:Primary Cell Culture; D046888:Tissue Array Analysis; D000097802:CD83 Antigen
chemical_list: D015703:Antigens, CD; D000956:Antigens, Viral; D018984:Epitopes, T-Lymphocyte; D007136:Immunoglobulins; D008562:Membrane Glycoproteins; D010455:Peptides; D000097802:CD83 Antigen
keywords: Antigen-specific T cells; B cells; CD4 help; CD8 cytotoxicity; CD83; Multiplex