title: High-density preculture of PBMCs restores defective sensitivity of circulating CD8 T cells to virus- and tumor-derived antigens.
abstract: Peripheral blood mononuclear cells (PBMCs) are the only source of human lymphoid cells routinely available for immunomonitoring of T-cell responses to microbial and tumor-associated antigens. However, previous work in mice and humans had indicated that CD4 T cells transiently lose antigen sensitivity when cellular contacts are lost (eg, by entering the circulation). Using the simple and robust protocol for resetting T cells to original reactivity (RESTORE; ie, preculturing PBMCs for 2 days at a high cell density before initiation of antigenic stimulation), we show that CD8 T-cell responses to viral and tumor-associated antigens are greatly underestimated in blood, and sometimes even remain undetected, if conventional, unprocessed PBMC cultures are used. The latter finding is particularly striking with regard to the appearance of Wilms tumor 1 protein-specific CD8 T-cell responses in leukemia patients after allogeneic bone marrow transplantation. The dramatic increase in antigen sensitivity of "restored" CD8 T cells is associated with phosphorylation of proximal T-cell receptor signaling components, and with the upregulation of genes involved in aerobic glycolysis, thereby increasing T-cell functionality. The RESTORE protocol permits a more meaningful monitoring of CD8 memory T-cell responses to viral infections and tumors and vaccination success. Furthermore, when generating T-cell lines for adoptive T-cell therapy, it avoids the loss of those clones, which strictly depend on the primed status conferred by cellular interactions in the tissue context for their initial reactivation by antigen. The data reported in this article have been deposited in the Gene Expression Omnibus database (accession number GSE63430).
authors.affiliation: Institute for Virology and Immunobiology, Department of Immunology, University of Würzburg, Würzburg, Germany;
authors.forename: Julia
authors.initials: J
authors.lastname: Wegner
authors.affiliation: Department of Oto-Rhino-Laryngology, Plastic, Aesthetic and Reconstructive Head and Neck Surgery, University Hospital of Würzburg, Würzburg, Germany;
authors.forename: Stephan
authors.initials: S
authors.lastname: Hackenberg
authors.affiliation: Core Unit Systems Medicine, University of Würzburg, Würzburg, Germany;
authors.forename: Claus-Jürgen
authors.initials: CJ
authors.lastname: Scholz
authors.affiliation: TheraMAB LLC, Würzburg, Germany;
authors.forename: Sergey
authors.initials: S
authors.lastname: Chuvpilo
authors.affiliation: TheraMAB LLC, Moscow, Russia;
authors.forename: Dmitry
authors.initials: D
authors.lastname: Tyrsin
authors.affiliation: TheraMAB LLC, Moscow, Russia;
authors.forename: Alexey A
authors.initials: AA
authors.lastname: Matskevich
authors.affiliation: Department of Internal Medicine II, University Hospital of Würzburg, Würzburg, Germany; and.
authors.forename: Götz Ulrich
authors.initials: GU
authors.lastname: Grigoleit
authors.affiliation: Interfaculty Institute for Cell Biology, Department of Immunology, University of Tübingen, Tübingen, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Stevanović
authors.affiliation: Institute for Virology and Immunobiology, Department of Immunology, University of Würzburg, Würzburg, Germany;
authors.forename: Thomas
authors.initials: T
authors.lastname: Hünig
mesh_terms: D000951:Antigens, Neoplasm / Q000276:immunology*; D000956:Antigens, Viral / Q000276:immunology*; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D002452:Cell Count; D018929:Cell Culture Techniques / Q000379:methods*; D002478:Cells, Cultured; D002648:Child; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000166:cytology* / Q000502:physiology; D037182:T-Cell Antigen Receptor Specificity
chemical_list: D000951:Antigens, Neoplasm; D000956:Antigens, Viral