title: Evaluation of suitable target antigens and immunoassays for high-accuracy immune monitoring of cytomegalovirus and Epstein-Barr virus-specific T cells as targets of interest in immunotherapeutic approaches.
abstract: Adoptive immunotherapy with donor-derived antiviral T cells can prevent viral complications such as with cytomegalovirus (CMV) and Epstein-Barr virus (EBV). In this context accurate monitoring of cellular immunity is essential and requires suitable quantitative and qualitative assays for high-throughput screening. We comparatively analyzed 57 HLA-typed healthy donors for memory T-cell responses to CMV- and EBV-derived proteins, peptide pools and single HLA-restricted peptides by five commonly used immunoassays in parallel: enzyme-linked immunospot (ELISPOT), cytokine secretion assay (CSA), intracellular cytokine staining (ICS), enzyme-linked immunosorbent assay (ELISA) and pMHC multimer staining. T-cell responses varied greatly between the different target antigens in the investigated assays. IFN-γ ELISPOT consistently detected the highest T-cell response levels against CMV and EBV. CMV-specific T cells were detected in 100% of CMV-seropositive donors tested using CMVpp65 protein and/or overlapping CMVpp65 peptide pool. CMV-specific T cells in HLA-A*02:01-positive/CMV-seropositive donors were identified directly by HLA-A02/CMVpp65 (A02pp65) multimer staining and, after short in vitro stimulation with HLA-A*02:01-restricted pp65 peptide, by ELISPOT, ELISA, ICS and CSA. A peptide-specific T-cell response was detected in only 4 HLA-A*02:01-positive donors (50%). Despite A02pp65 peptide negativity, T-cell responses to CMVpp65 protein and/or overlapping peptide pool were detected. Comparing the specific immune response against EBV antigens in healthy donors overall, BZLF1-specific T cells (<92.9% peptides, <56.3% peptide pool) were more frequent than EBNA-specific T cells (<64.3% peptides, <46.9% peptide pool) with higher percentage of positive findings for single HLA-restricted EBV peptides. T-cell response against HLA-B*08 peptide epitopes was predominant (multimer staining: EBNA3A: 9/14 and BZLF1: 7/14, IFN-γ ELISPOT: EBNA3A: 13/14 and BZLF1: 11/14). The fact that responses to EBV-specific antigens were not detected in every single EBV-seropositive donor as well as that the T-cell frequencies in response to the investigated EBV antigens differed strongly in the donor cohort indicates that these epitopes are less immunodominant than CMVpp65. Taken together, precise monitoring of T-cell immunity against infectious agents in potential T-cell donors and post-transplant recipients requires individual selection of antigens and immunoassays for the efficient detection and generation of clinically relevant T cells. Due to its lower detection limit and direct visualization of each IFN-γ-secreting cell we identified ELISPOT analysis to be preferable for high-throughput pre-screening. CSA was found to be advantageous for a more detailed analysis of antigen-specific T-cell subsets.
authors.affiliation: Institute for Transfusion Medicine, Hannover Medical School, Hannover, Germany; Integrated Research and Treatment Center (IFB-Tx), Hannover Medical School, Hannover Germany.
authors.forename: Sabine
authors.initials: S
authors.lastname: Tischer
authors.affiliation: Institute for Transfusion Medicine, Hannover Medical School, Hannover, Germany.
authors.forename: Daria
authors.initials: D
authors.lastname: Dieks
authors.affiliation: Institute for Transfusion Medicine, Hannover Medical School, Hannover, Germany; Integrated Research and Treatment Center (IFB-Tx), Hannover Medical School, Hannover Germany.
authors.forename: Cinja
authors.initials: C
authors.lastname: Sukdolak
authors.affiliation: Institute for Transfusion Medicine, Hannover Medical School, Hannover, Germany; Integrated Research and Treatment Center (IFB-Tx), Hannover Medical School, Hannover Germany.
authors.forename: Carola
authors.initials: C
authors.lastname: Bunse
authors.affiliation: Institute for Transfusion Medicine, Hannover Medical School, Hannover, Germany; Integrated Research and Treatment Center (IFB-Tx), Hannover Medical School, Hannover Germany.
authors.forename: Constanca
authors.initials: C
authors.lastname: Figueiredo
authors.affiliation: Institute for Transfusion Medicine, Hannover Medical School, Hannover, Germany.
authors.forename: Stephan
authors.initials: S
authors.lastname: Immenschuh
authors.affiliation: Integrated Research and Treatment Center (IFB-Tx), Hannover Medical School, Hannover Germany; Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School, Hannover, Germany.
authors.forename: Sylvia
authors.initials: S
authors.lastname: Borchers
authors.affiliation: Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School, Hannover, Germany.
authors.forename: Renata
authors.initials: R
authors.lastname: Stripecke
authors.affiliation: Integrated Research and Treatment Center (IFB-Tx), Hannover Medical School, Hannover Germany; Department of Pediatric Hematology and Oncology, Hannover Medical School, Hannover, Germany.
authors.forename: Britta
authors.initials: B
authors.lastname: Maecker-Kolhoff
authors.affiliation: Institute for Transfusion Medicine, Hannover Medical School, Hannover, Germany; Integrated Research and Treatment Center (IFB-Tx), Hannover Medical School, Hannover Germany.
authors.forename: Rainer
authors.initials: R
authors.lastname: Blasczyk
authors.affiliation: Institute for Transfusion Medicine, Hannover Medical School, Hannover, Germany; Integrated Research and Treatment Center (IFB-Tx), Hannover Medical School, Hannover Germany. Electronic address: eiz-vesper.britta@mh-hannover.de.
authors.forename: Britta
authors.initials: B
authors.lastname: Eiz-Vesper
mesh_terms: D019264:Adoptive Transfer; D000956:Antigens, Viral / Q000276:immunology*; D002478:Cells, Cultured; D016207:Cytokines / Q000378:metabolism; D003587:Cytomegalovirus / Q000276:immunology*; D003586:Cytomegalovirus Infections / Q000175:diagnosis* / Q000276:immunology / Q000628:therapy; D004797:Enzyme-Linked Immunosorbent Assay; D058501:Enzyme-Linked Immunospot Assay; D020031:Epstein-Barr Virus Infections / Q000175:diagnosis* / Q000276:immunology / Q000628:therapy; D006680:HLA Antigens / Q000235:genetics / Q000276:immunology; D004854:Herpesvirus 4, Human / Q000276:immunology*; D057166:High-Throughput Screening Assays; D006801:Humans; D007118:Immunoassay; D016056:Immunodominant Epitopes; D007156:Immunologic Memory; D059425:Interferon-gamma Release Tests; D015166:Monitoring, Immunologic / Q000379:methods*; D011237:Predictive Value of Tests; D016176:T-Lymphocyte Subsets / Q000276:immunology* / Q000637:transplantation / Q000821:virology
chemical_list: D000956:Antigens, Viral; D016207:Cytokines; D006680:HLA Antigens; D016056:Immunodominant Epitopes
keywords: Adoptive T-cell transfer; Antigen-specific T cells; Cytomegalovirus (CMV); Epstein–Barr virus (EBV); High-throughput immunoassay