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

J Immunol Methods. 2014 Jun;408:101-13. doi: 10.1016/j.jim.2014.05.011. Epub 2014 May 28.


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.

Keywords: Adoptive T-cell transfer; Antigen-specific T cells; Cytomegalovirus (CMV); Epstein–Barr virus (EBV); High-throughput immunoassay.

Publication types

  • Comparative Study
  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adoptive Transfer*
  • Antigens, Viral / immunology*
  • Cells, Cultured
  • Cytokines / metabolism
  • Cytomegalovirus / immunology*
  • Cytomegalovirus Infections / diagnosis*
  • Cytomegalovirus Infections / immunology
  • Cytomegalovirus Infections / therapy
  • Enzyme-Linked Immunosorbent Assay
  • Enzyme-Linked Immunospot Assay
  • Epstein-Barr Virus Infections / diagnosis*
  • Epstein-Barr Virus Infections / immunology
  • Epstein-Barr Virus Infections / therapy
  • HLA Antigens / genetics
  • HLA Antigens / immunology
  • Herpesvirus 4, Human / immunology*
  • High-Throughput Screening Assays
  • Humans
  • Immunoassay*
  • Immunodominant Epitopes
  • Immunologic Memory
  • Interferon-gamma Release Tests
  • Monitoring, Immunologic / methods*
  • Predictive Value of Tests
  • T-Lymphocyte Subsets / immunology*
  • T-Lymphocyte Subsets / transplantation
  • T-Lymphocyte Subsets / virology


  • Antigens, Viral
  • Cytokines
  • HLA Antigens
  • Immunodominant Epitopes