title: Rapid monitoring of immune reconstitution after allogeneic stem cell transplantation--a comparison of different assays for the detection of cytomegalovirus-specific T cells.
abstract: OBJECTIVE
  Cytotoxic T cells (CTLs) play an important role in controlling cytomegalovirus (CMV) infection after allogeneic hematopoietic stem cell transplantation (HSCT). The purpose of this study was to compare four different assays to detect CMV antigen-specific T cells for the monitoring of CMV-specific immune reconstitution after HSCT.
  
  
  METHODS
  Comparative monitoring of CMV-specific T cells was performed using HLA tetramer, interferon-gamma enzyme-linked immunospot (ELISPOT), intracellular IFN-γ (IC-FACS), and real-time polymerase chain reaction (RT-PCR) assays with immunodominant CMV pp65-HLA-A2-restricted peptide and recombinant pp65-protein in 18 patients after allogeneic HSCT.
  
  
  RESULTS
  Using pp65-peptide to stimulate T cells in HLA-A2-positive patients, antigen-specific T cells have been detected in 43% (10 of 23) by IC-FACS, 62% (13 of 21) by tetramer, 68% (15 of 22) by RT-PCR, and 53% (10 of 19) by ELISPOT. No T-cell responses were detected in HLA-A2-negative patients by all assays. Stimulating with combination of both pp65-peptide and pp65-protein detected 72% (18/25) by IC-FACS, 88% (22/25) by RT-PCR, and 59% (12/22) by ELISPOT. Comparing the different methods of T-cell detection we found a significant positive correlation between RT-PCR and tetramer analysis, IC flow cytometry and ELISPOT assay. Using pp65-protein for T-cell activation overcomes the HLA restriction of the HLA-A2-restricted pp65-peptide stimulation, thus increasing the detection limit of antigen-specific T cells.
  
  
  CONCLUSIONS
  CMV-specific T cells have been most frequently detected using RT-PCR suggesting a higher sensitivity compared with IC-FACS, tetramer, and ELISPOT assays. This technique allows rapid and sensitive monitoring of CMV-specific immune reconstitution after HSCT, which could guide prophylactic and therapeutic strategies to avoid active CMV disease.
authors: Department of Hematology and Oncology, University Hospital of Regensburg, University of Regensburg, Regensburg, Germany; Department of Medical Laboratory Sciences, Faculty of Allied Health Sciences, The Hashemite University, Zarqa, Jordan.
authors: Ahmad
authors: A
authors: Abu-Khader
authors: Stefan
authors: S
authors: Krause
mesh_terms: D000956:Antigens, Viral / Q000276:immunology; D003587:Cytomegalovirus / Q000276:immunology*; D003586:Cytomegalovirus Infections / Q000235:genetics / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005434:Flow Cytometry / Q000379:methods; D015789:HLA-A2 Antigen / Q000235:genetics / Q000276:immunology; D006239:Haplotypes; D018380:Hematopoietic Stem Cell Transplantation; D006801:Humans; D007371:Interferon-gamma / Q000096:biosynthesis; D010446:Peptide Fragments / Q000276:immunology; D010750:Phosphoproteins / Q000737:chemistry / Q000276:immunology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D016176:T-Lymphocyte Subsets / Q000276:immunology* / Q000378:metabolism; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology / Q000378:metabolism; D014184:Transplantation, Homologous; D014763:Viral Matrix Proteins / Q000737:chemistry / Q000276:immunology
chemical_list: D000956:Antigens, Viral; D015789:HLA-A2 Antigen; D010446:Peptide Fragments; D010750:Phosphoproteins; D014763:Viral Matrix Proteins; C065418:cytomegalovirus matrix protein 65kDa; D007371:Interferon-gamma
keywords: CMV-specific T cells; allogeneic hematopoietic stem cell transplantation; monitoring; real-time polymerase chain reaction