title: Multimer staining of cytomegalovirus phosphoprotein 65-specific T cells for diagnosis and therapeutic purposes: a comparative study.
abstract: BACKGROUND
  Cytomegalovirus (CMV) disease represents a serious complication after allogeneic peripheral blood stem cell (PBSC) transplantation. If possible, stem cell donors for transplantation are selected on the basis of their CMV serostatus. However, the cytomegalovirus-specific immune status can be further characterized by measuring CMV phosphoprotein 65-specific CD8(+) T cell frequencies using tetramers, pentamers, and streptamers. We therefore investigated the specificity and sensitivity of all 3 methods and compared the results to patient serostatus.
  
  
  METHODS
  Twenty-three samples from CMV-seropositive healthy volunteers and 15 samples from CMV-seropositive patients before and after allogeneic PBSC transplantation were stained with tetramers, pentamers, or streptamers and analyzed by flow cytometry.
  
  
  RESULTS
  Similar frequencies of CD8(+) and multimer(+) T cells could be measured by all 3 multimer technologies. The lowest background signals (< or =0.02%) were obtained using tetramer technology. Frequencies of 0.19%-2.48% of CMV phosphoprotein 65 495-503-specific CD8(+) T cells were detected in healthy volunteers. Antigen-specific T cells were detected in only 11 (48%) of 23 seropositive healthy volunteers. CMV antigenemia before day 100 after allogeneic PBSC transplantation occurred in 2 of 3 patients without any specific T cells.
  
  
  CONCLUSIONS
  These findings demonstrate the power of multimer staining and a certain limitation of serologic testing to define appropriate donors for transplantation. Therefore, whenever possible, CMV-seropositive donors of transplants to seropositive recipients should be screened for their CD8(+) T cell frequency. All 3 multimer technologies can be used, yielding similar results. The streptamer technology additionally offers the advantage of selecting CMV phosphoprotein 65-specific CD8(+) T cells at the good manufacturing practice level for adoptive T cell transfer.
authors.affiliation: Department of Internal Medicine III, University of Ulm, Ulm, Germany.
authors.forename: Junxia
authors.initials: J
authors.lastname: Yao
authors.forename: Clemens
authors.initials: C
authors.lastname: Bechter
authors.forename: Markus
authors.initials: M
authors.lastname: Wiesneth
authors.forename: Georg
authors.initials: G
authors.lastname: Härter
authors.forename: Marlies
authors.initials: M
authors.lastname: Götz
authors.forename: Lothar
authors.initials: L
authors.lastname: Germeroth
authors.forename: Philippe
authors.initials: P
authors.lastname: Guillaume
authors.forename: Ferishte
authors.initials: F
authors.lastname: Hasan
authors.forename: Stephanie
authors.initials: S
authors.lastname: von Harsdorf
authors.forename: Thomas
authors.initials: T
authors.lastname: Mertens
authors.forename: Detlef
authors.initials: D
authors.lastname: Michel
authors.forename: Hartmut
authors.initials: H
authors.lastname: Döhner
authors.forename: Donald
authors.initials: D
authors.lastname: Bunjes
authors.forename: Michael
authors.initials: M
authors.lastname: Schmitt
authors.forename: Anita
authors.initials: A
authors.lastname: Schmitt
mesh_terms: D000328:Adult; D000914:Antibodies, Viral / Q000097:blood; D000956:Antigens, Viral / Q000097:blood; D018414:CD8-Positive T-Lymphocytes / Q000737:chemistry* / Q000276:immunology*; D003586:Cytomegalovirus Infections / Q000175:diagnosis* / Q000276:immunology* / Q000628:therapy; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D016219:Immunotherapy, Adoptive; D016131:Lymphocyte Subsets / Q000737:chemistry / Q000276:immunology; D008297:Male; D008875:Middle Aged; D010750:Phosphoproteins / Q000032:analysis*; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods*; D014763:Viral Matrix Proteins / Q000032:analysis*
chemical_list: D000914:Antibodies, Viral; D000956:Antigens, Viral; D010750:Phosphoproteins; D014763:Viral Matrix Proteins; C065418:cytomegalovirus matrix protein 65kDa