Multimer Staining of Cytomegalovirus Phosphoprotein 65–Specific T Cells for Diagnosis and Therapeutic Purposes: A Comparative Study

• Published in: Clinical infectious diseases Vol. 46; no. 10; pp. e96 - e105
• Main Authors: Yao, Junxia, Bechter, Clemens, Wiesneth, Markus, Härter, Georg, Götz, Marlies, Germeroth, Lothar, Guillaume, Philippe, Hasan, Ferishte, von Harsdorf, Stephanie, Mertens, Thomas, Michel, Detlef, Döhner, Hartmut, Bunjes, Donald, Schmitt, Michael, Schmitt, Anita
• Format: Journal Article
• Language: English
• Published: United States The University of Chicago Press 15.05.2008
• Subjects: Adult; Antibodies, Viral - blood; Antigens, Viral - blood; CD8-Positive T-Lymphocytes - chemistry; CD8-Positive T-Lymphocytes - immunology; Cytomegalovirus; Cytomegalovirus Infections - diagnosis; Cytomegalovirus Infections - immunology; Cytomegalovirus Infections - therapy; Female; Flow Cytometry; Humans; Immunotherapy, Adoptive; Lymphocyte Subsets - chemistry; Lymphocyte Subsets - immunology; Male; Middle Aged; Phosphoproteins - analysis; Sensitivity and Specificity; Staining and Labeling - methods; Viral Matrix Proteins - analysis
• ISSN: 1058-4838; 1537-6591
• DOI: 10.1086/587749

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 (⩽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. Conclusion. 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.