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

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.