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 (< 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.
Yao
Junxia
J
Department of Internal Medicine III, University of Ulm, Ulm, Germany.
Bechter
Clemens
C
Wiesneth
Markus
M
Härter
Georg
G
Götz
Marlies
M
Germeroth
Lothar
L
Guillaume
Philippe
P
Hasan
Ferishte
F
von Harsdorf
Stephanie
S
Mertens
Thomas
T
Michel
Detlef
D
Döhner
Hartmut
H
Bunjes
Donald
D
Schmitt
Michael
M
Schmitt
Anita
A
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*
D000914:Antibodies, Viral; D000956:Antigens, Viral; D010750:Phosphoproteins; D014763:Viral Matrix Proteins; C065418:cytomegalovirus matrix protein 65kDa