Characterization of CMVpp65-specific CD8+ T lymphocytes using MHC tetramers in kidney transplant patients and healthy participants.
BACKGROUND
Cytomegalovirus (CMV) is a ubiquitous herpesvirus that infects 50-90% of individuals in different populations. After primary infection, the virus persists latently in myeloid cells under the control of specific T-cells. Reactivation of CMV infection may cause lethal organ dysfunction and is frequently seen in immunosuppressed individuals. CD8+ cytotoxic T-cells (CTL) have a primary role in suppressing CMV reactivation, and the dominating CTL response is directed against pp65.


METHODS
MHC tetramers, that is, complexes between HLA class I (or class II) molecules and antigenic peptides conjugated to fluorochromes allow the direct visualization of antigen-specific receptor-carrying T-cells using flow cytometry. We constructed a novel MHC tetramer for identification of CMVpp65-specific CD8+ T-cells using HLA-A2 molecules folded with the immunodominant NLVPMVATV peptide.


RESULTS
The A2/pp65 tetramer specifically stained CMV-directed T-cell lines, and sorted cells showed CMV-specific cytotoxicity. High proportions (0.1-9%) of the CD8+ T-cells were A2/pp65 tetramer+ in healthy HLA-A2+ CMV carriers and in immunosuppressed kidney transplant patients with latent infection. Patients with reactivated CMV infection exhibited up to 15% A2/pp65 tetramer+ cells, which seemed to correlate with CMV load over time. A2/pp65 tetramer+ cells expressed T-cell activation markers.


CONCLUSIONS
The construction of a novel A2/pp65 MHC tetramer enabled the design of a rapid and precise flow cytometric method allowing quantitative and qualitative analysis of CMV-specific T-cells. The number of A2/pp65 tetramer binding CTLs in blood may prove to be clinically relevant in assessing the immune response to CMV.
Engstrand
M
M
Division of Clinical Immunology & Transfusion Medicine, University Hospital, Uppsala, Sweden.
Tournay
C
C
Peyrat
M A
MA
Eriksson
B M
BM
Wadström
J
J
Wirgart
B Z
BZ
Romagné
F
F
Bonneville
M
M
Tötterman
T H
TH
Korsgren
O
O
D000328:Adult; D015415:Biomarkers / Q000032:analysis; D001773:Blood Cells / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D002460:Cell Line; D005260:Female; D015789:HLA-A2 Antigen / Q000276:immunology; D006801:Humans; D016030:Kidney Transplantation / Q000276:immunology*; D008285:Major Histocompatibility Complex / Q000235:genetics* / Q000276:immunology*; D010750:Phosphoproteins / Q000737:chemistry / Q000276:immunology*; D012016:Reference Values; D013194:Staining and Labeling; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology; D014763:Viral Matrix Proteins / Q000737:chemistry / Q000276:immunology*
D015415:Biomarkers; D015789:HLA-A2 Antigen; D010750:Phosphoproteins; D014763:Viral Matrix Proteins; C065418:cytomegalovirus matrix protein 65kDa