Journal
TRANSPLANTATION
Volume 69, Issue 11, Pages 2243-2250Publisher
LIPPINCOTT WILLIAMS & WILKINS
DOI: 10.1097/00007890-200006150-00005
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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 immunosupppressed 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.
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