Characterization of CMVpp65-specific CD8+T lymphocytes using MHC tetramersin kidney transplant patients and healthy participants

Background Cytomegalovirus (CMV) is a ubiquitous herpesvirus that infects 5 0-90% of individuals in different populations, After primary infection, the virus persists latently in myeloid cells under the control of specific T-c ells. 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 dominat ing 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 di rect visualization of antigen-specific receptor-carrying T-cells using flow cytometry. We constructed a novel MHC tetramer for identification of CMVpp 65-specific CD8+ T-cells using HLA-A2 molecules folded with the immunodomin ant NLVPMVATV peptide. Results. The A2/pp65 tetramer specifically stained CMV-directed T-cell line s, 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 carr iers and in immunosuppressed kidney transplant patients with latent infecti on, Patients with reactivated CMV infection exhibited up to 15% A2/pp65 tet ramer+ cells, which seemed to correlate with CMV load over time. A2/pp65 te tramer+ cells expressed T-cell activation markers, Conclusions, The construction of a novel A2/pp65 MHC tetramer enabled the d esign of a rapid and precise flow cytometric method allowing quantitative a nd qualitative analysis of CMV-specific T-cells. The number of A2/pp65 tetr amer binding CTLs in blood may prove to be clinically relevant in assessing the immune response to CMV.