CHARACTERIZATION OF CMVpp65-SPECIFIC CD8+ T LYMPHOCYTES USING MHC TETRAMERS IN KIDNEY TRANSPLANT PATIENTS AND HEALTHY PARTICIPANTS¹

Engstrand, Mats²; Tournay, Claire⁶; Peyrat, Marie A.⁷; Eriksson, Britt-Marie³; Wadström, Jonas⁴; Wirgart, Benita Zweygberg⁵; Romagné, Francois⁶; Bonneville, Marc⁷; Tötterman, Thomas H.^{2 8}; Korsgren, Olle²

Author Information

Divisions of Clinical Immunology & Transfusion Medicine, Infectious Diseases, and Transplantation Surgery, University Hospital, S 751 85, Uppsala, Sweden; Department of Clinical Microbiology, Karolinska Hopital, S 171 76, Stockholm, Sweden; Immunotech, Immunology Research Group, BP 177, 13276, Marseilles, France; INSERM U463, Institut de Biologie, 44035, Nantes, France

² Current affiliation: Division of Clinical Immunology & Transfusion Medicine, University Hospital, S 751 85, Uppsala, Sweden.

³ Current affiliation: Divisions of Infectious Diseases, University Hospital, S 751 85, Uppsala, Sweden.

⁴ Current affiliation: Division of Transplantation Surgery, University Hospital, S 751 85, Uppsala, Sweden.

⁵ Current affiliation: Department of Clinical Microbiology, Karolinska Hospital, S 171 76, Stockholm, Sweden.

⁶ Current affiliation: Immunotech, Immunology Research Group, 130 Avenue de Lattre de Tassigny, BP 177, 13276, Marseilles, France.

⁷ Current affiliation: INSERM U463, Institut de Biologie, 9 Quai Moncousu, 44035, Nantes, France.

Received 1 October 1999.

Accepted 21 March 2000.

⁸ Address correspondence to: Thomas Tötterman, MD. Professor, Clinical Immunology Section, Rudbeck Laboratory, Uppsala University, S-751 85 Uppsala Sweden. E-mail:[email protected].

¹ This study was supported by grants from the Swedish Medical Research Council (06P-11813 and 16X-12219).

Transplantation69(11):p 2243-2250, June 15, 2000.

Abstract

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.