Clonotype Selection and Composition of Human CD8 T Cells Specific for Persistent Herpes Viruses Varies with Differentiation but Is Stable Over Time 1 Emanuela M. Iancu,* Patricia Corthesy,* Petra Baumgaertner, † Estelle Devevre, † Verena Voelter,* Pedro Romero, † Daniel E. Speiser, † and Nathalie Rufer 2 * † Protection from reactivation of persistent herpes virus infection is mediated by Ag-specific CD8 T cell responses, which are highly regulated by still poorly understood mechanisms. In this study, we analyzed differentiation and clonotypic dynamics of EBV- and CMV-specific T cells from healthy adults. Although these T lymphocytes included all subsets, from early-differentiated (EM/CD28 pos ) to late-differentiated (EMRA/CD28 neg ) stages, they varied in the sizes/proportions of these subsets. In-depth clonal composition analyses revealed TCR repertoires, which were highly restricted for CMV- and relatively diverse for EBV-specific cells. Virtually all virus-specific clonotypes identified in the EMRA/CD28 neg subset were also found within the pool of less dif- ferentiated “memory” cells. However, striking differences in the patterns of dominance were observed among these subsets, because some clonotypes were selected with differentiation while others were not. Late-differentiated CMV-specific clonotypes were mostly characterized by TCR with lower dependency on CD8 coreceptor interaction. Yet all clonotypes displayed similar functional avidities, suggesting a compensatory role of CD8 in the clonotypes of lower TCR avidity. Importantly, clonotype selection and composition of each virus-specific subset upon differentiation was highly preserved over time, with the presence of the same dominant clonotypes at specific differentiation stages within a period of 4 years. Remarkably, clonotypic distribution was stable not only in late-differentiated but also in less-differentiated T cell subsets. Thus, T cell clonotypes segregate with differen- tiation, but the clonal composition once established is kept constant for at least several years. These findings reveal novel features of the highly sophisticated control of steady state protective T cell activity in healthy adults. The Journal of Immunology, 2009, 183: 319 –331. A s a result of antigenic challenge, naive (N) CD8 T cells undergo clonal expansion and differentiation into mem- ory and effector type lymphocytes (1) whose principal role is to eliminate virally infected and malignant cells. Whereas central-memory (CM) 3 T cells lack immediate effector function and express receptors allowing them to repeatedly circulate to lymph nodes, effector-memory (EM) and effector (EMRA) T lym- phocytes preferentially home to peripheral tissues and, upon stim- ulation, rapidly produce effector cytokines (e.g., IFN-) and cyto- toxic proteases (2, 3). Several technical improvements of T cell studies have demonstrated that primed Ag-experienced CD8 pos T lymphocytes are highly heterogeneous (4 –10), varying in terms of cell surface phenotype, function, and history of Ag encounter. Re- cently, we uncovered additional heterogeneity among EM and ef- fector subsets by studying the functional attributes of such T cells distinguished on the basis of expression of the costimulatory re- ceptors CD27 and CD28 (11, 12). There is increasing evidence that the immune response depends on the type and nature of infection (acute vs persistent), the amount of Ag present, the cycles of T cell rest and stimulation, and the site of replication of the pathogen (13–16). Although the existence of distinct subpopulations of CD8 pos T cells is now widely accepted, the lineage relationship and differentiation pathways that they follow remain a matter of controversy. EBV and CMV are common persistent viruses in the human population, infecting 90 and 60 –90% of individuals, respec- tively. In healthy individuals, there is an equilibrium maintained between viral and immune activity, which keeps the viruses in check and allows the vast majority of carriers to remain asymp- tomatic throughout their lives. However, these viruses are patho- genic in immunocompromised individuals. CMV can cause cyto- pathic damage in various organs, and EBV can induce malignancies. Previous studies have shown that following the res- olution of acute infectious mononucleosis and upon entering the chronic phase of EBV infection, CD8 pos T cells specific for EBV lytic Ags remain high in frequency (17–19) and carry an EM phe- notype with low levels of CCR7, variable expression of CD45RA (19, 20), and maintenance of the CD27 and CD28 coreceptor ex- pression (7, 21). Somewhat lower in frequency, the CD8 T cells specific for latent EBV Ags show higher CCR7 expression, lower CD45RA re-expression (19), and high CD27 and CD28 expression (19, 21). In contrast, CMV-specific T cells have a more differen- tiated phenotype with low CCR7, CD27, and CD28 expression, *Division of Experimental Oncology, Multidisciplinary Oncology Center, and † Di- vision of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, Lau- sanne Branch, Lausanne University Hospital, Lausanne, Switzerland Received for publication October 30, 2008. Accepted for publication May 1, 2009. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This study was sponsored and supported by the Swiss National Center of Compe- tence in Research Molecular Oncology, the Ludwig Institute for Cancer Research, and the Swiss National Science Foundation Grants 3100A0-105929 and 3200B0-118123. P.R. was funded in part by the FP6 European Union grant “Cancer Immunotherapy.” 2 Address correspondence and reprint requests to Dr. Nathalie Rufer, Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, Ho ˆpital Orthope ´- dique, niv 5, Aile Est, Avenue Pierre-Decker 4, CH-1011 Lausanne, Switzerland. E-mail address: Nathalie.Rufer@unil.ch 3 Abbreviations used in this paper: CM, central-memory; EM, effector-memory; EMRA, effector-memory and effector. Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 The Journal of Immunology www.jimmunol.org/cgi/doi/10.4049/jimmunol.0803647