Distinct Differences in the Expansion and Phenotype of TB10.4 Specific CD8 and CD4 T Cells after Infection with Mycobacterium tuberculosis Truc Thi Kim Thanh Hoang 1 , Anneline Nansen 2 , Sugata Roy 1 , Rolf Billeskov 1 , Claus Aagaard 1 , Tara Elvang 1 , Jes Dietrich 1 *, Peter Andersen 1 * 1 Department of Infectious Disease Immunology, Statens Serum Institut, Copenhagen, Denmark, 2 Department of Immunopharmacology, Novo Nordisk, Ma ˚løv, Denmark Abstract Background: Recently we and others have identified CD8 and CD4 T cell epitopes within the highly expressed M. tuberculosis protein TB10.4. This has enabled, for the first time, a comparative study of the dynamics and function of CD4 and CD8 T cells specific for epitopes within the same protein in various stages of TB infection. Methods and Findings: We focused on T cells directed to two epitopes in TB10.4; the MHC class I restricted epitope TB10.4 3–11 (CD8/10.4 T cells) and the MHC class II restricted epitope TB10.4 74–88 (CD4/10.4 T cells). CD4/10.4 and CD8/10.4 T cells displayed marked differences in terms of expansion and contraction in a mouse TB model. CD4/10.4 T cells dominated in the early phase of infection whereas CD8/10.4 T cells were expanded after week 16 and reached 5–8 fold higher numbers in the late phase of infection. In the early phase of infection both CD4/10.4 and CD8/10.4 T cells were characterized by 20–25% polyfunctional cells (IL-2 + , IFN-c + , TNF-a + ), but whereas the majority of CD4/10.4 T cells were maintained as polyfunctional T cells throughout infection, CD8/10.4 T cells differentiated almost exclusively into effector cells (IFN-c + , TNF-a + ). Both CD4/ 10.4 and CD8/10.4 T cells exhibited cytotoxicity in vivo in the early phase of infection, but whereas CD4/10.4 cell mediated cytotoxicity waned during the infection, CD8/10.4 T cells exhibited increasing cytotoxic potential throughout the infection. Conclusions/Significance: Our results show that CD4 and CD8 T cells directed to epitopes in the same antigen differ both in their kinetics and functional characteristics throughout an infection with M. tuberculosis. In addition, the observed strong expansion of CD8 T cells in the late stages of infection could have implications for the development of post exposure vaccines against latent TB. Citation: Hoang TTKT, Nansen A, Roy S, Billeskov R, Aagaard C, et al. (2009) Distinct Differences in the Expansion and Phenotype of TB10.4 Specific CD8 and CD4 T Cells after Infection with Mycobacterium tuberculosis. PLoS ONE 4(6): e5928. doi:10.1371/journal.pone.0005928 Editor: Elizabeth Didier, Tulane National Primate Research Center, United States of America Received February 20, 2009; Accepted April 27, 2009; Published June 16, 2009 Copyright: ß 2009 Hoang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported in part by the Bill and Melinda Gates foundation, and Danish Agency for Science Technology and Innovation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: JDI@ssi.dk (JD); PA@ssi.dk (PA) Introduction Mycobacterium tuberculosis (M.tb) continues to be a major threat to global health and immense efforts are currently invested in an attempt to understand the immune mechanisms involved in controlling this chronic disease. CD4 T cells are induced early during the acute phase whereas CD8 T cells have been reported to expand in the later stages of the infection [1,2]. However, the study of the dynamic development of antigen specific T cells at the clonal level has been complicated by the fact that CD8 T cell epitopes have only recently been identified within M.tb antigens. Therefore, examining the involvement of CD8 T cells in the defense against an infection with M.tb has in large part been based on the study of bulk CD8 T cells as in the study performed by Lazarevic et al. [1]. Recent studies have identified new CD8 T cell epitopes within M.tb derived proteins, such as CFP-10 and TB10.4 [3–8]. TB10.4 is a low molecular mass protein that belongs to the ESAT-6 family and was found to be highly immunogenic and to confer protection against an aerosol challenge with M.tb when administered to mice in a subunit vaccine composed of either TB10.4 alone or TB10.4 fused to another immune dominant M.tb protein, Ag85B [9,10]. Several T cell epitopes have been identified within TB10.4, namely the MHC class I restricted epitopes; the H2-K b TB10.4 3–11 , H2-K d TB10.4 20–28 and the MHC class II restricted epitope H2-K d TB10.4 74–88 [3,4,11,12]. With the recently emerging novel information on CD4 and CD8 T cells within the TB10.4 antigen it has now become possible to study the dynamics of the emergence, expansion and contraction of CD4 and CD8 T cell populations directed to epitopes derived from the same M.tb protein. This represents a unique opportunity to study the dynamic development of these subsets without the interference imposed by a temporal shift in the expression of different M.tb proteins during the course of a TB infection. In the present study we therefore focused on T cells directed against the two identified epitopes in TB10.4; the MHC- class I restricted H2-K b TB10.4 3–11 CD8 epitope [3] and the MHC class II restricted H2-K d TB10.4 74–88 CD4 epitope [11]. As these epitopes are restricted to different haplotypes we used the PLoS ONE | www.plosone.org 1 June 2009 | Volume 4 | Issue 6 | e5928