Bacillus Calmette Guerin Vaccination of Human Newborns Induces a Specific, Functional CD8  T Cell Response 1 Rose Ann Murray,* Nazma Mansoor, † Ryhor Harbacheuski,* Jorge Soler,* Virginia Davids, † Andreia Soares, † Anthony Hawkridge, † Gregory D. Hussey, † Holden Maecker, ‡ Gilla Kaplan,* and Willem A. Hanekom 2† Mounting evidence points to CD8  T cells playing an important role in protective immunity against Mycobacterium tuberculosis. The only available vaccine against tuberculosis, bacillus Calmette Gue ´rin (BCG), has traditionally been viewed not to induce these cells optimally. In this study, we show that vaccination of human newborns with BCG does indeed induce a specific CD8  T cell response. These cells degranulated or secreted IFN-, but not both, when infant blood was incubated with BCG. This stimulation also resulted in proliferation and up-regulation of cytotoxic molecules. Overall, the specific CD8  T cell response was quantita- tively smaller than the BCG-induced CD4  T cell response. Incubation of whole blood with M. tuberculosis also caused CD8  T cell IFN- expression. We conclude that BCG induces a robust CD8  T cell response, which may contribute to vaccination- induced protection against tuberculosis. The Journal of Immunology, 2006, 177: 5647–5651. T he World Health Organization declared tuberculosis a global health emergency in 1993. The situation is no less urgent today: one-third of the world’s population is in- fected with Mycobacterium tuberculosis, 8.9 million developed tuberculosis disease in 2003, and 1.75 million died (1). Control of the epidemic remains difficult because tuberculosis control pro- grams often lack adequate resources, prevalent HIV infection in- creases risk of disease, and because multidrug resistance is emerging. An effective vaccine that prevents lung tuberculosis would be an attractive and sustainable long-term intervention. Mycobacterium bovis bacille Calmette Gue ´rin (BCG) 3 remains the only available vaccine. BCG offers variable, but mostly poor, protection against pulmonary tuberculosis, at all ages (2). However, the vaccine is given to newborns soon after birth because 80% protection is afforded against severe forms of childhood tuberculosis, i.e., mil- iary disease and meningitis (2). The immune mechanisms under- lying this protection are not understood. Only a handful of reports have addressed immunity induced by newborn vaccination with BCG. Among more modern studies, Marchant et al. (3) reported that the vaccine induces a Th1-like response in newborns, charac- terized by IFN- production in CD4 + T cells following incubation of PBMC with soluble purified protein derivative of M. tubercu- losis. These results were confirmed by Hussey et al. (4), who dem- onstrated lymphoproliferation and predominant IFN- production after incubation of infant PBMC with purified protein derivative or with mycobacteria. Specific cytotoxic activity was also demon- strated; although it may be presumed that CD8 + T cells were re- sponsible for this function, this was not addressed. The aim of our study was to determine whether BCG vaccina- tion of human newborns induces specific and functional CD8 + T cells. Mycobacteria do indeed induce a CD8 + T cell response in humans, as evidenced by multiple reports of immunity induced by BCG vaccination at older ages, and of immunity in persons either latently infected or diseased by M. tuberculosis (5– 8). However, there is a widely held view that BCG itself induces suboptimal CD8 + T cell responses (9, 10). The overall importance of CD8 + T cells in protective immunity against mycobacteria remains in- completely understood. Multiple reports of murine studies suggest that CD8 + T cells participate in protection (11–14); however, overlap of functions with CD4 + T cells (such as IFN- produc- tion), dependency on CD4 + T cells (15), and complex kinetics of the induced CD8 + T cell response (13, 16 –18) have made an as- sessment of the relative contribution of the latter T cell subset to protection difficult to dissect. We propose that CD8 + T cells are important in BCG-induced protection against tuberculosis of hu- mans. Our study was the first to directly address this T cell pop- ulation in the context of newborn vaccination against tuberculosis. Materials and Methods Study participants and blood collection Healthy 10-wk-old infants, routinely vaccinated with intradermal BCG (Danish strain 1331; Statens Serum Institut) at birth, were enrolled from clinics in the Cape Town region of South Africa. Infants were excluded if known to be HIV-infected or born to HIV-infected mothers. Additionally, a rapid HIV test was performed on each infant on enrollment—positive results lead to exclusion. Infants with suspected or confirmed tuberculosis, or in contact with adults with pulmonary tuberculosis, were also excluded. Blood was collected and immediately incubated in whole blood assays (see below and Ref. 19), or PBMC were isolated from the blood and cryopre- served. Protocols for this study were approved by the Institutional Review Board of the University of Medicine and Dentistry of New Jersey and the *Public Health Research Institute, Newark, NJ 07103; † South African Tuberculosis Vaccine Initiative, Institute of Infectious Diseases and Molecular Medicine, Univer- sity of Cape Town, Cape Town, South Africa; and ‡ BD Biosciences, San Jose, CA 95131 Received for publication January 13, 2006. Accepted for publication July 20, 2006. 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 W.A.H. is supported by the National Institutes of Health Grant (NIH) AI 065653, the Dana Foundation, the European and Developing Countries Trials Partnership, and the Aeras Global Tuberculosis Vaccine Foundation. G.K. is supported by NIH Grants AI 054361 and AI 22616, and by the Heiser Foundation. 2 Address correspondence and reprint requests to Dr. Willem A. Hanekom, South African Tuberculosis Vaccine Initiative, Institute of Infectious Diseases and Molec- ular Medicine, Wernher Beit S2.01, University of Cape Town Health Sciences Fac- ulty, Anzio Road, Observatory, 7925 Cape Town, South Africa. E-mail address: Willem@rmh.uct.ac.za 3 Abbreviations used in this paper: BCG, Bacille Calmette Gue ´rin; WB-ICC, whole blood intracellular cytokine assay; MOI, multiplicity of infection; SEB, staphylococ- cal enterotoxin B. The Journal of Immunology Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00