An Anti-Inflammatory Role for V14 NK T cells in Mycobacterium bovis Bacillus Calmette-Gue ´rin-Infected Mice 1 Francesco Dieli, 2 * Masaru Taniguchi, † Mitchell Kronenberg, ‡ Stephane Sidobre, ‡ Juraj Ivanyi, § Lanfranco Fattorini, ¶ Elisabetta Iona, ¶ Graziella Orefici, ¶ Giacomo De Leo,* Domenica Russo,  Nadia Caccamo,* Guido Sireci,* Caterina Di Sano,  and Alfredo Salerno*  The possible contribution of NKT cells to resistance to Mycobacterium tuberculosis infection remains unclear. In this paper we characterized the V14 NKT cell population following infection with Mycobacterium bovis bacillus Calmette-Gue ´rin (BCG). BCG infection determined an early expansion of V14 NKT cells in liver, lungs, and spleen, which peaked on day 8 and was sustained until day 30. However, an NK1.1  V14 NKT population preferentially producing IFN- predominated at an early stage (day 8), which was substituted by an NK1.1  population preferentially producing IL-4 at later stages (day 30). Despite the fact that V14 NKT cell-deficient mice eliminated BCG as did control mice, they had significantly higher numbers of granulomas in liver and lungs. Additionally, while control mice developed organized small granulomas, those in V14 NKT-deficient mice had signs of caseation, large cellular infiltrates, and some multinucleated macrophages, suggesting that V14 NKT cells may actually work as anti-inflammatory cells by limiting excessive lymphocyte influx and tissue pathology. In agreement, we found an increased spon- taneous production and mRNA expression of TNF- in liver and lungs of V14 NKT-deficient mice, whose neutralization in vivo by anti-TNF- mAbs consistently reduced the number of granulomas in liver and lungs. Together, our results support a regulatory role for V14 NKT cells in the course of BCG infection through their ability to limit the extent of inflammatory response and point to an important role for this cell subset as a regulator of the balance between protective responses and immunopathology. The Journal of Immunology, 2003, 171: 1961–1968. P rotective immunity against mycobacterial infections is me- diated by interactions between specific T cells and acti- vated macrophage effector cells (1). These populations in- teract with each other by means of a complex network of chemokines, cytokines, and their receptors. The exact role and relative importance of each of these mediators, however, are not completely understood and are even somewhat controversial, al- though IFN- is thought to play a crucial role (2– 4). Mycobacterium bovis bacillus Calmette-Gue ´rin (BCG), 3 widely used as a human vaccine against tuberculosis, gives a poor or ambiguous protection to infection by Mycobacterium tuberculosis. The murine  and  T cell responses induced by BCG have been well defined (1, 5), but there is less knowledge about the response of smaller T cell subsets. NK T (NKT) cells are a heterogeneous population of T lym- phocytes that express the NK1.1 molecule and comprise at least four different categories of T cells (6, 7). Within NKT cells, the most abundant population expresses an invariant TCR -chain en- coded by the V14 and J281 gene segments and is usually double (i.e., CD4 and CD8) negative, although V14 NKT cells residing in the liver coexpress CD4 (8). Stimulation of V14 NKT cells by anti-CD3 mAbs or the synthetic ligand -galactosylceramide (- GalCer) in the context of CD1d promptly induces both IFN- and IL-4 production (9 –11). V14 NKT cells in the liver of BCG-infected mice rapidly pro- duce IFN- (12) and play an important role in granuloma forma- tion in response to phosphatidylinositol mannosides (PIMs) depro- teinized cell wall from M. tuberculosis (13). However, this does not seem to involve direct recognition of PIMs by the V14 TCR (14). Furthermore, studies in CD1d- and J281-deficient mice, which selectively lack V14 NKT cells, failed to show any role for protection against M. tuberculosis or BCG (15–19), although a recent study has reported that specific activation of NKT cells by -GalCer protects mice from tuberculosis (20). In view of the lack of knowledge about the significance of the several possible actions of V14 NKT cells, we examined their functions during BCG infection using V14 NKT-deficient mice. Materials and Methods Mice BALB/c and C57BL/6 mice were purchased from OLAC through Nossan (Correzzana, Italy). Generation of J281-deficient mice has been previ- ously described (21). Mice that lack the J281 gene segment are devoid of V14 NKT cells, but the other lymphoid cell lineages are intact (21). Populations that are more heterogeneous at the genetic level were estab- lished by backcrossing heterozygotes to C57BL/6 or BALB/c mice for more than five generations. The resultant heterozygous mice were mated to *Department of Biopathology, University of Palermo, Palermo, Italy; † Division of Molecular Immunology, Center for Biomedical Science, Chiba University School of Medicine, Chiba, Japan; ‡ La Jolla Institute for Allergy and Immunology, San Diego, CA 92121; § King’s College London at Guy’s Dental and Medical School, London, United Kingdom; ¶ Laboratory of Bacteriology and Clinical Mycology, National In- stitute of Health, Rome, Italy; and  Institute of Advanced Diagnostic Methodologies, National Research Council, Palermo, Italy Received for publication January 6, 2003. Accepted for publication June 5, 2003. 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 work was supported by grants from the Italian National Research Council (to F.D.), the Ministry for Education, University and Research (MIUR 60%, to A.S. and F.D.), the European Commission (Fifth Framework Program, Contract QLK2-1999- 00367), and National Institutes of Health Grant RO1KA52511 (to M.K.). 2 Address correspondence and reprint requests to Dr. Francesco Dieli, Dipartimento di Biopatologia, Universita ` di Palermo, Corso Tukory 211, I-90134 Palermo, Italy. E-mail address: dieli@unipa.it 3 Abbreviations used in this paper: BCG, Mycobacterium bovis bacillus Calmette- Gue ´rin; -GalCer, -galactosylceramide; PIM, phosphatidylinositol mannoside. The Journal of Immunology Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00