LETTERS CD1d is a major histocompatibility complex (MHC) class I–related molecule that functions in glycolipid antigen presentation to distinct subsets of T cells that express natural killer receptors and an invariant T-cell receptor-α chain (invariant NKT cells) 1–3 . The acquisition of glycolipid antigens by CD1d occurs, in part, in endosomes through the function of resident lipid transfer proteins, namely saposins 4–10 . Here we show that microsomal triglyceride transfer protein (MTP), a protein that resides in the endoplasmic reticulum of hepatocytes and intestinal epithelial cells (IECs) and is essential for lipidation of apolipoprotein B 11,12 , associates with CD1d in hepatocytes. Hepatocytes from animals in which Mttp (the gene encoding MTP) has been conditionally deleted, and IECs in which Mttp gene products have been silenced, are unable to activate invariant NKT cells. Conditional deletion of the Mttp gene in hepatocytes is associated with a redistribution of CD1d expression, and Mttp-deleted mice are resistant to immunopathologies associated with invariant NKT cell–mediated hepatitis and colitis. These studies indicate that the CD1d-regulating function of MTP in the endoplasmic reticulum is complementary to that of the saposins in endosomes in vivo. CD1d-restricted T cells regulate many immune disorders through the ability of CD1d to present lipid antigens to invariant NKT cells 13–15 . Because CD1d assembly occurs within the endoplasmic reticulum, in a manner similar to but distinct from that associated with other MHC class I–related molecules, it has been predicted that these in vivo functions of CD1d involve acquisition of glycolipid antigens in this cellular locale 5,16–18 . MTP is an endoplasmic reticulum–resident lipid transfer protein involved in the formation of apolipoprotein B–containing chylomicrons and very-low-density lipoproteins in hepatocytes and IECs, both of which are known to express CD1d 19–22 . Given these properties, as well as the ability of MTP to transfer glycolipids such as phosphatidylinositol 20 that are known to bind CD1d 18 , we reasoned that MTP could also be involved in CD1d function. To confirm a direct relationship between CD1d and MTP,we first assessed whether CD1d and MTP associate with each other in hepato- cytes. We immunoprecipitated protein lysates of hepatocytes from C57BL/6 (B6) mice with a monoclonal antibody to either CD1d or MHC class I (H–2D b ). We resolved the immunoprecipitates by SDS-PAGE, followed by western blotting for the presence of MTP. We detected a specific band of 97 kDa, consistent with MTP, in the CD1d, but not the MHC class I, immunoprecipitates (Fig. 1a). To further substantiate a direct biochemical relationship between CD1d and MTP, we examined CD1d expression and function in a conditional MTP-deficient mouse model. The mice, which had a ‘floxed’ Mttp gene (Mttp fl/fl ), had been intercrossed with mice expressing Cre recombinase under the control of the Mx1 promoter (Mx1Cre) 21,23 . The Mx1 promoter is induced by interferon-inducing substances, such as polyinosinic-polycytidylic acid (pIpC) 21,23,24 , such that pIpC treatment leads to deletion of Mttp (Mttp –/– ) prima- rily within the liver, spleen and intestine 24 . As previously reported 21,23 , treatment of Mttp fl/fl Mx1Cre mice with pIpC caused progressive hepatic steatosis (Supplementary Fig. 1 online), a decrease in serum triglyceride and cholesterol (Supplementary Table 1 online), and a decrease in MTP mRNA and protein (Supplementary Fig. 1) in the liver and colon. Consistent with this, the specific biochemical association observed between CD1d and MTP in hepatocytes of B6 mice was also observed in Mttp fl/fl Mx1Cre mice before, but not after, pIpC treatment and consequent deletion of Mttp (Fig. 1b). Given the biochemical association observed between CD1d and MTP (Fig. 1a,b), we surmised that the MTP deficiency induced by Mx-1Cre activity would affect CD1d expression, function or both in hepatocytes in vivo.We therefore examined the expression of CD1d in the liver of Mttp fl/fl Mx1Cre mice before and after pIpC treatment. Before pIpC treatment, CD1d was present diffusely throughout hepa- tocytes, with a membranous staining pattern, in both B6 and 1 Gastroenterology Division, Department of Medicine, Brigham and Women’s Hospital; 2 Program in Immunology; 3 Department of Pathology, Brigham and Women’s Hospital; and 4 Department of Anesthesia, Brigham and Women’s Hospital; Harvard Medical School, Boston, Massachusetts 02115, USA. 5 Gladstone Institute of Cardiovascular Disease, University of California-San Francisco, San Francisco, California 94110, USA. 6 Hematology-Oncology Division, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, USA. 7 These authors contributed equally to this work. Correspondence should be addressed to R.S.B. (rblumberg@partners.org). Published online 25 April 2004; doi:10.1038/nm1043 CD1d function is regulated by microsomal triglyceride transfer protein Suzana Brozovic 1,7 , Takashi Nagaishi 1,7 , Masaru Yoshida 1 , Stephanie Betz 2 , Azucena Salas 1 , Daohong Chen 1 , Arthur Kaser 1 , Jonathan Glickman 3 , Timothy Kuo 1 , Alicia Little 1 , Jamin Morrison 1 , Nadia Corazza 1 , Jin Yong Kim 1 , Sean P Colgan 4 , Stephen G Young 5 , Mark Exley 6 & Richard S Blumberg 1 NATURE MEDICINE VOLUME 10 | NUMBER 5 | MAY 2004 535 © 2004 Nature Publishing Group http://www.nature.com/naturemedicine