It is now established that CD1 molecules present lipid antigens to T cells, although it is not clear how the exchange of lipids between membrane compartments and the CD1 binding groove is assisted. We report that mice deficient in prosaposin, the precursor to a family of endosomal lipid transfer proteins (LTP), exhibit specific defects in CD1d-mediated antigen presentation and lack Vα14 NKT cells. In vitro, saposins extracted monomeric lipids from membranes and from CD1, thereby promoting the loading as well as the editing of lipids on CD1. Transient complexes between CD1, lipid, and LTP suggested a "tug-of-war" model in which lipid exchange between CD1 and LTP is on the basis of their respective affinities for lipids. LTPs constitute a previously unknown link between lipid metabolism and immunity and are likely to exert a profound influence on the repertoire of self, tumor, and microbial lipid antigens.CD1 molecules have evolved a unique hydrophobic binding groove that binds lipid antigens in both the secretory and endosomal compartments for presentation to T lymphocytes (1). In mice, the main population of CD1-restricted T cells, called Vα14 NKT cells, express a † These authors contributed equally to this work. To whom correspondence should be addressed. abendela@bsd.uchicago.edu (A.B.); lteyton@scripps.edu (L.T.). * These authors contributed equally to this work. CIHR Author Manuscript CIHR Author Manuscript CIHR Author Manuscriptsemi-invariant Vα14-Jα18/Vβ8 T cell receptor (TCR). These cells exhibit reactivity against CD1d in combination with endogenous ligands (2) that can be mimicked by α-galactosylceramide (αGC) (3). This population is conserved among mammalian species and regulates immune responses (4, 5). Like human CD1b-restricted T cells specific for mycobacterial glycolipids (6), Vα14 NKT cells are dependent on endosomal trafficking of CD1d for natural antigen recognition (7-10). Other endogenous or exogenous antigens do not require endosomal trafficking, however, suggesting that loading may be achieved in distinct cellular compartments depending on the nature of the antigen (9,11,12). CD1 endosomal trafficking is tightly controlled by cytoplasmic tail-encoded tyrosine-containing motifs binding adaptor protein 2 and 3 (AP-2 and AP-3) complexes, as well as by association with the invariant chain (Ii) or Ii/major histocompatibility (MHC) class II complexes (13-16).Because lipids are integral membrane components that might require lipid transfer proteins (LTP) (17) for extraction, we investigated whether various families of LTP might assist antigen presentation. We focused on the Vα14 NKT cell endosomal-dependent system to test the possible involvement of LTPs localized in the endocytic pathway, such as saposins (18) (Fig. 1D). These findings demonstrate a selective defect in the development of Vα14 NKT cells in the absence of prosaposin.Many NKT cell hybridomas can be activated in vitro to release interleukin-2 (IL-2) by coculture with fresh CD1d-expressing cells, such as thymocytes, which are...