Although several proteins involved in the uptake, exchange, or transmembrane transport of lipids have been clearly involved in various aspects of CD1-mediated antigen presentation (3
), the recently reported role of MTP remains somewhat elusive. The ER location of MTP, its coprecipitation with CD1d, and its well-established function in loading apoB with lipids have suggested that MTP might be a chaperone assisting the folding of nascent CD1d proteins through lipid loading. Indeed, chaperone-assisted folding and peptide loading are essential for MHC molecules, and it is logical to postulate that similar mechanisms might be associated with CD1 biosynthesis. However, some of the reported functional consequences of the genetic ablation or chemical inhibition of MTP were not fully consistent with this proposed function. For example, the decrease in CD1d surface expression was often modest and could not alone explain the profound defects in lipid antigen presentation seen upon genetic ablation of mttp
). Likewise, chemical inhibition of MTP in two different human cell lines led to a defect in NKT cell stimulation without notable decrease in surface CD1d levels (13
). In addition, when localization of CD1d was studied by confocal microscopy of liver sections, a majority of CD1d molecules were outside the ER, whether or not MTP was inactivated (14
). Finally, although purified MTP could transfer phosphatidylethanolamine onto plastic wells coated with CD1d, the lipid transfer assay was indirect, and the stoichiometry of CD1d loading was not determined (13
). Whether, like the well-studied saposins (3
), MTP can efficiently transfer lipids onto CD1d remains unclear.
We present direct cell biological and functional evidence suggesting an alternative explanation for the CD1d-mediated lipid presentation defects of MTP-deficient cells. Metabolic pulse-chase radiolabeling experiments established that the rates of CD1d biosynthesis and transit through ER and Golgi were unaltered in the absence of MTP. In addition, using a panel of well-characterized NKT hybridomas reactive to endogenous lipids, we found that, in the absence of MTP, CD1d-expressing cells stimulated the hybridomas responding to endogenous lipids loaded in the secretory compartment, whereas, in sharp contrast, they failed to stimulate those recognizing lysosomal antigens such as iGb3. Consistent with these in vitro findings, NKT cell development and stimulation by thymocytes were partially impaired in vivo in chimeric mice carrying a deletion of the first exon of MTP in their cortical thymocytes.
These surprising findings led us to consider the lysosomal stage of CD1d trafficking as a potential target of MTP. Previous studies have shown that, at steady state, most of the CD1d molecules normally reside in the lysosomal compartment, from which they undergo prolonged recycling back and forth to the plasma membrane. This movement is governed by a tyrosine motif in the intracytoplasmic tail of CD1d that binds AP-2 and AP-3 (19
). CD1d tail-truncated mutants fail to present many lipids, including the Vα14 NKT ligand iGb3, or the di- or triglycosylated derivatives of αGalCer that require processing by lysosomal glycosidases before recognition by Vα14 NKT cells. Thus, the functional defects associated with the tail truncation of CD1d resemble those associated with MTP deficiency.
The intracellular distribution patterns, however, are opposite. Whereas tail-truncated CD1d exhibits a higher level of surface expression and decreased intracellular accumulation, the intracellular/surface ratio is increased in MTP-deficient cells. Importantly, the intracellular site of accumulation is the late endosome/lysosome rather than the ER. Thus, the conserved internalization rate and markedly decreased recycling identify a block in the exit pathway that normally allows CD1d molecules to return to the plasma membrane after they have loaded lipid antigens in the lysosome. This anomaly provides a potential explanation for all the cell biological and functional changes of CD1d trafficking and antigen presentation observed in MTP-deficient cells or animals. Because little is known about the molecular mechanisms controlling recycling in normal cells, there is at present no clear suggestion as to the precise molecular target of MTP. Reports that MHC class II presentation is conserved in MTP-deficient cells and our own findings that lysosomal degradation of HEL and HRP proteins are unaltered, that PBS10, a fluorescently labeled αGC, traffics normally to late endosome and lysosome, and that granule exocytosis is conserved in the absence of MTP suggest that the lysosomal defects are relatively selective. However, the size of the Lysotracker+ compartment appeared to be significantly increased, and a lysosomal retention of BODIPY-LacCer, which normally reaches the Golgi after endocytosis to the late endosome, could be detected as well. Thus, changes in the lipid composition of some membranes or lipid storage may alter the dynamics of the CD1d recycling pathway. Alternatively, MTP deficiency may impact another unidentified protein involved in recycling. Notably, recent experiments investigating CD1d-mediated lipid presentation defects associated with herpes simplex virus-1 infection have uncovered a very similar dysregulation of CD1d recycling from lysosome to plasma membrane (22
), revealing the importance of this previously overlooked stage of CD1d trafficking and suggesting its relevance in host defense.
Our results do not rule out the possibility that, in the absence of MTP, aberrant lipid loading in the ER might prevent subsequent exchange with other lipid ligands in the lysosome and somehow alter CD1d recycling. This hypothesis, however, remains to be tested. Furthermore, although we found that 3/3 non-Vα14 autoreactive hybridomas (previously shown to respond to endogenous ligand loaded independently of lysosomal trafficking of CD1d) recognized CD1d in the absence of MTP, the response of another non-Vα14 hybridoma, 14S6, was reported to be altered (13
), suggesting that, in addition to the major lysosomal defects, lipid presentation in the secretory pathway might be partially impaired as well.
In conclusion, MTP deficiency induces severe defects of lipid antigen presentation by CD1d. Despite the ER location of MTP, convergent cell biological and functional experiments indicate that a major impact on CD1d-mediated antigen presentation is in the altered recycling of CD1d from the lysosome to the plasma membrane. This surprising finding reveals a previously ignored, long-range effect of MTP in an important, yet poorly understood, intracellular trafficking process.