The role of MTP in CD1d antigen presentation and NKT cell development

Microsomal triglyceride transfer protein (MTP), an ER lipid transfer protein critical for the secretion of apolipoprotein B (apoB), also regulates CD1d antigen presentation. MTP associates with CD1d in hepatocytes, and MTP silencing or gene deletion results in diminished hepatocyte CD1d presentation and protection from NKT cell-mediated hepatitis and colitis. Here we show by RT-PCR, northern blotting, and triglyceride transfer activity that MTP is present in mouse and human APCs. Inhibition of MTP in APCs, either by silencing or by chemical inhibition, results in decreased CD1d-restricted presentation of endogenous and exogenous lipids to NKT cells without affecting MHC II presentation. Furthermore, purified MTP directly transfers phospholipid to recombinant CD1d in vitro. Human mtp is transcriptionally upregulated in hematopoietic cells in response to inflammatory stimuli. In the mouse, a novel splice variant of mouse MTP ( mttp_v1 ) is found in non-apoB secreting tissues, including thymocytes and APCs, detectable by PCR and northern analysis. Mttp_v1 uses an alternate promoter allowing for low level, ubiquitous expression of MTPv1. Edman degradation of MTPv1 isolated from transferred cells revealed three unique residues. Recombinant MTP and MTPv1 were equivalent with respect to PDI association, subcellular localization, triglyceride transfer, phospholipid transfer, response to inhibitors, and ability to support apoB secretion. Both MTP and MTPv1 efficiently transferred phospholipid to CD1d in vitro. NKT cells are selected by CD1d expressed on cortical thymocytes. Using three different MTP inhibitors in fetal thymic organ culture (FTOC), we show that, without MTP function, CD1d levels on CD4+CD8+ FTOC cells were unaffected but NKT cell development was eliminated. MTP-inhibited FTOCs produced virtually no CD1d tetramer+ cells, and MTP-inhibited FTOC cells produced less IL-4 when stimulated with anti-CD3 or α-galactosylceramide pulsed APCs. FTOCs from MTP gene-deleted embryos did support NKT cell development, and these results are discussed. We conclude that MTP is an ER chaperone and lipid transfer protein for CD1d. MTP likely transfers endogenous lipids to nascent CD1d to ensure its proper folding and ability to interact with downstream lysosomal lipid transfer proteins. In the absence of MTP function, CD1d trafficks to the cell surface but is incapable of mediating NKT cell selection and activation.