It has been recognized that metabolism and immunity are highly integrated processes that regulate each other (31
). Innate and adaptive immune cells can modulate metabolic pathways, and, conversely, lipid mediators such as prostaglandins, lipoxins, and sphingolipid- and phosphatidylinositol-derived second messengers are crucial regulators of immune function (31
). In addition, eukaryotic and prokaryotic lipids can serve as antigens for CD1-restricted lipid antigen presentation, which can be modulated through cellular lipid metabolism (5
). It has recently been demonstrated that MTP, a protein critically involved in lipoprotein metabolism due to its role in the transfer of lipids onto APOB, is also a central regulator of CD1 function and can directly transfer lipids onto CD1 molecules based upon studies in model systems (6
). Here, we show that MTP deficiency in the rare human disease ABL is characterized by previously unrecognized immune defects, specifically by a loss of lipid antigen presentation by all group 1 and 2 CD1 molecules. Our studies therefore highlight MTP as a unique regulator of both metabolic and immune functions in humans and suggest that ABL is not only a primary disorder of lipid metabolism but also an immune disease involving CD1.
Importantly, CD1 dysfunction in ABL is caused specifically by deficiency in MTP and not by its downstream effects on the metabolism of APOB-containing lipoprotein particles, as demonstrated by unimpaired CD1 function in patients with FHBL caused by heterozygous and compound heterozygous mutations in APOB. In addition, it is noteworthy that expression of apolipoprotein E, an apolipoprotein critical for exogenous CD1-restricted antigen presentation, is unaltered in ABL (33
). Furthermore, MTP deficiency in ABL specifically affects CD1 lipid antigen presentation and does not lead to alterations in MHC class I– and class II–restricted peptide antigen presentation and the activation and phenotype of conventional T cells.
Mechanistically, the lack of functional MTP in ABL is associated with decreased expression of group 1 CD1, likely owing to proteasomal degradation, and leads to a pronounced defect in lipid antigen presentation. These results bear resemblance to classical MHC class I molecules, where interference with loading of self-derived peptides during biosynthesis in the ER causes proteasomal degradation (35
). Similarly, loss of MTP function also leads to increased proteasomal degradation of APOB (24
). Lack of ER retention of group 1 CD1 molecules in the absence of MTP is likely due to rapid proteasomal degradation preventing intracellular accumulation, similar to prior observations for APOB (36
). Our studies therefore demonstrate for the first time to our knowledge that MTP-mediated lipid transfer in the ER is required for unimpaired maturation and survival of group 1 CD1 molecules, which otherwise undergo proteasomal degradation. This is in accordance with the notion that endogenous lipid antigens might stabilize nascent CD1 molecules during biosynthesis (5
Interestingly, while group 1 CD1 expression is considerably reduced in ABL as shown here, the CD1 molecules that presumably escape proteasomal degradation exhibit a normal intracellular distribution of expression. It is therefore possible that these CD1 molecules are stabilized by endogenous ligands that are loaded in an MTP-independent manner, either spontaneously or facilitated by other, yet-to-be-characterized lipid transfer molecules. This might also explain why some CD1 family members, such as CD1b, appear to be less affected by MTP deficiency than others (Supplemental Figure 1).
CD1b maintains its ability to present at least some antigens that are acquired in late endosomal compartments such as GMM C80. These results suggest that the acidic pH and/or the presence of late endosomal and lysosomal lipid editing molecules might provide a mechanism for survival of a fraction of CD1b molecules. Thus, the defect in group 1 CD1 function appears to mainly affect antigens acquired in the secretory and more superficial compartments within the endolysosomal system.
Intriguingly, CD1d shows unimpaired cell surface expression despite a pronounced functional defect and inability to load exogenous antigens. MHC class II–restricted peptide processing is unaltered in ABL, and the CD1d defect is independent of endolysosomal trafficking. It is therefore unlikely that impaired loading of CD1d is simply the consequence of alterations in lysosomal processing. Alternatively, MTP deficiency during CD1d biosynthesis might lead to structural CD1d changes that prevent subsequent antigen loading. While lack of proteasomal degradation and unaltered CD1d expression seem to argue against this possibility, staining of murine MTP-deficient APCs with a panel of more than 10 antibodies indeed revealed lack of CD1d recognition by a selected subset of antibodies, consistent with structural CD1d changes (R.S. Blumberg, unpublished observations). Interestingly, these changes were also not associated with CD1d degradation. In addition, investigations of several lysosomal processing disorders including deficiencies in saposin, cathepsin, and glucocerebrosidase have demonstrated similar results, with unimpaired CD1d expression despite severe functional defects (37
). Finally, it was recently shown that only a minor subset of endogenous human CD1d-binding ligands are true antigens that lead to iNKT cell activation (40
). It is therefore possible that CD1d ligands and antigens differ in their requirement for MTP for loading onto CD1d. This could explain unaltered CD1d expression and subcellular distribution despite severe defects in CD1d-restricted antigen presentation, since MTP-independent ligands might stabilize CD1d during biosynthesis, assuring normal maturation and trafficking, but might lack antigenicity and thus the potential to activate NKT cells. In conclusion, the examination of CD1 function in ABL has revealed new insights into the manner in which MTP deficiency affects the expression and function of the various CD1 isoforms.
CD1-restricted lipid antigen presentation is involved in host antimicrobial defense, anticancer immunity, and the regulation of many autoinflammatory diseases (10
). Owing to the lack of known Mendelian disorders of CD1 and NKT deficiency in humans, most of this evidence is derived from the analysis of mouse models and has not been confirmed in humans. In addition, all in vivo studies have been limited to the analysis of CD1d, since mice lack expression of group 1 CD1 molecules. As a consequence, there is little information available on the biological significance of CD1-restricted lipid antigen presentation in humans (10
) Here, we describe the first human disease to our knowledge that is characterized by pronounced defects in all CD1 family members. As such, ABL provides unique insight into the biological role of human CD1. Interestingly, while only a minority of the 100 known ABL cases has been characterized in detail, several potentially immune-related clinical disorders have been described, including cardiac insufficiency caused by lesions resembling chronic interstitial myocarditis (1
), fatal recurrent pulmonary infections (44
), and malignancies that are otherwise rarely observed (45
). Although a causal relationship between these defects in ABL and impaired CD1 function is speculative, these phenomena are consistent with a CD1-associated defect, given the observed role of NKT cells in the defense against several cardiotropic (e.g., Coxsackie viruses) and pulmonary pathogens (e.g., Streptococcus pneumoniae
, Pseudomonas aeruginosa
, respiratory syncytial virus), as well as in anticancer immunity in mouse models (18
). Our studies therefore raise the possibility that the clinical observations described above in ABL are related to CD1-associated immune abnormalities, which warrants further investigation.
The selective nature of potentially immune-related defects reported in ABL is not surprising. First, it is well known that patients deficient in NK cells, TLRs, and TLR adaptors exhibit susceptibility to selected microorganisms with otherwise unaltered immune function (48
). This is in contrast to broad immunodeficiency reported for inbred mice with similar genetic defects and suggests that compensating mechanisms exist in humans that prevent generalized immunodeficiency (51
). Second, CD1 defects in ABL are not complete in the case of CD1b, which maintains its ability to present at least some antigens acquired in the deep endolysosomal system. Microorganisms such as M. tuberculosis
that express CD1b-restricted antigens loaded in these compartments (17
) might therefore elicit only minimally impaired CD1b-restricted immune responses in ABL. Finally, the low prevalence of ABL prevents the analysis of susceptibility to pathogens dependent upon CD1-mediated immune responses but that infrequently lead to infections in humans (e.g., Borrelia burgdorferi
). Notably, however, increased susceptibility to herpes viruses, a group of common pathogens, has not been reported in ABL. This is surprising given the dramatic defects in CD1d and CD1d-restricted iNKT cells in ABL and previous descriptions of EBV-induced X-linked lymphoproliferative syndrome (XLP) and disseminated Varicella zoster virus (VZV) infection in association with iNKT deficiency (55
). Since we were unable to examine antiviral immunity in ABL, this discrepancy is unexplained but may suggest that these previously described susceptibilities to herpes virus infections are due to other immune defects either alone or in combination with iNKT deficiency (55
In addition, abnormal CD1 function may not only be directly involved in the immune-related defects described in ABL but may also contribute significantly to the metabolic alterations observed in this disorder. For example, iNKT cells have been shown to drive hyperlipidemia and atherosclerosis but protect from hepatic steatosis (32
). Although the effects of MTP deficiency on lipoprotein metabolism are sufficient to explain both lack of atherosclerosis and frequent hepatic steatosis in ABL (1
), it is possible that CD1 and NKT dysfunction may contribute to these phenomena. Our findings therefore indicate that further investigation of the role of iNKT cells in the metabolic processes associated with ABL is warranted.
In conclusion, our studies show that the function of both of the known substrates for MTP, APOB (24
) and CD1 (6
), is dramatically disabled in ABL and highlight the importance of future studies investigating the role of CD1 defects in potentially immune-related phenomena associated with ABL. In addition, since CD1d-restricted NKT cells are implicated in diseases such as atherosclerosis (59
), inflammatory bowel disease (IBD) (63
), and autoimmune hepatitis (64
), our studies further raise the possibility that chemical inhibitors of MTP might be suitable not only for the treatment of hyperlipidemic disorders (65
) but also for interference with CD1-restricted lipid antigen presentation in human immune-mediated diseases.