In recent years, several studies demonstrated that CD1d presents pathogen-derived lipids capable of activating iNKT cells. The CD1d antigens α-glucuronosylceramide and α-galacturonosylceramide from Sphingomonas bacteria [32,33,34], α-galactosyldiacylglycerol from Borrelia burgdorferi [35], and α-glucosyldiacylglycerol from Streptococcus pneumonia [36] all contain α-linked glycans, a feature shared with α-GalCer. Since humans cannot synthesize α-linked glycolipids, an α-linked glycan could represent a specific antigenic determinant of CD1d-presented, pathogen-derived lipids. Besides these, different lipid species like cholesteryl α-glucoside from Helicobacter pylori [37] are presented by CD1d molecules and can activate iNKT cells. Since these lipid antigens are specific to microbes, they are non-self to the host’s immune system, which minimizes the chance of auto-reactivity.

The identity of physiologically relevant, stimulatory lipid antigens presented by CD1d in the context of viral infection remains, at this time, incompletely understood. As opposed to the above-mentioned microbial CD1d lipid antigens, virus-specific lipids do not exist. Therefore, the iNKT cell stimulatory lipids presented by CD1d during viral infection must be of host cell origin. This poses an intrinsic risk of undesired self-reactivity. To avoid this, self-lipids presented by CD1d should only be stimulatory towards iNKT cells during conditions of cellular stress, such as infection or carcinogenesis. Very recently, the cellular lipid profile was found to be altered during hepatitis B virus (HBV) infection, leading to increased activation of NKT cells. Whereas diverse NKT cells were stimulated by HBV-induced lysophosphatidylethanolamine, different lipid(s) were responsible for the activation of iNKT cells, although their nature was not identified [38].

Alterations in CD1d lipid presentation induced by viral infection appear linked to activation of pattern-recognition receptors, such as Toll-like receptors (TLRs). For example, the endosomal TLRs (TLR3, 7, 8, and 9) are involved in detecting viruses through the recognition of nucleic acid structures as pathogen-associated molecular patterns (PAMPs) [39,40]. TLR engagement could effectuate changes in CD1d antigen presentation in various ways, as discussed below.

First, increased synthesis of antigenic self-lipids affects CD1d-mediated lipid presentation. Stimulation of myeloid DCs with (human) TLR8 or (mouse) TLR9 ligands resulted in enhanced iNKT cell activation, which was dependent on both CD1d expression and cytokine secretion (IL-12 in humans, type I interferons in mice). Two observations strongly support the view that increased synthesis of glycosphingolipids upon TLR stimulation causes enhanced CD1d presentation to iNKT cells: i) chemical inhibition of glycosphingolipid synthesis reverted the added iNKT stimulatory capacity provided by TLR-ligand-treated DCs and ii) mRNA levels for enzymes involved in glycosphingolipid synthesis were enhanced in TLR-ligand-stimulated DCs [41,42]. Muindi et al. conducted a more detailed study on the effects of TLR engagement on the repertoire of glycosphingolipids presented by murine CD1d. Both total cellular glycosphingolipid content and the pool of CD1d-bound glycosphingolipids were markedly altered following TLR4 stimulation of DCs. Surprisingly, the relative abundance of glycosphingolipids associated with CD1d did not mirror that in the total cellular lipid pool, suggesting selective lipid loading of CD1d molecules [43]. Accumulation of the self-lipid β-glucopyranosylceramide occurred during bacterial infection or upon TLR4 stimulation, leading to iNKT cell activation in a CD1d-dependent manner [44]. Whether these results can be extrapolated to viral infection remains to be determined. Still, stimulation of various TLRs, among which were the virus-sensing TLR3, 7, and 9, altered the expression of transcripts favoring β-glucopyranosylceramide synthesis, indicating that β-glucopyranosylceramide-mediated iNKT cell activation could contribute to anti-viral defense [44].

Second, reduced degradation of antigenic self-lipids influences the lipid repertoire presented by CD1d molecules. The lysosome-resident enzyme α-galactosidase A (α-Gal-A), known to be involved in lipid metabolism, appears to affect the generation of self-antigens recognized by iNKT cells, at least in mice [45]. Both in vitro and in vivo, α-Gal-A-deficient DCs stimulated iNKT cells in a CD1d-dependent manner to a greater extent than wild-type DCs. Incubation of wild-type DCs with the TLR4 ligand LPS or the bacterium Listeria monocytogenes caused a temporary decrease in α-Gal-A activity. This effect was abrogated in DCs deficient for the TLR-adaptor protein MyD88, indicating that TLR signaling is required for the observed decrease in α-Gal-A enzymatic activity. Moreover, stimulation of DCs with TLR4 and TLR9 ligands resulted in accumulation of glycosphingolipids. Thus, the TLR-dependent inhibition of α-Gal-A activity would provide a mechanistic link between TLR-mediated pathogen recognition and the generation, and subsequent presentation, of antigenic self-lipids by CD1d [45].

Third, the trafficking route of CD1d molecules affects lipid presentation. A subset (5–10%) of CD1d molecules associates with MHC class II complexes [28]. In TLR-stimulated mature DCs, MHC class II molecules are dramatically relocalized from intracellular endosomal compartments to the cell surface. As a consequence, the MHC class II-associated pool of CD1d molecules would not encounter certain lipid species along the endolysosomal route. In line with this, the presentation of exogenous lipid antigens by CD1d molecules is reduced for mature DCs [46]. In further support, mice that lack the MHC class II-associated invariant chain exhibit defects in the localization of MHC class II molecules to the endolysosomal route [47]. In these same invariant chain-deficient mice, CD1d-mediated lipid presentation of endosome-derived model antigen α-GalGalCer is also much reduced [15].

Finally, CD1d upregulation improves lipid antigen presentation to iNKT cells. CD1d mRNA levels were found to be increased upon infection of DCs with herpes simplex virus type 1 (HSV-1) or human cytomegalovirus (HCMV). UV-inactivated viruses caused a similar, although slightly weaker effect, suggesting that viral PAMPs are mainly responsible for the higher CD1d mRNA expression. Indeed, stimulation of TLR7 also elevated CD1d mRNA expression levels. The increased CD1d mRNA levels were accompanied by enrichment of CD1d proteins at the cell surface and enhanced activation and proliferation of iNKT cells [48].

In conclusion, TLR-mediated recognition of viral infection leads to altered lipid presentation by CD1d molecules, thereby affecting the activation of iNKT cells. The exact identity of the lipids presented by CD1d at the surface of virus-infected cells awaits further elucidation.

Human immunodeficiency virus (HIV), the causative agent of acquired immune deficiency syndrome (AIDS), is a retrovirus that infects CD4⁺ cells (including T cells, NKT cells, macrophages and DCs). HIV infection is generally not cleared by the host immune system, resulting in chronic infection. To prevent elimination by T cells, HIV interferes with MHC class I- and class II-restricted antigen presentation. For instance, the HIV Nef protein causes downregulation of mature MHC class II complexes at the cell surface while display of immature MHC class II is increased [69]. More recently, HIV has been shown to also escape iNKT cell recognition (reviewed in [70]). A marked depletion of iNKT cells is observed after HIV infection, most likely resulting from cytolytic infection combined with activation-induced cell death. HIV further escapes iNKT cell recognition by downregulating CD1d surface display. Three viral proteins were shown to be involved in this process. Incubation of cells with recombinant HIV gp120 protein resulted in downregulation of CD1d surface levels [71], although the mechanism of action remains to be elucidated. HIV Nef accelerates the internalization of CD1d from the plasma membrane, retaining these lipid-presenting molecules in the trans-Golgi network [72]. Nef-induced downregulation acts via the tyrosine-based targeting motif located in the cytoplasmic tail of CD1d [72,73]. This is reminiscent of the requirement for the tyrosine residue in the cytoplasmic tail of MHC class I molecules that is involved in AP-1 binding underlying Nef-induced CTL evasion [74]. Whereas MHC class I molecules are then redirected to lysosomes [74], Nef targets CD1d molecules towards the trans-Golgi network [72]. Finally, HIV Vpu retains CD1d molecules in early endosomes, thereby impairing recycling of CD1d from endocytic compartments to the cell surface [75].

It is intriguing that a virus encoding few gene products like HIV codes for at least three proteins that inhibit CD1d-mediated lipid presentation, suggesting that iNKT cells are particularly important in the control of HIV infection.

Vaccinia virus (VV), the prototypic member of the family Poxviridae, has been successfully used as a vaccine to effectuate the eradication of smallpox. Poxviruses are large enveloped DNA viruses encoding ~200 proteins. In contrast, vesicular stomatitis virus (VSV) is a small single-stranded RNA virus belonging to the family Rhabdoviridae and codes for only five proteins. Interestingly, both viruses were found to interfere with CD1d-restricted lipid presentation by a similar mechanism.

Infection with either VV or VSV resulted in reduced activation of iNKT cells, although CD1d surface levels remained unchanged. The two viruses modulated MAPK signaling and subsequent intracellular CD1d trafficking, thereby presumably altering the lipid repertoire presented by CD1d for iNKT cell recognition [76]. The VV-encoded proteins B1R and H5R were found to be involved in evasion from CD1d-restricted iNKT cells [77]. Yet, B1R is a viral kinase that phosphorylates H5R, a transcription factor involved in late viral protein expression, and thus the effects of B1R and H5R on iNKT cell evasion may be indirect.

The use of a similar mechanism to thwart iNKT cell recognition by two unrelated viruses, like VV and VSV, could indicate this to be a general CD1d evasion strategy of viruses. However, infection with the small single-stranded RNA virus lymphocytic choriomeningitis virus (LCMV) had no direct effect on NKT cell activation [76] and thus interference with CD1d-restricted antigen presentation seems to be virus-specific.

Human papillomaviruses (HPV) are small DNA viruses infecting epithelia of the skin (cutaneous HPV subtypes) or mucous membranes (genital HPV subtypes). Whereas infection with low-risk cutaneous HPV types causes benign warts, the high-risk genital HPV types are associated with cervical cancer.

HPV type 16 inhibits MHC class I- and class II-restricted peptide presentation through expression of the small hydrophobic E5 protein [78,79,80]. In addition, HPV interferes with CD1d-resticted lipid presentation. In immunostained clinical samples, CD1d molecules were abundantly expressed by uninfected cervical epithelium, but undetectable in HPV-infected lesions. Expression of HPV E5 proteins in cell lines caused a reduction in both cell surface and total CD1d protein levels. HPV E5 interacts with calnexin and prevents exit of CD1d molecules from the ER. Although mechanistic details are lacking, upon cellular expression of HPV E5, CD1d molecules end up in the cytosol, where they are degraded by the proteasome. The ability to interfere with CD1d-mediated antigen presentation is conserved among the E5 proteins of a low-risk (HPV6) and a high-risk (HPV16) subtype [81].

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the most recently discovered human gamma-herpesvirus and is implicated in the development of malignancies, such as Kaposi’s sarcoma, primary effusion lymphoma, and multicentric Castleman’s disease [82]. Herpesviruses are large enveloped DNA viruses that persist for life in infected hosts, mostly as a latent infection with limited viral gene expression. Intermittent periods of reactivation allow transmission of herpesviruses to new hosts. The herpesvirus family is famous for its large variety of immune evasion tactics, affecting many aspects of the anti-viral immune response [83,84,85,86,87].

During productive KSHV infection, two immunoevasins, K3 and K5 (also known as modulator of immune recognition (MIR) 1 and 2, respectively), interfere with MHC class I-restricted antigen presentation. Both K3 and K5 are membrane-bound E3-ubiquitin ligases that ubiquitinate the cytosolic tails of MHC class I heavy chains, causing their enhanced endocytosis and subsequent lysosomal degradation [88,89]. Besides MHC class I, K5 reduces surface display of the co-stimulatory molecules B7.2 and ICAM-1 [90,91].

Likely based on similarities with MHC class I, CD1d molecules are also prone to K3- and K5-induced downregulation. Although CD1d molecules enter the endocytic pathway as a consequence of K5-mediated ubiquitination of their cytoplasmic tails, the total cellular levels of CD1d remain virtually unchanged. Thus, lysosomal degradation of CD1d is not increased in cells expressing KSHV K5 [92]. The difference in this aspect between MHC class I and CD1d most likely originates from their susceptibility to lysosomal degradation, with MHC class I molecules being susceptible and CD1d/β2m complexes being resistant. Finally, K5 expression inhibited CD1d-restricted iNKT cell activation. This, combined with observations that surface CD1d levels are reduced on B cells undergoing productive KSHV infection [92], might suggest that KSHV not only thwarts CTL, but also iNKT cell activation.

HSV-1 is a human alpha-herpesvirus and the causative agent of cold sores. Similar to other herpesviruses, HSV-1 hampers immune activation at multiple levels. For instance, HSV-1 inhibits MHC class I-restricted T cell recognition by combining a block in antigenic peptide import into the ER, exerted by ICP47, with a general reduction in host cell protein synthesis, imposed by the virion host shutoff (vhs) protein [93].

Downregulation of CD1d at the surface of HSV-1-infected cells is independent of host shutoff and total protein levels of CD1d are not reduced by HSV-1 infection. Instead, HSV-1 downregulates CD1d surface display by inhibiting recycling of CD1d molecules from endosomal compartments to the cell surface, leading to redistribution of CD1d to the limiting membranes of lysosomes. This HSV-1 induced downregulation is independent of the cytoplasmic tail of CD1d [94].

Two HSV-1 proteins were recently found to cooperatively hamper CD1d-mediated antigen presentation to iNKT cells, namely gB, a major viral glycoprotein, and US3, a viral serine-threonine kinase. gB was essential, but not sufficient, for CD1d downregulation during viral infection. Efficient CD1d downregulation required co-expression of an active US3 enzyme that modulates gB trafficking. In line with this, HSV-mediated inhibition of iNKT cell activation was reduced for US3-deficient viruses and abolished for gB-deficient viruses. HSV gB proteins directly bind to CD1d complexes within the ER and their association is maintained throughout intracellular trafficking. CD1d trafficking is altered by the concerted action of gB and US3, redirecting CD1d to the trans-Golgi network [95]. These observations seem in contrast with a previous study, reporting CD1d to be redirected to lysosomes [94]; the use of different HSV-1 strains may explain this apparent inconsistency. In addition to CD1d, HSV-1 gB is also involved in altered trafficking of MHC class II molecules [96,97]; whether similar mechanisms are underlying the manipulation of CD1d- and MHC class II-restricted antigen presentation will be an interesting topic for further research.

Reduced CD1d display was observed on DCs infected with high levels of HSV-1, but, unexpectedly, increased surface levels of CD1d were found when DCs contained low viral loads. Despite this, DCs infected with either low or high HSV titers were both crippled in their capacity to stimulate cytokine secretion by iNKT cells [98]. Therefore, additional mechanism(s), independent of surface CD1d downregulation, are likely employed by HSV to interfere with iNKT cell function. Interestingly, a recent study by Bosnjak et al. showed that HSV-1 infection of keratinocytes hampered the activation of iNKT cells independent of CD1d downregulation. Instead, coculture with HSV-1 infected cells did not impair TCR triggering but rather signaling downstream of the TCR within the iNKT cells. This inactivation of iNKT cells required direct contact between the HSV-1 infected cells and the iNKT cells and was sustained after removal of the infected cells [99].