Natural killer T cells: Know thyself
There are two main reasons why natural killer T (NKT) cells fascinate immunologists. First, they exhibit a unique mélange of properties found in innate immune cells as well as the properties of conventional T cells, part of adaptive immunity (1–4). Second, NKT cells exercise a determining influence on a variety of immune responses in mice, ranging from autoimmunity to their response to tumors and infections (1–4). Because NKT cells have a limited diversity of their T cell antigen receptor (TCR) α chains, they are often called invariant (i)NKT cells (3, 5). iNKT cells recognize glycolipid antigens from certain bacteria that are presented by CD1d, a nonpolymorphic antigen-presenting molecule (4), but this is not the entire story. Although students are taught that lymphocytes must be tolerant of self, this is not the case for iNKT cells, because there is evidence that iNKT cells are activated as a result of contact with self-antigens in the thymus (1–3, 6). Self-reactivity may allow iNKT cells to respond rapidly, not by specifically recognizing a panoply of microbial antigens but by responding to a secondary signal of infection with a very limited TCR diversity. It is widely believed that the same self-antigens expressed in the thymus activate the mature cells. These mature cells are in contrast to conventional T cells, which are positively selected by weak TCR interactions and would be negatively selected by TCR agonists. Therefore, although iNKT cells obey the ancient aphorism “to know thyself,” the chemical definition of the self-antigen has been elusive. It has been proposed that a single glycosphingolipid (GSL), isoglobotrihexosylceramide (iGb3), is required for iNKT cell differentiation in the thymus (7) and activation in the periphery (8, 9). GSLs are glycolipids that contain a ceramide lipid linked to one or more sugars (Fig. 1). Two papers in this issue of PNAS (10, 11) now cast doubt on the hypothesis that iGb3 is the unique self-antigen required for iNKT cells. In a biochemical analysis, Speak et al. (10) report that they could not detect iGb3 in the thymus or in dendritic cells (DCs) from mice and humans, whereas Porubsky et al. (11) find that mice lacking iGb3 synthase have normal iNKT cell number and function. Together, these studies suggest that there must be other self-antigens for iNKT cells.
Biosynthesis of the major classes of GSLs. Beginning with the formation of ceramide, GSLs are synthesized by the stepwise addition of monosaccharides, leading to the production of β-galactosylceramide (β-GalCer) or β-glucosylceramide (β-GlcCer). The lactosylceramide (LacCer) common precursor is formed by the action of lactosylceramide synthase, which transfers a galactose from UDP-galactose to glucosylceramide. The common lactosylceramide (Galβ1–4Glcβ1–1Cer) structure is then elongated by different glycosyltransferases, thereby defining the classes of GSLs. Enzymes involved in the synthesis and degradation of the various GSLs are indicated. Substrates of hexosaminidases A and B are depicted in green, and the iNKT cell ligand iGb3 is depicted in red.
The identification of iGb3 as a critical self-antigen was not based on biochemistry, but instead used genetics, immunology, and a series of deductions so incisive that the paper “unfolded like a detective story” (12). Because earlier work showed that cells that cannot produce β-glucosyl ceramide (see Fig. 1) cannot stimulate iNKT cell autoreactivity (13), Bendelac and coworkers (7) reasoned that the self-antigen was likely to be a GSL; therefore, they screened mice defective for enzymes involved in GSL metabolism. Mice lacking the gene encoding the β-subunit of lysosomal hexosaminidase A and B enzymes (hexb −/− mice) were found to be deficient for iNKT cells (7). GSLs with terminal N-acetyl galactosamine (GalNAc) sugars (shown in green in Fig. 1) therefore were thought to be potential precursors for the iNKT cell ligand because they are cleaved by β-hexosaminidases. The ganglio series was eliminated, because mice deficient for the GalNAc transferase (Galgt1 −/−) or GM3 synthase (Siat9 −/−) had normal iNKT cells (7). Among the remaining candidates, only iGb3 proved to be an agonist for iNKT cells and was the likely self-antigen if one assumed that the selecting thymic ligand was a TCR agonist. In a subsequent study (8), it was shown that DCs activated by Salmonella typhimurium could stimulate iNKT cells to produce IFN-γ. If hexb −/− DCs were exposed to S. typhimurium, however, they could not induce IFN-γ release from iNKT cells, implying that mature iNKT cell activation by DCs under inflammatory conditions required the presentation of a β-hexosaminidase-dependent antigen, presumably iGb3. The dependence on β-hexosaminidase enzymes is consistent with the hypothesis that iGb3, which is produced by β-hexosaminidase cleavage of iGb4 in lysosomes, is the major self-antigen required for iNKT development and activation of the mature cells. These findings raised two important questions. First, the synthetic antigen α-GalCer is known to be a potent agonist for the iNKT cell TCR. How does this NKT TCR cross-react with monosaccharidic ligands; with the galactose sugar in the α anomeric linkage to the lipid; and with trisaccharidic iGb3, in which the internal sugar linked to the ceramide is in the opposite, β conformation? Second, why was the specificity for iGb3 selected for antigen recognition by innate-like iNKT cells?
Subsequently, another group (14) provided a different interpretation for the absence of iNKT cells in hexb −/− mice and found that several other mouse strains with defects in GSL metabolism also have decreased iNKT cells, irrespective of the role of the mutant gene in the generation of iGb3. For example, mice deficient for β-galactosidase had decreased iNKT cells, despite the fact that β-galactosidase is not involved in iGb3 synthesis. According to Gadola et al. (14), the degree of iNKT cell deficiency in the mutant strains is related to the extent of their lipid storage disease and not to the role of the defective enzyme in iGb3 synthesis. This finding perhaps is the result of lipid storage defects disrupting the normal loading of glycolipids into CD1d. Missing, however, was evidence for or against the presence of iGb3 in DCs or thymus. As reported in this issue of PNAS, however, Speak et al. (10) used a sensitive HPLC assay to search for iGb3 and iGb4 in mouse and human tissues. The only mouse tissue in which iGb3 or iGb4 could be detected was the dorsal root ganglion (DRG); spleen, thymus, and DCs were negative. All human tissues were negative for both iGb3 and iGb4, consistent with other data (15, 16) suggesting that the human iGb3 synthase gene could be a pseudogene.
A critical issue is the detection limit, because, although it may be likely that mature iNKT cells require more than a few copies per cell of antigen, differentiating thymocytes are more sensitive to weak TCR signals. Therefore, it is possible that low levels of iGb3 could positively select iNKT cells. Speak et al. (10) estimated their detection limit to be ≈200 copies per cell in the thymus. Studies of mice deficient for enzymes in the catabolism of GSLs (Fig. 1), however, provided an even more sensitive assay. The enzyme α-galactosidase A degrades iGb3 to form the nonantigenic lactosyl ceramide. It therefore was expected that the absence of this enzyme would lead to increased iGb3, and a 250-fold increase was observed in the DRG in α-GalA −/− mice. Similarly, the iGb4 precursor of iGb3 was increased in the DRG of hexb −/− mice. Despite the expected accumulation of iGb3 and iGb4 in the DRG from the two mutant mouse strains, both isoglobo-series GSLs remained undetectable in thymus and DCs.
Also in this issue of PNAS, Porubsky et al. (11) generated and analyzed mice deficient for iGb3 synthase (iGb3S −/−). They found no difference in the number or phenotype of iNKT cells in these mice, and their iNKT cells responded normally by producing cytokines after stimulation with the potent agonist α-GalCer. HPLC analysis showed that the DRG of the iGb3S −/− mice lacked iGb3 and iGb4, indicating that there is no compensatory pathway for the synthesis of iGb3.
The conclusion from these studies (10, 11) is that iGb3 is not the sole ligand necessary for iNKT cells. It is possible that a compensatory pathway induced in the iGb3S −/− mice leads to the synthesis of another glycolipid that can positively select iNKT cells. The hypothetical compensatory pathway is not evident in hexb −/− mice, which could reflect the fact that β-hexosaminidase forms several GSLs, whereas iGb3 synthase is specific for the isoglobo-series. Alternatively, secondary effects in hexb −/− mice on lysosomes resulting from lipid storage could make the iNKT cell deficiency in hexb −/− mice difficult to overcome by presentation of an alternative antigen. Nevertheless, bacterial and autologous GSLs are the most potent iNKT cell antigens. Furthermore, iGb3 is an autologous GSL antigen in mice, and it remains the only known autologous iNKT cell antigen. It is difficult to believe that this is coincidental, and therefore perhaps the autologous ligand is closely related to iGb3. Gb3, which differs from iGb3 only for the linkage of the terminal galactose, is expressed in the mouse and human thymus, and a role for Gb3 is consistent with data from the hexb −/− mouse. Gb3 is not an agonist for the invariant TCR (7), however, which is not consistent with peripheral self-antigen-driven activation or with the still-unproven concept of agonist-mediated selection of iNKT cells in the thymus. Nevertheless, the fact that both iGb3 and α-GalCer activate iNKT cells illustrates the ability of this TCR to recognize diverse structures, raising the possibility that there could be a number of autologous glycolipids that contribute to iNKT cell positive selection, analogous to the diverse sets of self-peptides that positively select conventional T cells.
