Thinking Inside the Box: Endogenous α-Anomeric Lipid Antigens
Authors: Jessica Kim, Ji Hyung Kim, Florian Winau
Journal: Immunity (2014)

The most powerful iNKT cell antigen is α-galactosylceramide (α-GalCer), derived from the marine sponge. However, α-anomeric glycolipids are thought to be absent in mammals. In this issue of Immunity, Kain et al., 2014Kain L. Webb B. Anderson B.L. Deng S. Holt M. Constanzo A. Zhao M. Self K. Teyton A. Everett C. et al.Immunity. 2014; 41 (this issue): 543-554Abstract Full Text Full Text PDF Scopus (165) Google Scholar demonstrate the presence of mammalian α-linked glycosylceramides, such as α-GalCer. The most powerful iNKT cell antigen is α-galactosylceramide (α-GalCer), derived from the marine sponge. However, α-anomeric glycolipids are thought to be absent in mammals. In this issue of Immunity, Kain et al., 2014Kain L. Webb B. Anderson B.L. Deng S. Holt M. Constanzo A. Zhao M. Self K. Teyton A. Everett C. et al.Immunity. 2014; 41 (this issue): 543-554Abstract Full Text Full Text PDF Scopus (165) Google Scholar demonstrate the presence of mammalian α-linked glycosylceramides, such as α-GalCer. Innate-like T lymphocytes are able to rapidly respond to stimulation with the production of cytokines and other effector functions (Bendelac et al., 2007Bendelac A. Savage P.B. Teyton L. Annu. Rev. Immunol. 2007; 25: 297-336Crossref PubMed Scopus (1784) Google Scholar). Part of this group are invariant natural killer T (iNKT) cells, which express a restricted T cell receptor (TCR) repertoire and molecules associated with NK cells (Bendelac et al., 2007Bendelac A. Savage P.B. Teyton L. Annu. Rev. Immunol. 2007; 25: 297-336Crossref PubMed Scopus (1784) Google Scholar). The immediate mode of action of iNKT cells endows them with regulatory properties regarding innate and adaptive immune responses. Therefore, iNKT cells are involved in many disease processes from infection to cancer (Bendelac et al., 2007Bendelac A. Savage P.B. Teyton L. Annu. Rev. Immunol. 2007; 25: 297-336Crossref PubMed Scopus (1784) Google Scholar). In contrast to major-histocompatibility-complex-restricted T cells recognizing peptides, iNKT cells react with lipid antigens presented by CD1d molecules on the surface of antigen-presenting cells (APCs). The first iNKT cell antigen described was α-galactosylceramide (α-GalCer), initially derived from a marine sponge (Kawano et al., 1997Kawano T. Cui J. Koezuka Y. Toura I. Kaneko Y. Motoki K. Ueno H. Nakagawa R. Sato H. Kondo E. et al.Science. 1997; 278: 1626-1629Crossref PubMed Scopus (2157) Google Scholar). Moreover, several microbial species produce α-anomeric glycolipid antigens (Mattner et al., 2005Mattner J. Debord K.L. Ismail N. Goff R.D. Cantu 3rd, C. Zhou D. Saint-Mezard P. Wang V. Gao Y. Yin N. et al.Nature. 2005; 434: 525-529Crossref PubMed Scopus (952) Google Scholar, Kinjo et al., 2006Kinjo Y. Tupin E. Wu D. Fujio M. Garcia-Navarro R. Benhnia M.R. Zajonc D.M. Ben-Menachem G. Ainge G.D. Painter G.F. et al.Nat. Immunol. 2006; 7: 978-986Crossref PubMed Scopus (529) Google Scholar). To complicate matters, there is evidence that iNKT cells also recognize endogenous lipids (Chiu et al., 2002Chiu Y.H. Park S.H. Benlagha K. Forestier C. Jayawardena-Wolf J. Savage P.B. Teyton L. Bendelac A. Nat. Immunol. 2002; 3: 55-60Crossref PubMed Scopus (171) Google Scholar), and indeed, over the years several self-antigens with rather weak antigenicity have been proposed (Zhou et al., 2004Zhou D. Mattner J. Cantu 3rd, C. Schrantz N. Yin N. Gao Y. Sagiv Y. Hudspeth K. Wu Y.P. Yamashita T. et al.Science. 2004; 306: 1786-1789Crossref PubMed Scopus (841) Google Scholar, Brennan et al., 2011Brennan P.J. Tatituri R.V. Brigl M. Kim E.Y. Tuli A. Sanderson J.P. Gadola S.D. Hsu F.F. Besra G.S. Brenner M.B. Nat. Immunol. 2011; 12: 1202-1211Crossref PubMed Scopus (263) Google Scholar). However, current opinion holds that mammalian hosts are not able to synthesize glycosylceramides with the proximal sugar in an α-glycosidic bond. Notably, in this issue of Immunity, Kain et al., 2014Kain L. Webb B. Anderson B.L. Deng S. Holt M. Constanzo A. Zhao M. Self K. Teyton A. Everett C. et al.Immunity. 2014; 41 (this issue): 543-554Abstract Full Text Full Text PDF Scopus (165) Google Scholar demonstrate the presence of mammalian α-linked glycosylceramides, such as α-GalCer. Thus, the most powerful iNKT cell antigen, considered to be exclusively exogenous in nature, could be the long-searched-for endogenous antigen after all. A recent report demonstrated that the abundant self-lipid β-glucosylceramide (β-GluCer) serves as endogenous antigen for iNKT cell activation (Brennan et al., 2011Brennan P.J. Tatituri R.V. Brigl M. Kim E.Y. Tuli A. Sanderson J.P. Gadola S.D. Hsu F.F. Besra G.S. Brenner M.B. Nat. Immunol. 2011; 12: 1202-1211Crossref PubMed Scopus (263) Google Scholar). Kain et al. have now synthesized highly pure β-GluCer and found that it failed to stimulate iNKT cells. Then they tested commercially available β-GluCer from different sources, which restored iNKT cell activation. Subsequently, the authors treated those samples with the enzyme β-glucocerebrosidase (GBA) in order to fully digest β-GluCer. Surprisingly, the iNKT cell stimulatory capacity of the lipid samples did not change after substrate digestion. Thus, Kain et al. have shown that β-GluCer is not a veritable antigen and that minute contaminations with stimulatory lipid entities can trigger activation of iNKT cells. Following the idea of a possible role for α-linked glycolipids as endogenous antigens, the authors investigated the impact of a CD1d-α-GalCer antibody (L363) in functional iNKT cell experiments. In this context, autoreactivity of iNKT lymphocytes is known as the T cell response to APCs without the addition of exogenous lipid antigens (Bendelac et al., 2007Bendelac A. Savage P.B. Teyton L. Annu. Rev. Immunol. 2007; 25: 297-336Crossref PubMed Scopus (1784) Google Scholar). Interestingly, L363 antibody was able to abrogate the T cell response to untreated APCs, suggesting that endogenous α-linked glycosylceramides were present during antigen presentation. Next, Kain et al. studied the L363 antibody tool in depth by performing elegant affinity studies of CD1d-glycolipid recognition by surface plasmon resonance. Accordingly, L363 bound α-GalCer and α-GluCer in complex with CD1d molecules, although with lower affinity for the latter. By contrast, L363 failed to recognize any β-linked glycosylceramides in complex with CD1d. Subsequently, the CD1d-α-GalCer-specific antibody was used for immunoprecipitation of lipid complexes with CD1d in a dendritic cell (DC) line and CD1d transfectants (rat basophilic leukemia [RBL] cell line). Immunoprecipitates were analyzed by multiple reaction monitoring mass spectrometry and revealed a peak corresponding to standard C24:1 α-GalCer. These findings suggested the presence of monoglycosylceramides bound to CD1d in mammalian APC lines. However, the quantity of material recovered by immunoprecipitation was not sufficient for functional iNKT cell experiments. For the same reasons, the determination of α- and β-anomers by nuclear magnetic resonance (NMR) was also not possible. To overcome that obstacle, Kain et al. succeeded in generating an α-GalCer-specific antibody to be used for lipid immunoprecipitation. For this purpose, RBL-CD1d cells were metabolically labeled with C14-UDP-galactose prior to precipitation with anti-α-GalCer. Subsequently, immunoprecipitates were analyzed by thin-layer chromatography (TLC), which revealed a band that migrated at the height of reference C24:1 α-GalCer. As a prerequisite, Kain et al. established a TLC system that allows for the migratory separation of galactosylceramides only differing in their α- or β-anomeric configuration. Although this is not direct proof by NMR, the TLC results make the presence of endogenous α-GalCer likely. Furthermore, the authors examined a mechanism by which the availability of endogenous α-glycosylceramides is regulated. A previous study established the concept of threshold control, in which catabolic enzymes degrade endogenous lipid antigens under steady-state conditions and thus preclude efficient loading on CD1d and subsequent iNKT cell activation (Darmoise et al., 2010Darmoise A. Teneberg S. Bouzonville L. Brady R.O. Beck M. Kaufmann S.H. Winau F. Immunity. 2010; 33: 216-228Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Upon enzyme block in the degradative pathway, endogenous antigen accumulates and reaches a threshold for productive CD1d loading and stimulation of iNKT cells (Darmoise et al., 2010Darmoise A. Teneberg S. Bouzonville L. Brady R.O. Beck M. Kaufmann S.H. Winau F. Immunity. 2010; 33: 216-228Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Accordingly, α-galactosidase A (GLA) is able to cleave the terminal α-linked galactose from glycolipids such as globotriaosylceramide (Gb3), iGb3, and galabiaosylceramide. However, it remains unclear how GLA could regulate α-GalCer given that the enzyme is not capable of acting on the proximal α-galactose bound to ceramide. Kain et al. provide a solution to this problem. Accordingly, a two-step mechanism can regulate the abundance of endogenous α-glycosylceramides (Figure 1). First, acid ceramidase (ASAH1) removes the acyl chain, which leaves the sugar group connected to sphingosine and thus creates α-psychosine (or α-glucopsychosine). Second, GLA is now able to access α-psychosine for degradation to sphingosine and sugar (acid α-glucosidase [GAA] could replace GLA with regard to α-GluCer degradation). Thus, inhibition of GLA and/or ASAH1 can regulate the accumulation of endogenous α-GalCer for activation of iNKT cells (Figure 1). The study by Kain et al. lays the foundations for future challenges to elucidate the biological role of endogenous α-glycosylceramides. This will include the refinement of chemical and biochemical techniques in order to directly prove the α-anomeric entity by NMR. Improving the yield of endogenous α-GalCer will also facilitate functional antigen-presentation experiments to demonstrate its bioactivity. Another critical task would involve the isolation of endogenous α-glycosylceramides from mammalian host tissues. Taken together, these future achievements will allow the assessment of the in vivo role of endogenous α-GalCer and α-GluCer. Ultimately, every interesting study leads to new questions to be answered. In this context, which synthetic pathway promotes the generation of endogenous α-glycosylceramides? Maybe new enzymes that directly synthesize α-GalCer and α-GluCer will be discovered. In analogy to ceramide galactosyltransferase (CGT) and glucosylceramide synthase (GCS), which produce corresponding β-forms, one could propose the existence of CαGT and αGCS, respectively. Alternatively, the known enzymes CGT and GCS could work in an unfaithful fashion and generate minute amounts of the respective α-forms (Figure 1). Finally, we postulate the presence of glucose- or galactose-dependent anomerases that mediate the conversion of β-glycosylceramides into endogenous α-GluCer or α-GalCer for the development and activation of iNKT cells. The Identification of the Endogenous Ligands of Natural Killer T Cells Reveals the Presence of Mammalian α-Linked GlycosylceramidesKain et al.ImmunityOctober 16, 2014In BriefThe α-anomeric form of lipids is a potent trigger of NKT cell activation but is not thought to be synthesized by mammalian cells. Teyton and colleagues show that mammalian cells can produce small amounts of α-linked monoglycosylceramides. Full-Text PDF Open Archive