Adaptability of the semi-invariant natural killer T-cell receptor towards structurally diverse CD1d-restricted ligands
2009; Springer Nature; Volume: 28; Issue: 22 Linguagem: Inglês
10.1038/emboj.2009.286
ISSN1460-2075
AutoresWilliam C. Florence, Chengfeng Xia, Laura E. Gordy, Wenlan Chen, Yalong Zhang, James Scott‐Browne, Yuki Kinjo, Karl O. A. Yu, Santosh Keshipeddy, Daniel G. Pellicci, Onisha Patel, Lars Kjer‐Nielsen, James McCluskey, Dale I. Godfrey, Jamie Rossjohn, Stewart K. Richardson, Steven A. Porcelli, Amy R. Howell, Kyoko Hayakawa, Laurent Gapin, Dirk M. Zajonc, Peng George Wang, Sebastian Joyce,
Tópico(s)T-cell and B-cell Immunology
ResumoArticle8 October 2009free access Adaptability of the semi-invariant natural killer T-cell receptor towards structurally diverse CD1d-restricted ligands William C Florence William C Florence Department of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN, USA Search for more papers by this author Chengfeng Xia Chengfeng Xia Department of Chemistry and Biochemistry, Ohio State University, Columbus, OH, USA Search for more papers by this author Laura E Gordy Laura E Gordy Department of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN, USA Search for more papers by this author Wenlan Chen Wenlan Chen Department of Chemistry and Biochemistry, Ohio State University, Columbus, OH, USA Search for more papers by this author Yalong Zhang Yalong Zhang Department of Chemistry and Biochemistry, Ohio State University, Columbus, OH, USA Search for more papers by this author James Scott-Browne James Scott-Browne National Jewish Centre for Allergy and Immunology Research, Denver, CO, USA Search for more papers by this author Yuki Kinjo Yuki Kinjo Division of Developmental Immunology, La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA Search for more papers by this author Karl O A Yu Karl O A Yu Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY, USA Search for more papers by this author Santosh Keshipeddy Santosh Keshipeddy Department of Chemistry, University of Connecticut, Storrs, CT, USA Search for more papers by this author Daniel G Pellicci Daniel G Pellicci Department of Microbiology and Immunology, University of Melbourne, Melbourne, Victoria, Australia Search for more papers by this author Onisha Patel Onisha Patel Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Victoria, Australia Search for more papers by this author Lars Kjer-Nielsen Lars Kjer-Nielsen Department of Microbiology and Immunology, University of Melbourne, Melbourne, Victoria, Australia Search for more papers by this author James McCluskey James McCluskey Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Victoria, Australia Search for more papers by this author Dale I Godfrey Dale I Godfrey Department of Microbiology and Immunology, University of Melbourne, Melbourne, Victoria, Australia Search for more papers by this author Jamie Rossjohn Jamie Rossjohn Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Victoria, Australia Search for more papers by this author Stewart K Richardson Stewart K Richardson Department of Chemistry, University of Connecticut, Storrs, CT, USA Search for more papers by this author Steven A Porcelli Steven A Porcelli Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY, USA Search for more papers by this author Amy R Howell Amy R Howell Department of Chemistry, University of Connecticut, Storrs, CT, USA Search for more papers by this author Kyoko Hayakawa Kyoko Hayakawa Fox Chase Cancer Centre, Philadelphia, PA, USA Search for more papers by this author Laurent Gapin Laurent Gapin National Jewish Centre for Allergy and Immunology Research, Denver, CO, USA Search for more papers by this author Dirk M Zajonc Corresponding Author Dirk M Zajonc Division of Cell Biology, La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA Search for more papers by this author Peng George Wang Corresponding Author Peng George Wang Department of Chemistry and Biochemistry, Ohio State University, Columbus, OH, USA Search for more papers by this author Sebastian Joyce Corresponding Author Sebastian Joyce Department of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN, USA Search for more papers by this author William C Florence William C Florence Department of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN, USA Search for more papers by this author Chengfeng Xia Chengfeng Xia Department of Chemistry and Biochemistry, Ohio State University, Columbus, OH, USA Search for more papers by this author Laura E Gordy Laura E Gordy Department of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN, USA Search for more papers by this author Wenlan Chen Wenlan Chen Department of Chemistry and Biochemistry, Ohio State University, Columbus, OH, USA Search for more papers by this author Yalong Zhang Yalong Zhang Department of Chemistry and Biochemistry, Ohio State University, Columbus, OH, USA Search for more papers by this author James Scott-Browne James Scott-Browne National Jewish Centre for Allergy and Immunology Research, Denver, CO, USA Search for more papers by this author Yuki Kinjo Yuki Kinjo Division of Developmental Immunology, La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA Search for more papers by this author Karl O A Yu Karl O A Yu Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY, USA Search for more papers by this author Santosh Keshipeddy Santosh Keshipeddy Department of Chemistry, University of Connecticut, Storrs, CT, USA Search for more papers by this author Daniel G Pellicci Daniel G Pellicci Department of Microbiology and Immunology, University of Melbourne, Melbourne, Victoria, Australia Search for more papers by this author Onisha Patel Onisha Patel Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Victoria, Australia Search for more papers by this author Lars Kjer-Nielsen Lars Kjer-Nielsen Department of Microbiology and Immunology, University of Melbourne, Melbourne, Victoria, Australia Search for more papers by this author James McCluskey James McCluskey Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Victoria, Australia Search for more papers by this author Dale I Godfrey Dale I Godfrey Department of Microbiology and Immunology, University of Melbourne, Melbourne, Victoria, Australia Search for more papers by this author Jamie Rossjohn Jamie Rossjohn Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Victoria, Australia Search for more papers by this author Stewart K Richardson Stewart K Richardson Department of Chemistry, University of Connecticut, Storrs, CT, USA Search for more papers by this author Steven A Porcelli Steven A Porcelli Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY, USA Search for more papers by this author Amy R Howell Amy R Howell Department of Chemistry, University of Connecticut, Storrs, CT, USA Search for more papers by this author Kyoko Hayakawa Kyoko Hayakawa Fox Chase Cancer Centre, Philadelphia, PA, USA Search for more papers by this author Laurent Gapin Laurent Gapin National Jewish Centre for Allergy and Immunology Research, Denver, CO, USA Search for more papers by this author Dirk M Zajonc Corresponding Author Dirk M Zajonc Division of Cell Biology, La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA Search for more papers by this author Peng George Wang Corresponding Author Peng George Wang Department of Chemistry and Biochemistry, Ohio State University, Columbus, OH, USA Search for more papers by this author Sebastian Joyce Corresponding Author Sebastian Joyce Department of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN, USA Search for more papers by this author Author Information William C Florence1,‡, Chengfeng Xia2,‡, Laura E Gordy1, Wenlan Chen2, Yalong Zhang2, James Scott-Browne3, Yuki Kinjo4, Karl O A Yu5, Santosh Keshipeddy6, Daniel G Pellicci7, Onisha Patel8, Lars Kjer-Nielsen7, James McCluskey8, Dale I Godfrey7, Jamie Rossjohn8, Stewart K Richardson6, Steven A Porcelli5, Amy R Howell6, Kyoko Hayakawa9, Laurent Gapin3, Dirk M Zajonc 10, Peng George Wang 2 and Sebastian Joyce 1 1Department of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN, USA 2Department of Chemistry and Biochemistry, Ohio State University, Columbus, OH, USA 3National Jewish Centre for Allergy and Immunology Research, Denver, CO, USA 4Division of Developmental Immunology, La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA 5Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY, USA 6Department of Chemistry, University of Connecticut, Storrs, CT, USA 7Department of Microbiology and Immunology, University of Melbourne, Melbourne, Victoria, Australia 8Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Victoria, Australia 9Fox Chase Cancer Centre, Philadelphia, PA, USA 10Division of Cell Biology, La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA ‡These authors contributed equally to this work *Corresponding authors: Department of Microbiology and Immunology, Vanderbilt University School of Medicine, A4223 Medical Centre North, 1161 21st Avenue South, Nashville, TN 37232, USA. Tel.: +1 615 322 1472; Fax: +1 615 343 7392; E-mail: [email protected] of Cell Biology, La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037, USA. Tel.: +1 858 752 6605; Fax: +1 858 752 6994; E-mail: [email protected] of Chemistry and Biochemistry, Ohio State University, Columbus, OH 43210, USA. Tel.: +1 614 292 9884; Fax: +1 614 688 3106; E-mail: [email protected] The EMBO Journal (2009)28:3579-3590https://doi.org/10.1038/emboj.2009.286 Correction(s) for this article Adaptability of the semi-invariant natural killer T-cell receptor towards structurally diverse CD1d-restricted ligands10 September 2009 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The semi-invariant natural killer (NK) T-cell receptor (NKTcr) recognises structurally diverse glycolipid antigens presented by the monomorphic CD1d molecule. While the α-chain of the NKTcr is invariant, the β-chain is more diverse, but how this diversity enables the NKTcr to recognise diverse antigens, such as an α-linked monosaccharide (α-galactosylceramide and α-galactosyldiacylglycerol) and the β-linked trisaccharide (isoglobotriaosylceramide), is unclear. We demonstrate here that NKTcrs, which varied in their β-chain usage, recognised diverse glycolipid antigens with a similar binding mode on CD1d. Nevertheless, the NKTcrs recognised distinct epitopic sites within these antigens, including α-galactosylceramide, the structurally similar α-galactosyldiacylglycerol and the very distinct isoglobotriaosylceramide. We also show that the relative roles of the CDR loops within the NKTcr β-chain varied as a function of the antigen. Thus, while NKTcrs characteristically use a conserved docking mode, the NKTcr β-chain allows these cells to recognise unique aspects of structurally diverse CD1d-restricted ligands. Introduction The antigen receptor of αβ T cells faces a daunting recognition problem because each T-cell receptor (TCR) interfaces multiple ligands during its lifetime and yet maintains specificity. They recognise self-ligand (peptide or lipid) in the context of a self-antigen-presenting molecule (major histocompatibility complex (MHC) class I, class II or CD1) in the thymus; foreign ligand in the context of a self-MHC/CD1 molecule in the periphery; and in some cases, non-self-ligands in the context of non-self-MHC molecules. This recognition conundrum is much more daunting for the semi-invariant natural killer (NK) T-cell receptor (NKTcr) because its recognition landscape is predominantly germline-encoded and, hence, highly conserved (Borg et al, 2007; Scott-Browne et al, 2007; Wun et al, 2008), while the antigenic landscapes of CD1d–lipid complexes are highly diverse (Koch et al, 2005; Zajonc et al, 2005, 2008; Kinjo et al, 2006). Understanding how NKT cells see extremely diverse lipid antigens is important because this innate T lymphocyte regulates early immune responses to pathogens and tumours, and is a target for immunotherapy against some autoimmune diseases (Van Kaer, 2005). The TCR α and β-chains each comprise variable (V) and constant (C) domains, in which the V domains contain the complementarity determining region-1 (CDR1), CDR2 and CDR3, that interact with the peptide-laden MHC (pMHC) molecules. While variations within V-gene segments confer diversity to CDR1 and CDR2, imprecise joining and non-templated nucleotide additions during V(D)J recombination generate the much more diverse CDR3 (Rudolph et al, 2006). TCR specificity is not limited to peptidic antigens. NKT cells express a semi-invariant TCR that recognises lipid-based antigens presented by the monomorphic CD1d molecule. The NKTcr is made up of an invariant TCR α-chain, which is generated by TRAV11*02 (mouse and rat Vα14) to TRAJ18 (Jα18) or orthologous TRAV10 (human Vα24) to TRAJ18 rearrangement. Despite the stochastic nature of V(D)J recombination, stringent intrathymic selection steps permit only one CDR3α for NKT cells. Thus, NKT cells express a completely invariant TCR α-chain that is highly conserved across species (Koseki et al, 1990; Porcelli et al, 1993; Dellabona et al, 1994; Lantz and Bendelac, 1994). In contrast, CDR3β of TRBV13-2*01 (mouse Vβ8.2) and the orthologous TRBV25-1 (human Vβ11), which pair with the invariant Vα14 and Vα24 α-chains, respectively, are highly diverse (Porcelli et al, 1993; Lantz and Bendelac, 1994). So too are the CDR3β of TRBV29*02 (Vβ7) and TRBV1 (Vβ2) β-chains, which also pair with Vα14 and Vα24 α-chains in mice (Lantz and Bendelac, 1994; Capone et al, 2003). Thus, NKT cells have an invariant TCR α-chain, but maintain some diversity in their TCR β-chain, which is why they are referred to as semi-invariant. The NKTcr binds structurally diverse CD1d-restricted ligands that range from α-linked monohexosylceramides such as the prototypic NKT-cell antigen α-galactosylceramide (αGalCer; Kawano et al, 1997; Burdin et al, 1998) and the closely related α-galacturonosylceramide (αGalACer; Sphingomonas spp.; Kinjo et al, 2005; Mattner et al, 2005; Sriram et al, 2005); α-galactosyldiacylglycerol (αGalDAG; Borrelia burgdorferi) (Kinjo et al, 2006), to the trihexosylceramide isoglobotriaosylceramide (iGb3) Zhou et al, 2004). Despite this apparent diversity, most mammalian glycolipids are not recognised by the NKTcr, including lactosylceramide (LacCer), globotriaosylceramide (Gb3; α1,4Gal-LacCer), which is closely related to iGb3 (α1,3Gal-LacCer), monosialylganglioside GM1 (α2,3sialyl-LacCer) or isoglobotetraosylceramide (iGb4) (Zhou et al, 2004). A small subset of NKTcrs also recognise phosphatidylethanolamine (Rauch et al, 2003), phosphatidylinositol (Gumperz et al, 2000), β-galactosylceramide (Ortaldo et al, 2004; Parekh et al, 2004), the disialylganglioside GD3 (Wu et al, 2003) and microbial phosphatidylinositol-mannoside (PIM4; Mycobacterium tuberculosis (Fischer et al, 2004). Crystal structures of several CD1d–antigen complexes showed that these lipids bind to CD1d through their long-chain base (LCB) and/or fatty acyl chain(s) so that the polar head groups protrude out of the CD1d antigen-binding site (ABS). Furthermore, the disposition of the head group depends on the unique pattern of hydrogen-bond network formed by residues of CD1d α1 and α2-helices with the polar aspects of the bound ligand (Giabbai et al, 2005; Koch et al, 2005; Zajonc et al, 2005, 2006). Thus, the α-linked galactose of αGalCer lies just above the entrance, flat and parallel to the plain of CD1d's ABS (Koch et al, 2005; Zajonc et al, 2005). In striking contrast, the β-anomeric linkage of the first sugar orients the glycan α1,3Gal–β1,4Gal–β1,1′Glc of iGb3 perpendicular to the ABS (Zajonc et al, 2008). How the semi-invariant NKTcr recognises multiple diverse ligands in the context of CD1d remains an enigma. A major advance in understanding how the NKTcr recognises its cognate antigen came from the crystal structure of the human NKTcr/CD1d–αGalCer co-complex (Borg et al, 2007). It showed that the Vα24 NKTcr interfaces CD1d–αGalCer in an unusual parallel docking mode by engaging germline-encoded CDR1α, CDR3α and CDR2β residues above the F' pocket (Borg et al, 2007; Wun et al, 2008). Further, alanine-scanning mutagenesis of mouse and human NKTcrs revealed that they interact with CD1d–αGalCer similarly by using germline-encoded hotspots within CDR1α, CDR3α and CDR2β (Scott-Browne et al, 2007; Wun et al, 2008). One of these studies also revealed that the Vα14 NKTcr bound to CD1d–αGalCer and the very different CD1d–iGb3 landscapes in a conserved manner, using similar germline-encoded hotspots (Scott-Browne et al, 2007). The NKTcr showed very little conformational change upon ligation (Gadola et al, 2006; Kjer-Nielsen et al, 2006; Borg et al, 2007; Zajonc et al, 2008), consistent with a 'lock and key' recognition model where neither temperature nor ionic strength affected the CD1d–αGalCer interaction (Cantu et al, 2003). These findings suggest that the NKTcr acts like a pattern-recognition receptor and also explain why the NKTcr ligand recognition is CD1d-restricted. Yet, these studies provide little insight into ligand specificity and the molecular features of the diverse epitopes recognised, and do not explain the basis for distinct TCR β-chain usage for antigen recognition by the NKTcr. As an approach to understand the role of β-chain in CD1d-restricted antigen recognition, we probed a panel of NKT-cell-derived hybridomas that express different TCR β-chains, as well as a panel of NKTcr point mutants that have altered CDR, with a variety of CD1d-restricted glycolipid ligands and variants with modifications of their sugar moieties. From the recognition patterns that emerged, we concluded that the NKTcr was highly adaptable, depending on the β-chain used, to recognise unique aspects of structurally diverse ligands within a conserved binding footprint on CD1d molecules. Results TCR β-chain usage dictates diverse glycolipid antigen recognition by the NKTcr Unlike the CDR3α of the NKTcr, the CDR3β is highly diverse, both in length and primary structure (e.g., Supplementary Table S1). Additionally, CDR1β and CDR2β of distinct β-chains differ from each other. Because mouse NKTcr use multiple different β-chains (Vβ8.2, Vβ7 and Vβ2, as well as Vβ14 and Vβ6; Porcelli et al, 1993; Lantz and Bendelac, 1994), we hypothesised that differences within CDR1β, CDR2β and CDR3β account for the ability of NKTcr to recognise diverse lipid antigens. Using a panel of NKT hybridomas expressing identical Vα14 α-chain paired with four commonly used β-chains (Vβ8.2, Vβ14, Vβ7 and Vβ6; Lantz and Bendelac, 1994; Gui et al, 2001), we determined whether TCR β-chain usage influences recognition of diverse glycolipid ligands (Supplementary Figure S1A). Note that Vβ8.2, Vβ14, Vβ7 and Vβ6 have diverse CDR1β, but partly conserved, CDR2β sequences (e.g., Y46 and/or Y48 residues that were critical for interaction with the α1-helix of CD1d; Borg et al, 2007; Scott-Browne et al, 2007; Wun et al, 2008; Mallevaey et al, 2009; Pellicci et al, 2009). Additionally, the NKT hybridomas expressed diverse CDR3β regions in association with the same Vβ-region and, hence, the same CDR1β and CDR2β (Supplementary Table S1). Bone marrow-derived dendritic cells (BMDCs) pulsed with αGalCer, iGb3 or αGalDAG were co-cultivated with the above NKT hybridoma panel and IL-2 secretion was assessed as a measure of activation. The data showed that the Vβ8.2+, Vβ14+ and Vβ7+ hybridomas recognised αGalCer whereas the Vβ6+ hybridoma did not (Figure 1A), despite the fact that these hybridomas showed comparable response to antigen-independent stimulation through CD3ε (Figure 1D). These data are consistent with previous reports, which demonstrated that several NKTcr β-chains are permissive to αGalCer recognition (Mallevaey et al, 2009). The Vβ8.2Jβ2.6+ hybridoma was autoreactive (i.e., in the absence of exogenously added lipid) to BMDCs and was included as a positive control for the assay (Figure 1C). Figure 1.TCRβ usage impacts Vα14 TCR antigen recognition. (A) BMDCs pulsed with 0.1–100 ng/ml αGalCer overnight were co-cultivated with a panel of NKT hybridomas expressing Vα14 α-chain paired with unique TCR Vβ-chains (Supplementary Table S1). After 24 h, ELISA measured IL-2 secreted into the culture medium in response to NKT hybridoma activation. The data are represented as the half-maximal response (top panel) or half-maximal stimulatory concentration (EC50; bottom panel) of αGalCer. (B, C) BMDCs pulsed with 10 μg/ml iGb3 (B) or 20 μg/ml αGalDAG (C) overnight were co-cultivated with the hybridoma panel and activation was measured by ELISA after 24 h stimulation. (D) NKT hybridomas expressing different β-chains were stimulated with 0.1–27 μg (in three-fold dilution) plate-bound anti-CD3ε mAb. After ∼24 h, IL-2 ELISA was performed on culture supernatants to determine the sensitivity of each hybridoma to direct TCR stimulation. The data are represented as the EC50 of anti-CD3ε mAb. Data are representative of duplicate (B–D) or triplicate (A) experiments, each performed in triplicate. Download figure Download PowerPoint Compared with the αGalCer recognition pattern, the recognition of iGb3 and αGalDAG by this hybridoma panel was clearly distinct (Figure 1). With the exception of the Vβ8.2Jβ2.2+ hybridoma, most Vβ8.2+ NKT hybridomas recognised iGb3 (Figure 1B). While the Vβ7+ hybridoma also recognised iGb3, the Vβ14+ and Vβ6+ hybridomas did not (Figure 1B). Note that a titration experiment showed that the hybridoma panel required a minimum of 10 μg of iGb3 and 20 μg of αGalDAG for recognition (data not shown). Therefore, because micelle formation and bioavailability influence lipid antigen presentation at higher concentrations, the functional avidity of the different Vβ-expressing NKTcrs for iGb3 and αGalDAG could not be determined. As the aforementioned experiment used phytosphingosine-containing iGb3 (Supplementary Figure S1A), we determined whether the chemical make-up of the LCB influenced the recognition pattern. iGb3 made up of sphingosine-, sphinganine (4,5-dihydro)- and 4,5-dibromosphinganine-containing LCB were recognised just as well as phytosphingosine-based iGb3 (data not shown), suggesting that the chemical composition of the sphingoid backbone had very little influence on iGb3 recognition. Finally, all Vβ8.2+, the Vβ7+ and only the Vβ14Jβ1.2i+ hybridomas recognised αGalDAG (Figure 1C), whereas the remaining three Vβ14+ and the Vβ6+ hybridomas were unresponsive to this antigen (Figure 1C). The varied antigen-recognition pattern could have resulted from variation in TCR expression and differences in the activation threshold of the different NKT hybridomas. Therefore, we determined the IL-2 response of each NKT hybridoma to antigen-independent stimulation using anti-CD3ε monoclonal antibody (mAb). All hybridomas responded similarly to this stimulation (Figure 1D). This result suggests that the recognition pattern of NKT hybridomas was intrinsic to the NKTcr and not due to variation in TCR expression levels or activation threshold. Taken together, the above data suggest that CDR3β together with CDR1β and CDR2β dictate NKTcr's ability to recognise structurally diverse ligands. Alternatively, although less likely, other cell-surface molecules differentially expressed by the hybridomas could result in the observed differences in responses. Vβ usage impacts the recognition of 3′ and 4′ αGalCer analogues To gain insight into how the NKTcr recognises αGalCer, we used αGalCer analogues, which contain modifications at the 3′- and 4′-hydroxyls of galactose (Supplementary Figure S1B). Note that the αGalCer analogues contained C8 acyl chain, which does not alter the conformation of CD1d upon binding (Zajonc et al, 2005; Pellicci et al, 2009). The 3′- and 4′-hydroxyls of αGalCer interact with NKTcr's CDR1α and/or CDR3α residues (Supplementary Figure S2) (Pellicci et al, 2009) yet their deoxy forms were reported not to affect recognition (Wun et al, 2008). Therefore, we determined whether chemical modifications at the 3′ (-amino, -azido and -N-acetyl) and 4′ (-O-methyl, -O-ethanol and -N-acetyl) of galactose are recognised by NKTcr, and whether Vβ usage influences this recognition using the approach described above. Replacement of the 3′-hydroxyl with an -amino, -azido or -N-acetyl group prevented recognition by the NKT hybridoma panel regardless of the NKTcr's β-chain composition (Figure 2A). Therefore, although the 3′-hydroxyl itself is dispensable (Wun et al, 2008), chemical substitutions at this position are detrimental for NKTcr recognition. Figure 2.NKTcrs recognise 4′-hydroxy variants in a distinct manner. (A) BMDCs pulsed with 500 ng/ml of 3′-hydroxy variants (Supplementary Figure S1B) overnight were co-cultivated with the same panel of NKT hybridomas described in Figure 1. After 24 h, ELISA measured IL-2 secreted into the culture medium in response to NKT hybridoma activation. (B) BMDCs were pulsed with the indicated concentrations of αGalCer or its 4′-hydroxy variants and used to stimulate NKT hybridomas. Top row: Vβ8.2Jβ1.1 (circles); Vβ8.2Jβ2.1 (diamonds); Vβ8.2Jβ2.5 (triangles) and Vβ8.2Jβ2.6 (squares); middle row: Vβ14Jβ1.2i (circles); Vβ14Jβ1.2ii (diamonds); Vβ14Jβ2.5 (triangles) and Vβ14Jβ2.6 (squares); bottom row: Vβ7 (diamonds) and Vβ6 (squares). ELISA measured IL-2 secreted into the culture medium in response to NKT hybridoma activation. (C) Schematic rendition of NKTcr recognition pattern. Amino-acid sequence of CDR1β, CDR2β and CDR3β (upper case) of each Vα14 TCR is indicated on the left; lowercase indicates residues flanking each CDR. Vβ sequences were obtained from IMGT (http://www.imgt.org/textes/IMGTrepertoire/). Data in panels A and B are representative of two independent experiments performed in triplicate. Download figure Download PowerPoint On the other hand, the recognition of the 4′ α-GalCer analogues was quite varied. All Vβ8.2+ hybridomas recognised these αGalCer variants, but to different degrees, indicating that CDR3β sequences might influence this recognition (Figure 2B, top row panel 1). The Vβ8.2+ hybridomas recognised 4′-O-methyl and 4′-O-ethanol αGalCer analogues to a similar extent (Figure 2B, top row panels 2 and 3), whereas the 4′-N-acetyl variant was less antigenic when compared with the recognition of αGalCer (Figure 2B, top row panel 4). All Vβ14+ NKT hybridomas recognised αGalCer but not to the same extent as the Vβ8.2+ hybridomas (Figure 2B, middle row panel 1). Recognition of the 4′ analogues by Vβ14+ NKT hybridomas was Jβ-dependent, such that only the Vβ14Jβ2.6+ hybridoma recognised both 4′-O-methyl and 4′-O-ethanol analogues, whereas the Vβ14Jβ1.2i+ hybridoma recognised only the 4′-O-ethanol variant (Figure 2B, middle row panels 2 and 3). The 4′-N-acetyl variant was not recognised by any of the Vβ14+ hybridomas (Figure 2B, middle row panel 4). Furthermore, the Vβ7+ NKT hybridoma recognised αGalCer and each of the 4′ variants to similar extent, whereas the Vβ6+ hybridoma failed to respond to any of these lipid antigens (Figure 2B, bottom row). These data suggest that the 4′-N-acetyl variant is a less potent antigen than the 4′-O-methyl and -O-ethanol analogues, thereby revealing an agonistic hierarchy in NKTcr ligands: αGalCer>4′-O-methyl∼4′-O-ethanol≫4′-N-acetyl. As the 4′-N-acetyl analogue is not recognised by Vβ14+ hybridomas (Figure 3B), we conclude that the recognition of weak NKTcr ligands is influenced not only by CDR3β but also by CDR1β and CDR2β loops. Figure 3.NKTcr interfaces CD1d–iGb3 with a distinct recognition logic. (A) BMDCs pulsed with 10 μg/ml iGb3, 2″′-deoxy-iGb3, 3″′-deoxy-iGb3, 4″′-deoxy-iGb3 or 6″′-deoxy-iGb3 were used to stimulate a panel of NKT hybridomas described in Figure 1. After 24 h, ELISA measured IL-2 secreted into the culture medium in response to NKT hybridoma activation. Data are representative of two independent experiments performed in triplicate. (B) Schematic rendition of NKTcr recognition pattern. Amino-acid sequence of CDR1β, CDR2β and CDR3β (upper case) of each Vα14 TCR is indicated on the left; lowercase indicates residues flanking each CDR (for sequences see http://www.imgt.org/textes/IMGTrepertoire/). Download figure Download PowerPoint Vβ usage also impacts iGb3 recognition Low affinity of the CD1d–iGb3/NKTcr interaction has complicated structural, biochemical and functional studies of this ligand–receptor complex (Zhou et al, 2004; Zajonc et al, 2008). We approached the structural analyses of this complex using a combination of iGb3 variants and the aforementioned hybridoma panel expressing Vα14 α-chain paired with Vβ8.2, Vβ14, Vβ7 or Vβ6 β-chain. For this, 2″′, 3″′, 4″′ or 6″′ deoxy variants of the terminal galactose were incorporated into the phytosphingosine-based ceramide backbone during syntheses (Supplementary Figure S1C). As described above, we found that only Vβ7+ and Vβ8.2+, with the exception of Vβ8.2Jβ2.2+, NKT hybridomas recognised iGb3 (Figure 1A), a pattern similar to 4′-N-acetyl-αGalCer recognition (Figure 2B). This recognition required the 2″′-hydroxyl group within the terminal galactose by most (Vβ8.2Jβ2.5, Vβ8.2Jβ2.2, Vβ8.2Jβ2.4 and Vβ8.2Jβ2.2), and the 3″′-hydroxyl by some (Vβ8.2Jβ2.5 and Vβ8.2Jβ2.2 only), Vβ8.2 NKT hybridomas (Figure 3). None of the Vβ14+ hybridomas recognised iGb3 or its deoxy variants (Figure 3). Therefore, despite their obvious structural differences, the three CDRβ loops impact iGb3 recognition in a manner similar to 4′-N-acetyl αGalCer recognition. CDR1β and CDR3β residues, in addition to previously identified 'hotspots' influence
Referência(s)