Adaptability of the semi-invariant natural killer T-cell receptor towards structurally diverse CD1d-restricted ligands

Abstract

The semi-invariant natural killer (NK) T-cell receptor (NKTcr) recognises structurally diverse glycolipid antigens presented by the monomorphic CD1d molecule. While the alpha-chain of the NKTcr is invariant, the beta-chain is more diverse, but how this diversity enables the NKTcr to recognise diverse antigens, such as an alpha-linked monosaccharide (alpha-galactosylceramide and alpha-galactosyldiacylglycerol) and the beta-linked trisaccharide (isoglobotriaosylceramide), is unclear. We demonstrate here that NKTcrs, which varied in their beta-chain usage, recognised diverse glycolipid antigens with a similar binding mode on CD1d. Nevertheless, the NKTcrs recognised distinct epitopic sites within these antigens, including alpha-galactosylceramide, the structurally similar alpha-galactosyldiacylglycerol and the very distinct isoglobotriaosylceramide. We also show that the relative roles of the CDR loops within the NKTcr beta-chain varied as a function of the antigen. Thus, while NKTcrs characteristically use a conserved docking mode, the NKTcr beta-chain allows these cells to recognise unique aspects of structurally diverse CD1d-restricted ligands.

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-CD3ɛ 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-CD3ɛ mAb. Data are representative of duplicate (B–D) or triplicate (A) experiments, each performed in triplicate.

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.

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/).

Figure 4

The 4′-N-acetyl αGalCer variant interacts with Vα14 TCR with a unique register. (A) BMDCs pulsed with 500 ng/ml αGalCer, 4′-O-methyl, 4′-O-ethanol or 4′-N-acetyl variants were used to stimulate a panel of NKTcr mutants consisting of a single point mutation within CDR1, CDR2 and CDR3 of both TCR α- and β-chains. The Vα3.2+ NKT hybridoma was used as the negative control. After 24 h, ELISA measured IL-2 secreted into the culture medium in response to NKT hybridoma activation. As the wt hybridoma and the derived mutants were equally sensitive to antigen-independent stimulation (Scott-Browne et al, 2007), the data were normalised to wt IL-2 response to αGalCer-pulsed BMDCs and represented as mean±s.e.m. of three independent experiments performed in triplicate. (B) Schematic rendition of NKTcr recognition pattern.

Figure 5

NKTcr recognises CD1d–iGb3 with a distinct recognition logic. (A) BMDCs were pulsed with iGb3 or its variants (as described in Figure 3A) and used to stimulate a panel of NKTcr mutants, and activation was monitored (as described in Figure 3A). Data were normalised as in Figure 4A and represented as mean±s.e.m. of at least three independent experiments performed in triplicate. (B) Schematic rendition of NKTcr recognition pattern.

Figure 6

The αGalDAG recognition logic is distinct from that of αGalCer or iGb3 recognition by NKTcr. BMDCs pulsed with 20 μg/ml αGalDAG were used to stimulate a panel of NKTcr mutants and activation was measured as described in Figure 4A. Data were normalised as described in Figure 4A and represent the mean±s.e.m. of at least three independent experiments performed in triplicate.

Figure 7

The recognition logic. (A) iGb3 modelled onto the Vα14Vβ8.2/CD1d–αGalCer crystal structure shows clashes with the CDR1α and CDR3α loops. CDR1α (orange); CDR2α (green); CDR3α (blue); CD1d (grey) and iGb3 (yellow). (B) The Vα14Vβ8.2 NKTcr surface showing CDR loops (top panel). Mutations that decreased ligand binding are shown in red (middle panel). The NKTcr surface with αGalCer (left), iGb3 (middle) and αGalDAG (right). Mutations that increased ligand binding are shown in dark blue (bottom panel). The NKTcr surface with 4′-N-acetyl analogue of αGalCer (left) and 2″′-deoxy analogue of iGb3 (right). TCR surface (pale cyan); CDR1α (orange); CDR2α (green); CDR3α (blue); CDR1β (pale green); CDR2β (pink) and CD1d helices (dark grey); CDR3β is not shown.