Atypical natural killer T-cell receptor recognition of CD1d-lipid antigens

Abstract

Crucial to Natural Killer T (NKT) cell function is the interaction between their T-cell receptor (TCR) and CD1d-antigen complex. However, the diversity of the NKT cell repertoire and the ensuing interactions with CD1d-antigen remain unclear. We describe an atypical population of CD1d-α-galactosylceramide (α-GalCer)-reactive human NKT cells that differ markedly from the prototypical TRAV10-TRAJ18-TRBV25-1(+) type I NKT cell repertoire. These cells express a range of TCR α- and β-chains that show differential recognition of glycolipid antigens. Two atypical NKT TCRs (TRAV21-TRAJ8-TRBV7-8 and TRAV12-3-TRAJ27-TRBV6-5) bind orthogonally over the A'-pocket of CD1d, adopting distinct docking modes that contrast with the docking mode of all type I NKT TCR-CD1d-antigen complexes. Moreover, the interactions with α-GalCer differ between the type I and these atypical NKT TCRs. Accordingly, diverse NKT TCR repertoire usage manifests in varied docking strategies and specificities towards CD1d-α-GalCer and related antigens, thus providing far greater scope for diverse glycolipid antigen recognition.

Figure 1. Identification of CD1d–α-GalCer reactive atypical NKT cells.

(a) Flow cytometry of CD1d–α-GalCer reactive cells enriched and expanded from PBMCs from three healthy human donors. TRDV1⁻ γδTCR⁻ cells were analysed for the expression of TRBV25-1 versus CD1d-endogenous tetramer or CD1d–α-GalCer tetramer (left-hand density plots). TRDV1⁻ γδTCR⁻ CD1d–α-GalCer tetramer⁺ cells were analysed for the expression of TRAV10 (middle density plots). CD1d–α-GalCer tetramer⁺ TRBV25-1⁺ type I cells and CD1d–α-GalCer tetramer⁺ TRBV25-1⁻ cells were analysed for the expression of CD4 and CD8α (right-side density plots). (b) The mean percentage of double negative (DN), CD4⁺ and CD8⁺ cells among CD1d–α-GalCer tetramer⁺ TRBV25⁺ Type I cells (dark grey) and CD1d–α-GalCer tetramer⁺ TRBV25⁻ cells (light grey). Each symbol represents cells from a different donor (n=6). (c) The mean percentage of CD1d–α-GalCer tetramer⁺ TRBV25⁻ cells of total CD1d–α-GalCer reactive NKT cells, from 19 individual donors. Donors that showed no clear population of atypical NKT cells were given an arbitrary value of 0.01%.

Figure 2. Lipid reactivity of atypical TRBV25-1⁻ NKT cell lines.

(a) CD1d tetramer staining of CD1d–α-GalCer-reactive cells enriched and expanded from PBMCs from four healthy human donors. Plots show TRBV25-1 versus CD1d tetramers loaded with α-GalCer (C24:1), ‘endogenous' antigen, α-GlcCer, 3′-deoxy-α-GalCer, 4′-deoxy-α-GalCer or OCH. Data show one of two representative experiments. (b) Histograms depicting human CD1d–lipid antigen tetramer staining (white histograms) of CD3⁺ Jurkat T-cell lines transduced with the 9C1, 9B1, 9B2, 9B3 atypical NKT cell TCRs or with the NKT15 type I NKT cell TCR or an irrelevant pHLA-specific TCR control, overlaid with ‘endogenous' tetramers (grey histograms). Numbers in each histogram represent CD1d–lipid tetramer mean fluorescence intensity. Data are representative of two separate experiments. (c) Graphs depict the mean IFN-γ, IL-2, IL-4 and IL-13 concentrations in culture supernatants of 4–5 × 10³ in vitro-expanded/purified CD1d–α-GalCer tetramer⁺ TRBV25-1⁺ (type I NKT, white bars), CD1d–α-GalCer tetramer⁺ TRBV25-1⁻ (atypical NKT, black bars), CD1d–α-GalCer tetramer⁺ TRDV1⁺ γδTCR⁻ (δ/αβ NKT, grey bars), and CD1d–α-GalCer tetramer⁻ (control T cells, hashed bars), with different lipid Ag (0.5 μg ml⁻¹) in the presence of K562.CD1d APCs or PMA/ionomycin for 24 h. Data are representative of n=5–7 donors, with each symbol depicting a separate donor (each symbol derived from n=1–2 technical replicates). Data are pooled from two independent experiments.

Figure 3. Functional reactivity of atypical TRBV25-1⁻ NKT cell lines to CD1d–lipid Ag.

(a) Histograms depicting CD69 expression on gated Jurkat T-cells lines transduced with the 9C1, 9B1, 9B2, 9B3, NKT15 or control pHLA-specific TCRs, after overnight in vitro co-culture with either WT (CD1d⁻), CD1d-intermediate (CD1d^int) or CD1d-high (CD1d^hi)-expressing C1R APCs (left-hand columns), or (b) with CD1d^int C1R APCs plus graded concentrations of α-GalCer (C_26:0) (right-hand columns). Numbers in each histogram represent CD69 mean fluorescence intensity (MFI). Data in a (three left-hand columns) are representative of two separate experiments; data in b (right-hand columns) are each representative of one experiment.

Figure 4. Affinity of non-canonical TRBV25-1⁻ NKT cell TCRs to CD1d–Ag.

The affinity of TCR-CD1d–Ag interactions were determined by surface plasmon resonance, by measuring the binding of graded concentrations of soluble 9C1 (19–0.038 μM), 9B2 (34–0.067 μM), and a type I NKT cell control (NKT15, 10–0.02 μM), to human CD1d loaded with α-GalCer, α-GlcCer, 3′-deoxy α-GalCer, 4′-deoxy α-GalCer or CD1d-endogenous. Saturation plots for 9C1 (red), 9B2 (blue) and NKT15 (black) versus each respective ligand are shown in the lower panels. K_D, dissociation constant; K_a, association rate; t_1/2, half-life. Results are representative of two similar experiments.

Figure 5. Overview of the docking of atypical NKT TCR ternary complexes.

Ternary complexes of (a) human 9C1 TCR–CD1d–α-GalCer, (b) 9B2 TCR–CD1d–α-GalCer, (c) NKT15 TCR–CD1d–α-GalCer (PDB code 2PO6 (ref. 27) and (d) mouse XV19 TCR–CD1d–sulfatide (PDB code 4EI5 (ref. 36). Top panels depict an overview of each structure, middle panels illustrate the TCRs docking onto CD1d and lower panels show the TCR footprints on the CD1d–Ag molecular surface. The CD1d and β2-microglobulin molecules are coloured in light blue and light brown, respectively. 9C1 TCRα, brown; 9C1 TCRβ, light pink; 9B2 TCRα, light green; 9B2 TCRβ, purple; NKT15 TCRα, green; NKT15 TCRβ, cyan; XV19 TCRα, yellow; XV19 TCRβ, grey. The CDR loops are coloured as follows: CDR1α, aqua; CDR2α, purple; CDR3α, red; CDR1β, green; CDR2β, orange; CDR3β, blue. The α-GalCer and sulfatide are coloured in black and light brown sticks (top panel), or black and light brown spheres (middle and lower panels), respectively. In the middle panels, the centre of mass of the respective TRAV and TRBV variable domains are shown as black spheres. In the bottom panels, the molecular surface of CD1d is coloured in light grey.

Figure 6. Interactions at the CD1d–Ag–TCR interface.

(a) Left panel, 9C1 TCR α-chain interactions with CD1d; middle panel, 9C1 TCR β-chain interactions with CD1d; right panel, 9C1 TCR interactions with α-GalCer; (b) Left panel, 9B2 TCR α-chain interactions; middle panel, 9B2 TCR β-chain interactions with CD1d; right panel, 9B2 TCR interactions with α-GalCer; (c) Left panel, NKT15 TCR α-chain interactions with CD1d; middle panel, NKT15 TCR β-chain interactions with CD1d; right panel, NKT15 TCR interactions with α-GalCer. For clarity, only the hydrogen bonds are shown as black dashed lines and the α1- and α2-helices of CD1d are shown as cartoon representation and coloured in light blue. CDR loops are coloured according to Fig. 5; spheres represent water molecules.

Figure 7. Molecular mimicry between the 9B2 and 9C1 TCR-CD1d–α-GalCer complexes.

Superposition of the 9B2 and 9C1 TCR ternary complexes, coloured in grey and light blue, respectively. The superposition is based on the CD1d molecules of each complex. For clarity, only the CDR1α/CDR3β of 9C1 and the CDR2α/CDR3α 9B2 are shown.