A molecular basis for the exquisite CD1d-restricted antigen specificity and functional responses of natural killer T cells

Abstract

Natural killer T (NKT) cells respond to a variety of CD1d-restricted antigens (Ags), although the basis for Ag discrimination by the NKT cell receptor (TCR) is unclear. Here we have described NKT TCR fine specificity against several closely related Ags, termed altered glycolipid ligands (AGLs), which differentially stimulate NKT cells. The structures of five ternary complexes all revealed similar docking. Acyl chain modifications did not affect the interaction, but reduced NKT cell proliferation, indicating an affect on Ag processing or presentation. Conversely, truncation of the phytosphingosine chain caused an induced fit mode of TCR binding that affected TCR affinity. Modifications in the glycosyl head group had a direct impact on the TCR interaction and associated cellular response, with ligand potency reflecting the t(1/2) life of the interaction. Accordingly, we have provided a molecular basis for understanding how modifications in AGLs can result in striking alterations in the cellular response of NKT cells.

Fig. 1. Schematic of α-GalCer and the AGLs

(A) α-GalCer (B) OCH (C) α-GalCer (C20:2) (D) α-GlcCer (C20:2) (E) 3′4″-deoxy-α-GalCer (F) 4′4″-deoxy-α-GalCer (G) 4′-deoxy-α-GalCer (H) 4″-deoxy-α-GalCer (I) α-GalCer (C24) (J) α-GlcCer (C24)

Fig. 2. Analysis of the interaction between Vβ8.2 NKT TCR and the CD1d-analogues as assessed by surface plasmon resonance (SPR)

SPR sensograms showing the interaction between Vβ8.2 NKT TCR and CD1d loaded with (A) α-GalCer (B) OCH (C) C20:2 (D) α-GlcCer (E) 3′4″-deoxy α-GalCer (F) 4′4″-deoxy-α-GalCer (G) 4′-deoxy-α-GalCer (H) α-GalCer (C24) (I) α-GlcCer (C24). NKT TCR were injected over streptavidin immobilised CD1d-Ag and simultaneously over a control cell containing unloaded CD1d. (Top) Sensograms show the binding (response units, RU) of increasing concentrations of TCR to CD1d-Ag following baseline subtraction, displaying data points overlaid with curve fits. (Bottom) Saturation plots demonstrating equilbrium binding of NKT TCR to immobilised CD1d-Ag. The affinities derived by equilibrium analysis (K_Deq) were equivalent to those derived by kinetic analysis.

Fig. 3. NKT cell proliferative response to AGLs

For (A) and (B), CFSE labelled thymocytes enriched for NKT cells were cultured for 3d with glycolipid-pulsed Tcra-J^tm1Tg (Jα18^−/−) splenocytes. (A) A selection of representative flow cytometry profiles showing percent proliferated cells based on decreased CFSE intensity of α-GalCer-CD1d tetramer⁺ NKT cells at each AGL dose. (B) Graph depicts percentage divided cells (mean ± SEM) from 2-6 independent experiments carried out as above. (C) MACS enriched CD1d-α-GalCer tetramer⁺ thymic NKT cells (2×10⁴) were CFSE-labelled and cultured in plates pre-coated with either 10 or 1 μg/ml CD1d-glycolipid complex. After 72h CFSE dilution was measured by flow cytometry. Graphs depict the percentage of divided NKT cells ± SEM of n=3 replicates per group from a single experiment.

Fig. 4. NKT cell cytokine response to AGLs

For (A) and (B), thymocytes enriched for NKT cells were cultured for 8h with Tcra-J^tm1Tg (Jα18^−/−) splenocytes previously pulsed with glycolipid (100ng/ml). GolgiStop (BD Biosciences) was added for the last 4h (A) The percentage IFN-γ⁺ α-GalCer-CD1d tetramer⁺ NKT cells was detected by ICS. Data is representative of 4 similar experiments. (B) Culture supernatants were collected at 8h and cytokine amounts were quantified using CBA (BD Biosciences). Data is taken from one of at least 3 representative experiments, (n=5 replicates), graphs depict mean and SEM. (C) Supernatants from cells stimulated by plate bound CD1d-glycolipid complex, as described in Fig. 3C, were harvested at 72hr and cytokines analysed by CBA. Graphs depict the concentration of cytokines ± SEM of n=3 replicates per group from a single expt.

Fig. 5. Structure of the NKT TCR in complex with different α-GalCer AGLs

Ribbon representation of the NKT TCR-CD1d-Ag structures with the corresponding footprints shown underneath. (A) NKT TCR in complex with CD1d-α-GalCer. (B) NKT TCR-CD1d-OCH. (C) NKT TCR-CD1d-C20:2. (D) NKT TCR-CD1d-α-GlcCer. (E) NKT TCR-CD1d-3′4″-deoxy-α-GalCer. (F) NKT TCR-CD1d-4′4″-deoxy-α-GalCer. Footprints are color-coded based on the CDR loop contributions. TCRα, cyan; TCRβ, green; CDR1α loop, purple; CDR3α loop, yellow; CDR2β loop, orange; CD1d heavy chain, grey; β₂m, dark grey; α-GalCer, magenta; C20:2, pink; α-GlcCer, red; 3′4″-deoxy-α-GalCer, brown; 4′4″-deoxy-α-GalCer, blue; OCH, light blue; Spacer lipid, light green. (G) NKT TCR induced fit of CD1d-OCH within the F′-pocket. Comparison of the binary CD1d-OCH (yellow; spacer lipid, cyan) with the ternary NKT TCR-CD1d-OCH complex(light blue; spacer lipid, light green). α-GalCer is shown in magenta.

Fig. 6. Comparison of the glycsoyl head group NKT TCR interactions

(A) α-GalCer (B) α-GalCer. Additional view of CDR3α loop interactions with CD1d. (C) C20:2 (D) C20:2. Additional view of CDR3α loop interactions with CD1d. (E) OCH. (F) 3′4″-deoxy-α-GalCer (G) 4′4″-deoxy-α-GalCer. (H) α-GlcCer. CDR1α loop, purple; CDR3α loop, yellow; CD1d α-helices, grey; α-GalCer, magenta; C20:2, pink; OCH, light blue; spacer lipid, light green; 3′4″-deoxy-α-GalCer, brown; 4′4″-deoxy-α-GalCer, blue; α-GlcCer, red; site of modification to analogues, cyan. H-bonds shown as dashed black lines.

Fig. 7. Vβ7 usage and the CD1d-AGLs

For (A-C) CFSE or eFluor 670 labelled thymocytes enriched for NKT cells were cultured for 3d with glycolipid-pulsed Tcra-J^tm1Tg (Jα18^−/−) splenocytes and Vβ expression of α-GalCer-CD1d tetramer⁺ NKT cells was analysed by flow cytometry at distinct division cycles. (A) an example of the approach to determine the percentage of undivided, or division 6 cells that were Vβ7⁺ after culture in the presence of glycolipid. (B) percentage of Vβ7 or Vβ8.1-8.2⁺ cells within each division peak for 3 representative glycolipids. (C) Summary of results showing the percentage of Vβ7⁺ NKT cells within the most proliferated cells (from the last major generation) from 2-6 independent experiments. (D) Dose response analysis of NKT cell hybridoma, engineered to express the NKT TCR used in SPR and crystallography studies (see methods), stimulated overnight with BMDCs plus the various AGLs at concentrations from 125 nM to 125 pM. Each data point represents the mean of triplicate IL-2 readings ± SD from a single experiment. BMDCs derived from Cd1d^−/− mice used in the same conditions did not stimulate the hybridoma (data not shown).