Extended Data Fig. 4: Bispecific properties of VHH1D12 stabilize the CD1d-type I NKT-TCR interface.
a, Enlarged view of VHH1D12 CDR1 (green), CDR3 (blue), and CDR1 and CDR3 loop interactions with CD1d(α-GalCer). Residues involved in contacts are represented as sticks, with hydrogen bonds represented as red dashed lines. Nitrogen, oxygen, and sulphate are coloured in blue, red, and yellow respectively. VHH1D12 residues are labelled in purple, CD1d and β2m residues in black. b, Sensorgrams and saturation plot of concentrations series for VHH1D12 passed over CD1d(endogenous). Sensorgram data are representative of 2 independent experiments performed in duplicate, saturation plot indicates data points from both independent experiments.. RU, response unit, KD, equilibrium dissociation constant, Ka, association rate constant, Kd, dissociation rate constant. c, Footprint of NKT12 TCR CDR regions on CD1d(α-GalCer) molecular surface and enlarged view of CDR2β (orange), CDR3β (yellow), CDR1α (teal), CDR2α (green) and CDR3α (purple) loop interactions with CD1d(α-GalCer) (grey). Residues and bonds are represented as above. d, Expression (%) of CD107a on type I NKT cells after 4 h co-culture with MM.1S.mCherry/luc.CD1d cells plus 10 nM VHH1D12 wild type (WT) or indicated alanine substitution mutants (n = 4 independent experiments), and e, histogram depicting binding of biotinylated VHH1D12 and alanine substitution mutants to C1R.GFP.CD1d, analysed by flow cytometry. f, Histogram depicting binding of lipid loaded CD1d-tetramer, whether or not pre-incubated with VHH1D12 (red histogram) or VHH1D12 R96A (blue histogram) (~5 times molar excess), to type I NKT cells. Data are representative of 3 separate experiments with type I NKT cells obtained from individual donors. g,h, Sensorgrams and saturation plots of concentrations series for NKT12 TCR passed over VHH1D12 R45A-CD1d(α-GalCer) (g) or VHH1D12 R96A (i). Sensorgram data are representative of 2 independent experiments performed in duplicate, saturation plot indicates data points from both independent experiments. d, The bars indicate mean +s.d.
