Atypical Natural Killer T-cell receptor recognition of CD1d-lipid antigens supplementary Information.
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1 Atypical Natural Killer T-cell receptor recognition of CD1d-lipid antigens supplementary Information. Supplementary Figure 1. Phenotypic analysis of TRBV and TRBV CD1d-α-GalCerreactive cells. Four donors with a detectable population of TRBV cells after enrichment and in vitro expansion of CD1d-α-GalCer tetramer + TRDV1 - γδtcr - cells from the blood were analysed for their expression of a panel of cell surface markers. White histograms depict staining for NKG2D, CD158A/B/F/G/H, CD56 and CD161 on TRBV type I NKT cells (left-hand columns) and TRBV cells (right-hand columns), overlayed with isotype controls (gray histograms). Numbers indicate percentage of cells within the indicated gate. Data are representative of two independent experiments for donors 1 and 2, and one experiment for donors 5 and 6. 1
2 Supplementary Figure 2. CD1d tetramer staining of atypical NKT cell lines. Histograms depicting human CD1d-lipid antigen or mouse CD1d-α-GalCer (C 24:1 analogue) tetramer staining (red 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 unloaded tetramers (grey histograms). Numbers in each histogram represent CD1d-lipid tetramer mean fluorescence intensity. Data are representative of one experiment. 2
3 Supplementary Figure 3. Enrichment of Type I, atypical and δ/αβ NKT cells. Density plots show gated subsets of type I (TRBV TRDV1 - ), atypical (TRBV TRDV1 - ) and δ/αβ (TRDV1 + ) NKT cell subsets amongst CD1d-α-GalCer tetramer + CD3 + γδtcr - cells that were enriched using magnetic beads and cell sorting, and subsequently expanded in vitro. Numbers on plots represent the percentage of gated events. Data shown was performed over 2 independent experiments. 3
4 Supplementary Figure 4. Electron density map corresponding to α-galcer. (a) Fo-Fc electron density maps contoured at the 2.5σ level of the lipid antigen α-galcer in (a) the 9C1 TCR-CD1d-α- GalCer ternary complex and (b) the 9B2 TCR-CD1d-α-GalCer ternary complex. (c) Superposition of the 9C1 TCR-CD1d-α-GalCer (magenta), 9B2 TCR-CD1d-α-GalCer (grey), NKT15 TCR-CD1d-α- GalCer (red) and XV19 TCR-CD1d-sulfatide (cyan) ternary complexes. For clarity, only the α1- and α2- helices of CD1d and the center of mass (spheres) of the respective TCRs are shown. (d) Superposition of the unligated 9C1 TCR (in yellow) and the 9C1 TCR in complex with CD1d-α- GalCer (in red). For clarity, only the CDRs are shown. 4
5 Supplementary Table 1. Data collection and refinement statistics 9B2 TCR-CD1d-α- GalCer 9C1 TCR-CD1d-α- GalCer 9C1 TCR Data collection Temperature 100K 100K 100K Resolution limits (Å) ( ) ( ) ( ) Space Group P C2 P2 1 Cell dimensions (Å) a=56.4, b=76.1, c=255.7 α=β=γ=90.00 a=210, b=46.3, c=124.7 α=γ=90.00 β=120 a=43.1, b=73.1, c=65.6, α=γ=90.00 β=94.49 Total N o. observations (9779) (36325) (46559) N o. unique (2732) (3953) (11965) observations Multiplicity 3.8 (3.6) 4.6 (4.6) 4.1 (3.9) Data completeness 94.2 (92.6) 99.5 (97.3) 99.7 (98.8) Wilson B-factors (Å 2 ) I/σ I 7.5 (2.4) 8.2 (1.6) 9.8 (2.1) 1 R p.i.m (%) 8.6 (35.6) 4.5 (30.9) 4 (30.4) Refinement statistics 2 R factor (%) R 3 free (%) Non hydrogen atoms - Protein - Water - Heterogen Ramachandran plot (%) - Most favoured - Allowed Molprobity Score 99 th percentile 96 th percentile 97 th percentile B-factors (Å 2 ) - Average main chain - Average side chain rmsd bonds (Å) rmsd angles ( ) R p.i.m = Σ hkl [1/(N-1)] 1/2 Σ i I hkl, i - <I hkl > / Σ hkl <I hkl > 2 R factor = ( Σ F o - F c ) / ( Σ F o ) - for all data except as indicated in footnote % of data was used for the R free calculation Values in parentheses refer to the highest resolution bin. 5
6 Supplementary Table 2. 9C1 TCR contacts with α-galcer and CD1d. TCR gene TCR residues CD1d residues Bond type CDR1α Tyr31 Trp153, Glu156 VDW CDR1α Ala29 Trp160 VDW CDR2α Ser52 Glu156 VDW CDR2α Ser52-Oγ Glu156-Oε2 HB CDR2α Ser52-Oγ Glu156-Oε1 HB CDR3α Thr109 Trp153, Glu156, Thr157, Trp160 VDW CDR3α Thr109-O Thr157-Oγ1 HB CDR3α Gly110 His68, Ile69, Trp153, Thr157 VDW CDR3α Phe111 Thr65, His68, Trp153 VDW CDR3α Phe111-O Trp153- Nε1 HB CDR3α Gln112 His68, Val72, Trp153 VDW CDR3α Gln112-Oε1 His68-Nε2 HB CDR1β Val30 Arg79 VDW CDR2β Gln57-Oε1 Ser76-Oγ HB CDR2β Gln57 Arg79 VDW CDR2β Asn58-Oδ1 Arg79-Nη1 HB CDR2β Asn58 Arg79 VDW CDR3β Leu111 Trp153 VDW FWβ Leu66 His68, Arg71, Val72, Ser75 VDW FWβ Asp67 His68 VDW TCR gene TCR residues α-galcer atoms Bond type CDR1α Tyr31-Oη O3A Water mediated H-bond CDR3α Gln112 C6A, O6A VDW CDR3α Gln112-Nε2 O6A HB CDR1β Ser31-Oγ O4A Water mediated H-bond CDR2β Gln57 C5M, C1A, O5A, C6A, O6A VDW CDR2β Gln57-Nε2 O6A HB CDR3β Arg109 C3A, O3A, C4A, O4A VDW CDR3β Arg109-O O3A, O4A HB CDR3β Arg109-O O3A Water mediated H-bond CDR3β Leu111 O4A VDW FWβ Tyr55 O6A VDW HB: Hydrogen bond, VDW: Van der Waals, SB: salt bridge. Cut-off at 4 Å for VDW interactions and 3.5 Å for HB. FW, framework. 6
7 Supplementary Table 3. 9B2 TCR contacts with α-galcer and CD1d TCR gene TCR residues CD1d residues Bond type CDR1α Gln31 Trp160 VDW CDR1α Tyr32 Trp153 VDW CDR1α Tyr32 Trp160 VDW CDR1α Tyr32-Oη Thr157-Oγ1, Trp160-Nε1 HB CDR2α Tyr57 Glu156, Trp153 VDW CDR2α Ser58 Glu156 VDW CDR3α Leu110-O Asn62-Oδ1 HB CDR3α Leu110 Thr165, Leu66, Thr65, Trp160 VDW CDR3α Asn111-Nδ2 Gln168-Nε2 VDW, HB CDR3α Ala114 Asn62, Gln61 VDW CDR3α Ala114-O Thr65-Oγ1 HB FWα Tyr55 Trp153 VDW FWα Tyr55-Oη Trp153-Nε1 HB FWα Lys82 Glu156 SB CDR2β Ile61 Glu64 VDW CDR3β Gly109 His68 VDW CDR3β Pro110 His68 VDW CDR3β Phe111 Trp160 VDW CDR3β Phe111 Ile69 VDW TCR gene TCR residues α-galcer atoms Bond type CDR3β Gln112 O6A VDW CDR3β Gln112 C6A VDW HB: Hydrogen bond, VDW: Van der Waals, SB: salt bridge. Cut-off at 4 Å for VDW interactions and 3.5 Å for HB. FW, framework 7
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