Nature Immunology: doi:10.1038/ni.3098

Supplementary Figure 1

BK6 TCR variable sequence and SDS-PAGE from size-exclusion chromatography formation of complexes of the BK6 TCR and CD1a-lipid.

(a) Protein sequence for the BK6 TCR highlighting the CDR from germline encoded regions and recombination. (b) A non-reducing SDS-PAGE gel indicating bands associated with the BK6 TCR alone, CD1a-endo alone and fractions from the SEC chromatogram displayed in Fig. 1b. Elution volumes are indicated for the start of the fraction, collection the subsequent 1 ml elution volume and STD

Nature Immunology: doi:10.1038/ni.3098

indicates a protein molecular weight standard ladder.

Nature Immunology: doi:10.1038/ni.3098

Supplementary Figure 2

Mass spectrometry analysis of CD1a monomers and CD1a loaded with PE-rhodamine.

(a) Normal phase (HPLC-QToF-MS) from CD1a monomers that did not complex with the BK6 TCR using size exclusion chromatography. (b, c) CD1a proteins loaded with endogenous lipids (CD1a-endo) were or were not treated with rhodamine labeled phosphatidylethanolamine(PE-rhodamine) followed by elution with organic solvents as in Fig. 1 as well as negative mode nanospray electrospray ionization mass spectrometry (b) or HPLC-QTof-MS (c). Lipids were identified as phosphatidylinositol (PI), phosphatidylcholine (PC) or sphingomyelin (SM) based on CID-MS and comparison to authentic standards as shown in Fig. 1.

Nature Immunology: doi:10.1038/ni.3098

Supplementary Figure 3

Electron density maps for the lipids in the CD1a ternary and CD1a binary structures.

(a-d) Representation of the CD1a lipid binding cleft showing σ-weighted (a-d) 2Fo-Fc refined density peaks above an r.m.s.d. of 0.8 in blue and (e-h) Fo-Fc simulated annealing omit maps contoured at an r.m.s.d. of 2.5 in green around the (a, e) oleic acid (OLA), (b, f)LPC from the BK6 TCR-CD1a ternary complexes or (c, g) sphingomyelin (SM), (d, h)LPC) CD1a binary complexes. CD1a is shown in white in ribbon representation and lipids in stick format colored according to atom type with cyan carbons for LPC, salmon pink carbons for oleic acid and white carbons for sphingomyelin.

Nature Immunology: doi:10.1038/ni.3098

Supplementary Figure 4

Fatty acids eluted from BK6 TCR–CD1a–endo complexes.

Fatty acyl chain composition revealed by HPLC-QToF-MS from (a) purified BK6 TCR-CD1a-endo ternary crystals, (b) BK6 TCR-CD1a-endo complexes from size exclusion chromatography.

Nature Immunology: doi:10.1038/ni.3098

Supplementary Figure 5

CD1a-sulfatide tetramer staining of Jurkat.BK6 cells.

BK6.Jurkat cells or control MR1 restricted Jurkat cells were labeled with CD1a-endo tetramer or tetramer loaded with sulfatide at a 1:6 protein:lipid molar ratio. Cells were gated for similar levels of GFP expression and Mean Fluorescence Intensity (MFI) of CD1a tetramer is shown in the upper right corner. FACS plots are representative of 3 independent experiments.

Nature Immunology: doi:10.1038/ni.3098

Supplementary Figure 6

Collision-induced dissociation (CID-MS) analysis of lipids eluted from BK6 TCR–CD1a–endo complexes.

Negative mode nanospray ESI-MS (a) of lipid eluting from CD1a-TCR complexes using methods shown in Fig. 1. After lipids were tentatively identified as PG (b), PI (c), PC (d) and SM (e) based on mass, CID-MS of each molecule confirmed the expected fragments corresponding to those presented in Fig. 2c.

Nature Immunology: doi:10.1038/ni.3098

Supplementary Table 1 BK6 TCR-CD1a-LPC contacts.

CDR TCR CD1a1 Bond CDR1α Gln37α Asn160 VDW Tyr38α Asn160 VDW Tyr38αOη Asn160Oδ H-bond CDR2α Tyr57α Asp156, His159, Asn160 VDW Tyr57αOη His159Nδ1 H-bond Tyr57αOη Asn160N H-bond CDR2α framework

Tyr55α His153 VDW

CDR3α Ser109αOγ Asn160Nδ, Asp164Oδ2 H-bond Ser109α Ile157, Asn160, Asp164, Arg168 VDW Leu110αN Asn160Oδ H-bond Leu110αO Thr165Oγ H-bond Leu110α Ile157, Asn160, Thr165, Arg168 VDW Pro111α Phe58, Glu62, Glu65, Arg168 VDW Asn112αN Glu65Oε2 H-bond Asn112α Glu65 VDW Ala113αN Glu65Oε1 H-bond Ala113α Glu65 VDW CDR3β Phe109β Glu65, Thr68, Leu69 VDW Leu110βO Arg76Nη2 H-Bond Leu110β Leu69, Arg73, Arg76, Ile157 VDW Thr111βO Asn151Nδ H-Bond Thr111β Arg76, Asn151, His153 VDW Gln112β Asn151, His153 VDW Gly113β His153 VDW

• Atomic contacts determined using the CCP4i implementation of CONTACT and a cutoff of 4Å.

• Van der Waals interactions defined as non-hydrogen bond contact distances of 4Å or less.

• Hydrogen bond interactions are defined as contact distances of 3.3Å or less. • Salt bridge interactions are defined as contact distances of 4.5Å or less.

Nature Immunology: doi:10.1038/ni.3098

Supplementary Table 2 Alignment of CDR3α and CDR3β regions of BK6 with those of two additional CD1a-autoreactive T-cell clones.

T-cell clones were isolated from peripheral blood from blood bank donors by limiting dilution (see methods), and screened for CD1a-autoreactivity using CD1a-transfected K562 cells. We sequenced the TCR α and β chains of CD1a autoreactive T-cell lines, BC5cl78 and BCTcl81. Depicted are the CDR1, CDR2 and CDR3 regions shown in comparison to BK6, which highlight conserved tyrosine and hydrophobic amino acids. Germline-encoded tyrosines in the α-chain that interact with CD1a in the BK6 structure or have the same sequence number in the newly derived clones are highlighted in yellow, and other residues from BK6 that interact with CD1a are underlined. The amino acids of the CDR3 regions are color-coded based on their physicochemical properties; hydrophobic - aliphatic/unique, hydrophobic - aromatic, neutral - polar side chains, acidic, basic.

Nature Immunology: doi:10.1038/ni.3098