Abstract
The hallmark function of αβ T cell antigen receptors (TCRs) involves the highly specific co-recognition of a major histocompatibility complex molecule and its carried peptide. However, the molecular basis of the interactions of TCRs with the lipid antigen–presenting molecule CD1c is unknown. We identified frequent staining of human T cells with CD1c tetramers across numerous subjects. Whereas TCRs typically show high specificity for antigen, both tetramer binding and autoreactivity occurred with CD1c in complex with numerous, chemically diverse self lipids. Such extreme polyspecificity was attributable to binding of the TCR over the closed surface of CD1c, with the TCR covering the portal where lipids normally protrude. The TCR essentially failed to contact lipids because they were fully seated within CD1c. These data demonstrate the sequestration of lipids within CD1c as a mechanism of autoreactivity and point to small lipid size as a determinant of autoreactive T cell responses.
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Acknowledgements
We thank S. A. Porcelli (Albert Einstein College of Medicine, New York) for the 3C8 T cell clone; the Monash Macromolecular crystallization Facility staff for assistance with crystallization and the staff at the Australian synchrotron for assistance with data collection; D. Ly for advice on tetramer staining patterns; and L. L. Tan, H. Halim and N. Williams for technical assistance. Supported by the US National Institutes of Health (R01 AR048632, R01 AI049313 and AI U19111224), the Australian Research Council (Laureate Fellowship to J.R.), the National Health and Medical Research Council (Early Career Fellowship to K.S.W.) and the Wellcome Trust (Senior Investigator Award to D.A.P.).
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K.S.W. undertook research, including the structural biology and surface plasmon resonance experiments, produced CD1c-lipid and TCR-CD1c-lipid complexes and analyzed data; J.F.R. produced CD1c-lipid complexes and isolated and analyzed T cell clones; T.-Y.C. eluted lipids for identification by MS; J.F.R., K.L., A.P.U., K.L.M., J.E.M., S.S., S.I., J.D.A., J.J.M. and I.V.R. generated tetramers or conducted tetramer staining experiments; K.L., J.J., S.R.L. and M.M. recruited and carried out clinical studies of healthy donors; J.L.N., E.J.G., O.L.H., T.S.W., R.C., K.L.T. and J.D.A. either provided key reagents or analyzed data; J.A.M. provided statistical analysis; K.L., A.W.P., D.I.G., S.G., D.A.P. and I.V.R. provided oversight for experiments and/or contributed to writing the paper; and D.B.M. and J.R. conceived of the project, provided oversight and wrote the paper.
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Supplementary Figure 1 Gating ancestry for tetramer stains in Fig. 1 and Supplementary Fig. 2.
(a) Gating ancestry for the CD1c-endo tetramer stains shown in Figure 1a (CD3+CD14–CD19–) and Supplemental Figure 2 (CD3–CD14–CD19–). (b) Gating ancestry for the CD1-endo and CD1b-endo tetramer stains and graphed data shown in Figure 1b and 1c.
Supplementary Figure 2 CD1c-endo tetramer binding to CD3– cells
CD1c-endo tetramer staining of live CD3–CD14–CD19– cells, pre-gated as shown in Supplementary Figure 1a. The corresponding CD1c-endo tetramer stains for CD3+CD14–CD19– cells are shown in Figure 1a.
Supplementary Figure 3 Sequence alignment of the different human CD1 isoforms
Residues on the CD1c α-helices that interacted with the 3C8 TCR are highlighted. Alanine-substituted residues are highlighted to indicate effects on CD69 expression by J76.3C8 cells: > 50% decrease, red; < 25% decrease, green. Residues that were not substituted are colored purple. * identical residue; : conserved residue;. conserved residue in 3 CD1 isoforms.
Supplementary Figure 4 Collision induced dissociation mass spectrometry of the CD1c ligand
Collision induced dissociation mass spectrometry of the CD1c ligand of m/z 359.316 is identified as monoacylglycerol (MAG) based on fragments corresponding to a single fatty acid (m/z 285.279) and a mass interval corresponding to glycerol. This pattern matches that derived from an authentic MAG standard.
Supplementary Figure 5 Reversed-phase analysis of lipids eluted from CD1c or 3C8 TCR–CD1c in HPLC-TOF-MS
Ions matching the expected retention time and m/z value of monoacylglycerol (MAG), sphingomyelin (SM), or phosphatidylcholine (PC) with a lipid moiety of CX:Y, where X is the total number of carbon atoms in the alkyl chain(s) and Y is the total number of unsaturations.
Supplementary Figure 6 Absolute quantification of lipids detected in association with CD1c or 3C8 TCR–CD1c using HPLC-TOF-MS
Lipids present in the eluents of the indicated CD1c or CD1c-TCR complex were compared to authentic standards of (a) MAG, (b) PC and (c) SM measured under the same conditions and expressed as area under the curve of the ion chromatogram (count-seconds). These values were used to calculate molar ratios of lipid ligands shown in Figure 5 (right). Data are representative of two independent experiments with similar results.
Supplementary Figure 7 Close up view of the CD1c antigen-binding pockets
(a) Superposition of CD1c-SL (cyan) onto the 3C8 TCR-CD1c-MAG (pink) structure showing the decanes and lauric acids occupying a position near the F′- and G′-portals 1. Despite occupying the same A′-pocket, the density modeled as MAG penetrated further into the antigen-binding cleft. (b) Electron density maps of MAG C16:0 and (c) stearic acid modeled into the 3C8 TCR-CD1c-endo crystal structure (blue, 2Fo-Fc = 1.0 σ), and (d) PC modeled into the CD1c-endo crystal structure (blue, 2Fo-Fc = 0.7 σ). (e) Close-up view of the interactions between residues in the CD1c binding pocket and MAG. (f) Spacer lipids in the F′-pocket housed within a network of hydrophobic residues. (g) Superposition of the previously solved CD1c-SL1, CD1c-MPM2 and CD1c-PM3 crystal structures onto the 3C8 TCR-CD1c-lipid and CD1c-lipid complexes. The CD1c molecule is displayed as gray surfaces, and residues that participated in forming the F′-roof are highlighted in violet. CD1c α-helices are colored as: CD1c-MAG, pink; CD1c-SL, cyan; CD1c-SM, green; CD1c-MPM, orange; and CD1c-PM, teal. MAG, pink sticks; Decane, purple sticks; Stearic acid, cyan sticks; PC, yellow sticks.
Supplementary Figure 8 Sorting of HD1CD4+ and HD1CD4– cells and treatment of MAG rescues staining with a SM-treated CD1c tetramer
(a) HD1CD4– cells and HD1CD4+ cells were stained with CD1c tetramer-APC or not, two weeks after sorting and stimulation with irradiated feeder cells and anti CD3 antibody. (b) CD1c monomers were first treated overnight with SM, or mock treated. Subsequently, ceramide, MAG, or diacylglycerol (DAG) was added and the CD1c-ligand mixtures were incubated overnight for a second time. The tetramerized monomers were used to stain the cell line HD1CD4+ (top), or HD1CD4– (bottom). Data are representative of 2 independent experiments with similar results.
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Wun, K.S., Reijneveld, J.F., Cheng, TY. et al. T cell autoreactivity directed toward CD1c itself rather than toward carried self lipids. Nat Immunol 19, 397–406 (2018). https://doi.org/10.1038/s41590-018-0065-7
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