Research ArticleAUTOREACTIVITY

A molecular basis of human T cell receptor autoreactivity toward self-phospholipids

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Science Immunology  20 Oct 2017:
Vol. 2, Issue 16, eaao1384
DOI: 10.1126/sciimmunol.aao1384
  • Fig. 1 TCRs bind to CD1b-PG complexes formed in cells.

    (A) Cellular phospholipids share a PA core structure. PG, PC, PE, PS, and PI are distinguished by head groups above the dotted line. (B) C1R cells transfected with CD1a or CD1b were pulsed with the indicated lipid and stained with PG90 or GEM42 TCR tetramers. Gating strategy is shown in fig. S2. (C) Total cellular lipids were extracted from human monocyte-derived DCs, C1R cells, and K562 cells, separated using normal-phase HPLC, and detected as ion chromatograms that match the mass values for PG (m/z 775.5 and 747.5), which were previously established from TLC-MS. (D) MS signals for the abundant species of PG, PE, and PC. Experiments in (B) and (C) were performed at least twice, with similar results. Measurements in (D) were performed once in each of the three cell types.

  • Fig. 2 CD1b lipid complexes formed in cells.

    (A) CD1b protein complexes containing endogenous lipids (CD1b-endo) were extracted and analyzed using negative mode shotgun nanoelectrospray ionization ion trapping MS (nanospray). (B) Proteins prepared as in (A) were also analyzed by normal-phase HPLC-MS using QToF detection (HPLC-MS) measuring ions nearly coeluting with a PG standard at ~11-min retention time. The bottom panel shows signals magnified by 200-fold. (C) The identity of the compound as PG was established on the basis of its mass (m/z 747.511) and collision-induced dissociation MS. (D) Absolute lipid concentrations of cellular (CD1b-endo) and PG-treated proteins (CD1b-PG) were determined using authentic external standards (fig. S5).

  • Fig. 3 Steady-state affinity measurements of TCRs against CD1b-presenting phospholipids.

    SPR steady-state affinity measurements for the (A) PG90 and (B) PG10 TCRs, against CD1b presenting a range of phospholipid ligands, including CD1b in complex with endogenous lipids (CD1b-endo), CD1b-PG, CD1b-PC, CD1b-PE, CD1b-PI, CD1b-PA, and CD1b-PS. SPR experiments were conducted as duplicate in two independent experiments; CD1b-endo against PG10 and PG90 TCRs was carried out in three independent experiments. Steady-state KD values (μM), as well as error bars, represent mean ± SEM. SPR sensograms (top) and equilibrium curves (bottom) were prepared in GraphPad Prism 7.0. For equilibrium curves for which n ≤ 2, data points from both independent experiments are presented with no error bars. The (A) PG90 and (B) PG10 TCR CDR amino acid loop sequences and gene usage are included. See table S3 for raw data.

  • Fig. 4 Comparison of ternary TCR-lipid-CD1 complex structures.

    Crystal structures of self-lipid presentation by CD1b (PG90 TCR-CD1b-PG) (A, D, and G) and CD1a [BK6 TCR–CD1a–lysophosphatidylcholine (LPC)] (B, E, and H) (29) and foreign antigen presentation by CD1b (GEM42 TCR-CD1b-GMM) (C, F, and I) (16). Blue and purple spheres, representing the center of mass of TCR α and β, respectively (D to F), footprints (red, bottom panels), and extent of antigen interaction (cyan), with antigen atoms not involved in the interaction in black (G to I). Ternary complexes (A to C) are shown in ribbon diagrams and bound lipid antigens as spheres. Blue, scaffold lipids; dark green, PG; orange, LPC; yellow, GMM.

  • Fig. 5 Docking footprint of PG90 TCR on CD1b-PG.

    (A) Footprint of PG90 CDR loops on CD1b. CD1b is represented as a white surface, with PG represented as spheres. Points of contact with TCR CDRs shown in the indicated color. Contacts between (B) CDR1α (teal), (C) CDR2α (green), (D) CDR3α (purple), (E) CDR1β (red) and CDR2β (orange), and (F) CDR3β (yellow) and CD1b are highlighted. Hydrogen bonds and salt bridges are represented as red and black dashes, respectively. Amino acid residues are represented as sticks, with the exception of glycine residues, which are represented as spheres. The spheres in (B) and (F) represent the Cα atoms of the glycine residues. No side chain is represented for residue Glu68 (*) because of the lack of electron density.

  • Fig. 6 The functional and structural basis of phospholipid recognition by the PG90 TCR.

    (A) Staining of the PG90 cell line by CD1b tetramers with a single alanine mutant at the indicated site with or without bound PG. Residues interacting with the PG90 TCR are highlighted in blue. MFI, mean fluorescence intensity. (B) Color coding summary: white, not tested; dark gray, no effect (<25% reduction); orange, moderate effect (25 to 75% reduction); red, markedly reduced binding (>75% reduction). (C) Electrostatic potential of the cationic cup. The potential contours are shown on a scale from +10.0 (positive charge, blue) to −10.0 kBT e−1 (negative charge, red); white indicates a value close to 0 kBT e−1 (neutral charge). (D) Side view of hydrogen bond network between PG (green) and PG90 TCR, with CDR1α, CDR3α, and CDR3β loops represented in teal, purple, and yellow, respectively. Contacts with the head group of PG (E) and GMM (F). The interactions are represented as red dashes, whereas the cyan dash represents the hydrogen bond (E), and salt bridge (F), that forms the arginine capstone.

  • Fig. 7 CD1b presentation of phospholipids and movement of PG upon PG90 TCR docking.

    (A) Top view shows superposition of CD1b-PG (green), CD1b-PA (dark blue), and CD1b-PS (pink), with scaffold lipids colored black. Oxygen, nitrogen, and phosphate groups are colored red, blue, and orange, respectively. The CD1b antigen-binding cleft is represented as a light blue transparent surface. (B) Overlay of the separately solved structures of CD1b-PG (light green) and the PG90 TCR (light purple and yellow) with the ternary CD1b-PG-PG90 TCR (dark green, dark purple, and dark yellow) structure. (C) CD1b is represented as a cartoon in white, and PG and scaffold lipids are represented as sticks in blue (CD1b-PG) and green (PG90 TCR-CD1b-PG). Hydrophobic interactions between Tyr169 and Phe49, and PG and scaffold lipids are represented as blue (CD1-PG) and green (PG90 TCR-CD1b-PG) dashes. Direction of movement is indicated by a black arrow.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/2/16/eaao1384/DC1

    Fig. S1. TCR tetramer validation.

    Fig. S2. Representative gating strategy for Fig. 1B.

    Fig. S3. Major lipid classes produced by C1R and K562 cells.

    Fig. S4. TLC-MS analysis of C1R lipids nearly comigrating with a PG standard.

    Fig. S5. Quantification of PG in cells or eluted from CD1b.

    Fig. S6. Nanoelectrospray analysis of protein eluates.

    Fig. S7. Loading of phospholipids in CD1b molecule.

    Fig. S8. Electron density maps for the phospholipid antigens presented by CD1b.

    Fig. S9. Phospholipid anchoring residues within CD1b pockets.

    Fig. S10. Sequence alignments of TCR Vα and Vβ regions.

    Table S1. Data collection and refinement statistics.

    Table S2. Contacts between PG90 TCR and CD1b-PG.

    Table S3. Raw data for Fig. 3A.

  • Supplementary Materials

    Supplementary Material for:

    A molecular basis of human T cell receptor autoreactivity toward self-phospholipids

    Adam Shahine, Ildiko Van Rhijn, Tan-Yun Cheng, Sarah Iwany, Stephanie Gras,* D. Branch Moody,* Jamie Rossjohn*

    *Corresponding authors. Email: stephanie.gras{at}monash.edu (S.G.); bmoody{at}partners.org (D.B.M.); jamie.rossjohn{at}monash.edu (J.R.)

    Published 20 October 2017, Sci. Immunol. 2, eaao1384 (2017)
    DOI: 10.1126/sciimmunol.aao1384

    This PDF file includes:

    • Fig. S1. TCR tetramer validation.
    • Fig. S2. Representative gating strategy for Fig. 1B.
    • Fig. S3. Major lipid classes produced by C1R and K562 cells.
    • Fig. S4. TLC-MS analysis of C1R lipids nearly comigrating with a PG standard.
    • Fig. S5. Quantification of PG in cells or eluted from CD1b.
    • Fig. S6. Nanoelectrospray analysis of protein eluates.
    • Fig. S7. Loading of phospholipids in CD1b molecule.
    • Fig. S8. Electron density maps for the phospholipid antigens presented by CD1b.
    • Fig. S9. Phospholipid anchoring residues within CD1b pockets.
    • Fig. S10. Sequence alignments of TCR Vα and Vβ regions.
    • Table S1. Data collection and refinement statistics.
    • Table S2. Contacts between PG90 TCR and CD1b-PG.

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    Other Supplementary Material for this manuscript includes the following:

    • Table S3 (Microsoft Excel format). Raw data for Fig. 3A.

    Files in this Data Supplement:

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