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αβ T cell antigen receptor recognition of CD1a presenting self lipid ligands

Nature Immunology volume 16, pages 258–266 (2015)Cite this article

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Abstract

A central paradigm in αβ T cell–mediated immunity is the simultaneous co-recognition of antigens and antigen-presenting molecules by the αβ T cell antigen receptor (TCR). CD1a presents a broad repertoire of lipid-based antigens. We found that a prototypical autoreactive TCR bound CD1a when it was presenting a series of permissive endogenous ligands, while other lipid ligands were nonpermissive to TCR binding. The structures of two TCR-CD1a-lipid complexes showed that the TCR docked over the A′ roof of CD1a in a manner that precluded direct contact with permissive ligands. Nonpermissive ligands indirectly inhibited TCR binding by disrupting the TCR-CD1a contact zone. The exclusive recognition of CD1a by the TCR represents a previously unknown mechanism whereby αβ T cells indirectly sense self antigens that are bound to an antigen-presenting molecule.

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Figure 1: Analysis of BK6 TCR–CD1a–lipid complexes.
Figure 2: Identification of lipid ligands eluted from CD1a.
Figure 3: Lipids presented by CD1a modulate interactions with the BK6 TCR.
Figure 4: The BK6 TCR binds to CD1a independently of direct antigen contact.
Figure 5: Molecular details of the BK6 TCR–CD1a interface.
Figure 6: Mutational analysis defining key interactions between the BK6 TCR and CD1a.
Figure 7: Comparison of CD1a-antigen structures from binary and ternary complexes.

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Acknowledgements

We thank L. Tan, M. Bhati, J. Waddington, B. O'Sullivan, M. Ciula and staff at the Australian synchrotron and the Monash macromolecular crystallization facility for technical assistance; P. Savage (Brigham Young University) for α-galactosylceramide; S.A. Porcelli (Albert Einstein College of Medicine) for the BK6 clone; and J. Altman for the design of CD1a protein expression constructs. Supported by NIAID (R01 AR048632 and AI049313 to D.B.M.), the National Health and Medical Research Council of Australia (1013667; Early Career Fellowship to D.G.P.; Senior Principal Research Fellowship 1020770 to D.I.G.; and NHMRC Australia Fellowship AF50 to J.R.), the Australian Research Council (CE140100011 and LE110100106; Future Fellowships to S.G. and A.P.U.) and the Dermatology Foundation (A.d.J.).

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Author notes

  1. Richard W Birkinshaw and Daniel G Pellicci: These authors contributed equally to this work.

  2. D Branch Moody, Dale I Godfrey and Jamie Rossjohn: These authors jointly directed this work.

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, Australia

    Richard W Birkinshaw, Andrew N Keller, Maria Sandoval-Romero, Stephanie Gras & Jamie Rossjohn

  2. ARC Centre of Excellence in Advanced Molecular Imaging, Monash University, Clayton, Australia

    Richard W Birkinshaw, Andrew N Keller, Stephanie Gras & Jamie Rossjohn

  3. Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, University of Melbourne, Parkville, Australia

    Daniel G Pellicci, Adam P Uldrich & Dale I Godfrey

  4. ARC Centre of Excellence in Advanced Molecular Imaging, University of Melbourne, Parkville, Australia

    Daniel G Pellicci, Adam P Uldrich & Dale I Godfrey

  5. Brigham and Women's Hospital Division of Rheumatology, Immunology and Allergy and Harvard Medical School, Boston, Massachusetts, USA

    Tan-Yun Cheng & D Branch Moody

  6. Department of Dermatology, Columbia University, New York, New York, USA

    Annemieke de Jong

  7. Institute of Infection and Immunity, Cardiff University, School of Medicine, Heath Park, Cardiff, UK

    Jamie Rossjohn

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Contributions

R.W.B. and D.G.P. performed experiments, provided intellectual input and analyzed data; T.-Y.C., A.N.K., M.S.-R., S.G., A.d.J. & A.P.U. performed experiments, analyzed data, and/or provided intellectual input; and D.B.M., D.I.G. and J.R. led the investigation, devised the project and wrote the manuscript together.

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Correspondence to D Branch Moody, Dale I Godfrey or Jamie Rossjohn.

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Integrated supplementary information

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 indicates a protein molecular weight standard ladder.

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.

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.

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.

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.

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.

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Birkinshaw, R., Pellicci, D., Cheng, TY. et al. αβ T cell antigen receptor recognition of CD1a presenting self lipid ligands. Nat Immunol 16, 258–266 (2015). https://doi.org/10.1038/ni.3098

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