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Butyrophilin 3A1 binds phosphorylated antigens and stimulates human γδ T cells

Abstract

Human T cells that express a T cell antigen receptor (TCR) containing γ-chain variable region 9 and δ-chain variable region 2 (Vγ9Vδ2) recognize phosphorylated prenyl metabolites as antigens in the presence of antigen-presenting cells but independently of major histocompatibility complex (MHC), the MHC class I–related molecule MR1 and antigen-presenting CD1 molecules. Here we used genetic approaches to identify the molecule that binds and presents phosphorylated antigens. We found that the butyrophilin BTN3A1 bound phosphorylated antigens with low affinity, at a stoichiometry of 1:1, and stimulated mouse T cells with transgenic expression of a human Vγ9Vδ2 TCR. The structures of the BTN3A1 distal domain in complex with host- or microbe-derived phosphorylated antigens had an immunoglobulin-like fold in which the antigens bound in a shallow pocket. Soluble Vγ9Vδ2 TCR interacted specifically with BTN3A1-antigen complexes. Accordingly, BTN3A1 represents an antigen-presenting molecule required for the activation of Vγ9Vδ2 T cells.

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Figure 1: Chromosomal location of the gene(s) encoding the molecule(s) that present phosphorylated antigens.
Figure 2: BTN3A1 is essential for the presentation of phosphorylated antigens to Vγ9Vδ2 T cells.
Figure 3: Recombinant immunoglobulin V-like region of BTN3A1 stimulates γδ-transgenic cells.
Figure 4: Binding of IPP to bacterial BTN3A1.
Figure 5: Structure of BTN3A1 in complex with IPP.
Figure 6: Structure of BTN3A1 in complex with HMBPP.
Figure 7: Specific interaction of the Vγ9Vδ2 TCR with BTN3A1.

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Acknowledgements

We thank H.-J. Gober, M. Kistowska, A. Donda and Y. Uematsu for 'pioneer' experiments; S. Sansano, L. Angman and J.Y. Siew for technical assistance; F. Zolezzi, J. Lum, H.H. Jang, J. Chen and M. Poidinger for microarray data and analysis; P. Cresswell (Yale University) for the 721.174, 721.221 and 721.220 cell lines; K.-H. Grzeschik (Philipps University of Marburg) for the Call A9.1.9.9 and Call A9.1.13.10 cell lines; E. Mellins (Stanford University) for the 5.2.4 cell line; M. Heim (University of Basel) for the HuH7 line; G. Spagnoli (University of Basel) for the BS125-3 cell line and the MART-1-specific, HLA-2-restricted T cell clone; E. Palmer (University of Basel) for OT-II mice; C.-H. Chiang and G. Grüber for assistance with structural studies; and N. McCarthy for revising the manuscript. Supported by Singapore Immunology Network of the Agency for Science, Technology and Research, the Swiss National Foundation (G.D.L.), Oncosuisse (L.M. and G.D.L.), the Wellcome Trust (D.A.P.) and Medical Research Council Australia (J.R.).

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S.V. and A.K. designed studies, did experiments and analyzed data; G.S.W. did protein purification and SPR studies; G.S.R. did mass spectrometry; M.C. and S.E.D. did cellular experiments; T.B., A.T. and N.K.W. generated soluble TCRs and contributed to SPR studies; E.G. generated TCR-encoding cDNA; D.U.S. and K.K.V. did SERS experiments; M.O. revised the manuscript; D.A.P. and J.R. supervised TCR production and SPR studies and revised the manuscript; L.M. generated γδ-transgenic mice and supervised the work; G.D.L. conceived of the experiments and oversaw study design and data analysis; S.V., L.M. and G.D.L. wrote the manuscript; and all authors discussed results and commented on the manuscript.

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Correspondence to Gennaro De Libero.

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Vavassori, S., Kumar, A., Wan, G. et al. Butyrophilin 3A1 binds phosphorylated antigens and stimulates human γδ T cells. Nat Immunol 14, 908–916 (2013). https://doi.org/10.1038/ni.2665

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