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Inhibition of T cell receptor signaling by cholesterol sulfate, a naturally occurring derivative of membrane cholesterol

Nature Immunology volume 17, pages 844–850 (2016)Cite this article

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Abstract

Most adaptive immune responses require the activation of specific T cells through the T cell antigen receptor (TCR)–CD3 complex. Here we show that cholesterol sulfate (CS), a naturally occurring analog of cholesterol, inhibits CD3 ITAM phosphorylation, a crucial first step in T cell activation. In biochemical studies, CS disrupted TCR multimers, apparently by displacing cholesterol, which is known to bind TCRβ. Moreover, CS-deficient mice showed heightened sensitivity to a self-antigen, whereas increasing CS content by intrathymic injection inhibited thymic selection, indicating that this molecule is an intrinsic regulator of thymocyte development. These results reveal a regulatory role for CS in TCR signaling and thymic selection, highlighting the importance of the membrane microenvironment in modulating cell surface receptor activation.

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Figure 1: CS blocks TCR signaling.
Figure 2: CS disrupts TCR nanoclusters.
Figure 3: CS associates with TCR and disrupts cholesterol–TCR interaction.
Figure 4: Developmental regulation of CS abundance in thymocytes.
Figure 5: Increasing CS abundance induces apoptosis in DP thymocytes.
Figure 6: Effects of SULT2B1 deficiency on mature T cells recognizing the HY self-antigen.
Figure 7: Development of HY-specific from SULT2B1-deficient bone marrow.

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References

  1. Davis, M.M. et al. Ligand recognition by αβ T cell receptors. Annu. Rev. Immunol. 16, 523–544 (1998).

    CAS  PubMed  Google Scholar 

  2. Schamel, W.W. & Alarcón, B. Organization of the resting TCR in nanoscale oligomers. Immunol. Rev. 251, 13–20 (2013).

    Article  PubMed  Google Scholar 

  3. Chakraborty, A.K. & Weiss, A. Insights into the initiation of TCR signaling. Nat. Immunol. 15, 798–807 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Malissen, B., Grégoire, C., Malissen, M. & Roncagalli, R. Integrative biology of T cell activation. Nat. Immunol. 15, 790–797 (2014).

    CAS  PubMed  Google Scholar 

  5. Davis, M.M. et al. T cells as a self-referential, sensory organ. Annu. Rev. Immunol. 25, 681–695 (2007).

    CAS  PubMed  Google Scholar 

  6. Huang, J. et al. A single peptide-major histocompatibility complex ligand triggers digital cytokine secretion in CD4+ T cells. Immunity 39, 846–857 (2013).

    CAS  PubMed  Google Scholar 

  7. Schamel, W.W. et al. Coexistence of multivalent and monovalent TCRs explains high sensitivity and wide range of response. J. Exp. Med. 202, 493–503 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Schamel, W.W., Risueño, R.M., Minguet, S., Ortíz, A.R. & Alarcón, B. A conformation- and avidity-based proofreading mechanism for the TCR-CD3 complex. Trends Immunol. 27, 176–182 (2006).

    CAS  PubMed  Google Scholar 

  9. Lillemeier, B.F. et al. TCR and Lat are expressed on separate protein islands on T cell membranes and concatenate during activation. Nat. Immunol. 11, 90–96 (2010).

    CAS  PubMed  Google Scholar 

  10. Lillemeier, B.F., Pfeiffer, J.R., Surviladze, Z., Wilson, B.S. & Davis, M.M. Plasma membrane-associated proteins are clustered into islands attached to the cytoskeleton. Proc. Natl. Acad. Sci. USA 103, 18992–18997 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Molnár, E. et al. Cholesterol and sphingomyelin drive ligand-independent T-cell antigen receptor nanoclustering. J. Biol. Chem. 287, 42664–42674 (2012).

    PubMed  PubMed Central  Google Scholar 

  12. Alarcón, B., Swamy, M., van Santen, H.M. & Schamel, W.W. T-cell antigen-receptor stoichiometry: pre-clustering for sensitivity. EMBO Rep. 7, 490–495 (2006).

    PubMed  PubMed Central  Google Scholar 

  13. Martínez-Martín, N. et al. Cooperativity between T cell receptor complexes revealed by conformational mutants of CD3ɛ. Sci. Signal. 2, ra43 (2009).

    PubMed  Google Scholar 

  14. Huppa, J.B. et al. TCR-peptide-MHC interactions in situ show accelerated kinetics and increased affinity. Nature 463, 963–967 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Sherman, E. et al. Functional nanoscale organization of signaling molecules downstream of the T cell antigen receptor. Immunity 35, 705–720 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Valitutti, S., Müller, S., Cella, M., Padovan, E. & Lanzavecchia, A. Serial triggering of many T-cell receptors by a few peptide-MHC complexes. Nature 375, 148–151 (1995).

    CAS  PubMed  Google Scholar 

  17. Davey, G.M. et al. Preselection thymocytes are more sensitive to T cell receptor stimulation than mature T cells. J. Exp. Med. 188, 1867–1874 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Hogquist, K.A. et al. T cell receptor antagonist peptides induce positive selection. Cell 76, 17–27 (1994).

    CAS  PubMed  Google Scholar 

  19. Pircher, H., Rohrer, U.H., Moskophidis, D., Zinkernagel, R.M. & Hengartner, H. Lower receptor avidity required for thymic clonal deletion than for effector T-cell function. Nature 351, 482–485 (1991).

    CAS  PubMed  Google Scholar 

  20. Kumar, R. et al. Increased sensitivity of antigen-experienced T cells through the enrichment of oligomeric T cell receptor complexes. Immunity 35, 375–387 (2011).

    CAS  PubMed  Google Scholar 

  21. Kaech, S.M., Wherry, E.J. & Ahmed, R. Effector and memory T-cell differentiation: implications for vaccine development. Nat. Rev. Immunol. 2, 251–262 (2002).

    CAS  PubMed  Google Scholar 

  22. Kersh, E.N. et al. TCR signal transduction in antigen-specific memory CD8 T cells. J. Immunol. 170, 5455–5463 (2003).

    CAS  PubMed  Google Scholar 

  23. Fahmy, T.M., Bieler, J.G., Edidin, M. & Schneck, J.P. Increased TCR avidity after T cell activation: a mechanism for sensing low-density antigen. Immunity 14, 135–143 (2001).

    CAS  PubMed  Google Scholar 

  24. Li, Q.J. et al. miR-181a is an intrinsic modulator of T cell sensitivity and selection. Cell 129, 147–161 (2007).

    CAS  PubMed  Google Scholar 

  25. Shevchenko, A. & Simons, K. Lipidomics: coming to grips with lipid diversity. Nat. Rev. Mol. Cell Biol. 11, 593–598 (2010).

    CAS  PubMed  Google Scholar 

  26. Liu, J. & Rost, B. Comparing function and structure between entire proteomes. Protein Sci. 10, 1970–1979 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Coskun, Ü., Grzybek, M., Drechsel, D. & Simons, K. Regulation of human EGF receptor by lipids. Proc. Natl. Acad. Sci. USA 108, 9044–9048 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Xu, C. et al. Regulation of T cell receptor activation by dynamic membrane binding of the CD3ɛ cytoplasmic tyrosine-based motif. Cell 135, 702–713 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Gagnon, E., Schubert, D.A., Gordo, S., Chu, H.H. & Wucherpfennig, K.W. Local changes in lipid environment of TCR microclusters regulate membrane binding by the CD3ɛ cytoplasmic domain. J. Exp. Med. 209, 2423–2439 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Shi, X. et al. Ca2+ regulates T-cell receptor activation by modulating the charge property of lipids. Nature 493, 111–115 (2013).

    PubMed  Google Scholar 

  31. Strott, C.A. & Higashi, Y. Cholesterol sulfate in human physiology: what's it all about? J. Lipid Res. 44, 1268–1278 (2003).

    CAS  PubMed  Google Scholar 

  32. Bacia, K., Schwille, P. & Kurzchalia, T. Sterol structure determines the separation of phases and the curvature of the liquid-ordered phase in model membranes. Proc. Natl. Acad. Sci. USA 102, 3272–3277 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Kalland, M.E., Oberprieler, N.G., Vang, T., Taskén, K. & Torgersen, K.M. T cell-signaling network analysis reveals distinct differences between CD28 and CD2 costimulation responses in various subsets and in the MAPK pathway between resting and activated regulatory T cells. J. Immunol. 187, 5233–5245 (2011).

    CAS  PubMed  Google Scholar 

  34. Fuda, H., Lee, Y.C., Shimizu, C., Javitt, N.B. & Strott, C.A. Mutational analysis of human hydroxysteroid sulfotransferase SULT2B1 isoforms reveals that exon 1B of the SULT2B1 gene produces cholesterol sulfotransferase, whereas exon 1A yields pregnenolone sulfotransferase. J. Biol. Chem. 277, 36161–36166 (2002).

    CAS  PubMed  Google Scholar 

  35. Bergner, E.A. & Shapiro, L.J. Increased cholesterol sulfate in plasma and red blood cell membranes of steroid sulfatase deficient patients. J. Clin. Endocrinol. Metab. 53, 221–223 (1981).

    CAS  PubMed  Google Scholar 

  36. Epstein, E.H. Jr., Krauss, R.M. & Shackleton, C.H. X-linked ichthyosis: increased blood cholesterol sulfate and electrophoretic mobility of low-density lipoprotein. Science 214, 659–660 (1981).

    CAS  PubMed  Google Scholar 

  37. Beck-García, E., Beck-García, K., Schlosser, A. & Schamel, W.W. Analysis of interactions between proteins and fatty acids or cholesterol using a fatty acid/cholesterol pull-down assay. Anal. Biochem. 436, 75–77 (2013).

    PubMed  Google Scholar 

  38. Thiele, C., Hannah, M.J., Fahrenholz, F. & Huttner, W.B. Cholesterol binds to synaptophysin and is required for biogenesis of synaptic vesicles. Nat. Cell Biol. 2, 42–49 (2000).

    CAS  PubMed  Google Scholar 

  39. Hogquist, K.A. & Jameson, S.C. The self-obsession of T cells: how TCR signaling thresholds affect fate 'decisions' and effector function. Nat. Immunol. 15, 815–823 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Hogquist, K.A., Jameson, S.C. & Bevan, M.J. The ligand for positive selection of T lymphocytes in the thymus. Curr. Opin. Immunol. 6, 273–278 (1994).

    CAS  PubMed  Google Scholar 

  41. Yu, W. et al. Clonal deletion prunes but does not eliminate self-specific αβ CD8+ T lymphocytes. Immunity 42, 929–941 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Surh, C.D. & Sprent, J. T-cell apoptosis detected in situ during positive and negative selection in the thymus. Nature 372, 100–103 (1994).

    CAS  PubMed  Google Scholar 

  43. Daniels, M.A. et al. Thymic selection threshold defined by compartmentalization of Ras/MAPK signalling. Nature 444, 724–729 (2006).

    CAS  PubMed  Google Scholar 

  44. Au-Yeung, B.B. et al. Quantitative and temporal requirements revealed for Zap70 catalytic activity during T cell development. Nat. Immunol. 15, 687–694 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Dong, B. et al. Activation of nuclear receptor CAR ameliorates diabetes and fatty liver disease. Proc. Natl. Acad. Sci. USA 106, 18831–18836 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Moon, J.J. et al. Tracking epitope-specific T cells. Nat. Protoc. 4, 565–581 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Schaffert, S.A. et al. mir-181a-1/b-1 Modulates tolerance through opposing activities in selection and peripheral T cell function. J. Immunol. 195, 1470–1479 (2015).

    CAS  PubMed  Google Scholar 

  48. Yang, W. et al. Potentiating the antitumour response of CD8+ T cells by modulating cholesterol metabolism. Nature 531, 651–655 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Williams, M.L. & Elias, P.M. Stratum corneum lipids in disorders of cornification: increased cholesterol sulfate content of stratum corneum in recessive X-linked ichthyosis. J. Clin. Invest. 68, 1404–1410 (1981).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Swamy, M., Siegers, G.M., Minguet, S., Wollscheid, B. & Schamel, W.W. Blue native polyacrylamide gel electrophoresis (BN-PAGE) for the identification and analysis of multiprotein complexes. Sci. STKE 2006, pl4 (2006).

    PubMed  Google Scholar 

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Acknowledgements

We thank Y.-H. Chien, A. Habtezion, Y. Wong and M. Lozano for helpful discussions; X. Zeng, E.W. Newell, W.O. Gorman, L. Wagar, J. Xie, K.H. Roh, A. Morath, M. Swamy, J. Huppa and J. Huang for experimental assistance; D. Russell for providing Sult2b1−/− mouse embryos. Supported by the US National Institutes of Health (R01 AI022511, U19 AI 090019 and U19-AI057229 to M.M.D.) and the Howard Hughes Medical Institute (M.M.D.). W.W.A.S. received grants from the Deutsche Forschungsgemeinschaft through EXC294 and SCHA 976/2-1. This study was also supported in part by the Excellence Initiative of the Deutsche Forschungsgemeinschaft (GSC-4, Spemann Graduate School).

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Authors and Affiliations

  1. The Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, California, USA

    Feng Wang & Mark M Davis

  2. Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USA

    Feng Wang & Mark M Davis

  3. Center for Biological Signaling Studies (BIOSS) and Faculty of Biology, University of Freiburg, Freiburg, Germany

    Katharina Beck-García, Carina Zorzin & Wolfgang W A Schamel

  4. Centre for Chronic Immunodeficiency (CCI), University of Freiburg, Freiburg, Germany

    Katharina Beck-García, Carina Zorzin & Wolfgang W A Schamel

  5. Spemann Graduate School of Biology and Medicine (SGBM), University of Freiburg, Freiburg, Germany

    Katharina Beck-García

  6. Institute for Immunity, Transplantation and Infection, Stanford University School of Medicine, Stanford, California, USA

    Mark M Davis

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Contributions

F.W. and M.M.D. conceived the project. F.W. designed and conducted the experiments. F.W., K.B.-G. and C.Z. worked on TCR proteoliposome reconstitution and BN-PAGE. K.B.-G. and C.Z. performed cholesterol-beads pulldown assay. W.W.A.S. contributed ideas and technical support. F.W. and M.M.D. wrote the manuscript. W.W.A.S. and other authors edited the manuscript.

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Correspondence to Mark M Davis.

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Integrated supplementary information

Supplementary Figure 1 Increasing CS inhibits TCR signaling.

(a) Lipids were extracted from T cells incubated with 100 μM CS in vitro (left), and thymocytes after intrathymic injection of CS (right). CS and cholesterol were quantified by small molecule mass spectrometry. The ratio of CS/ cholesterol is shown.

(b) Human αβ T cells were pre-treated with DMSO and CS. Non-stimulated cells are shown in red. Cells stimulated by anti-CD3ɛ (clone OKT3) are shown in blue. Phosphorylation of CD3ζ was measured by phospho-flow cytometry. Representative data of three independent experiments are shown.

Supplementary Figure 2 CS inhibits upregulation of CD69.

5C.C7 T cell blasts were pre-treated treated with DMSO (left) and cholesterol sulfate (right). Staining of CD69 surface expression was performed after 4h stimulation. Non-stimulated T cells were shown in red. Cells stimulated with anti-CD3ɛ antibody and PMA/Ionomycin were shown in blue and yellow, respectively. Representative data of three independent experiments are shown.

Supplementary Figure 3 Overexpression of SULT2B1 inhibits TCR signaling.

5C.C7 T cell blasts were transduced with SULT2B1 or control adenovirus with or without supplementation of the SULT2B1 substrate, 3'-phosphoadenosine-5'-phosphosulfate (PAPS). (a) Expression of SULT2B1 mRNA was determined by real-time PCR with the gene-specific TaqMan assay (Life technologies). (b) T cells were stimulated with the anti-CD3ɛ antibody 145-2C11 and TCR-induced phosphorylation of S6 protein was measured by phospho-flow cytometry. (c) T cells were stimulated with MCC-pulsed CH27 APC cells, and secreted IL-2 was determined by ELISA and presented in arbitrary units (AU). Data are from one experiment representative of three independent experiments. Unpaired t tests were performed resulting in the p values shown (n=5, mean with SEM).

Supplementary Figure 4 Digitonin disrupts TCR nanocluster.

(a) The purified TCRs were reconstituted in liposomes with indicated lipid compositions. The proteoliposomes were lysed with 1% digitonin and the lysates were subjected to Blue-Native PAGE and immune-blotted with anti-CD3ζ antibodies. The ferritin markers, f1 and f2 corresponding to 440 kDa and 880 kDa, are shown. Band intensities of nanoclustered and monomeric TCR were quantified with the Li-Cor Odyssey infrared imager and shown as a ratio of the two forms. Quantification data were collected from three independent experiments shown on the right.

(b) After DMSO or CS-treatment, the M.mζ-SBP cells were lysed with 1% digitonin. Purified protein complexes were subjected to Blue-Native PAGE and anti-CD3ζ immuno-blotting. Band intensities of nanoclustered and monomeric TCR were quantified with the Li-Cor Odyssey infrared imager and shown as a ratio of the two forms. Quantification data were collected from three independent experiments shown on the right.

Supplementary Figure 5 TCR signaling in DP thymocytes is more sensitive to CS than in SP thymocytes.

CD4+ SP (a), CD8+ SP (b) and DP thymocytes (c) treated with DMSO or CS at indicated concentrations. Pre-treated thymocytes were fluorescently labeled and mixed at a 1:1 ratio. Labeled cells were loaded with Indo-1 calcium-sensitive dye, and stimulated by adding anti-CD3ɛ 145-2C11 and anti-hamster IgG crosslinking antibodies. Indo-1 ratio indicating calcium flux was assessed by flow cytometry. Representative data of three experiments are shown. Black arrows indicate the time points of adding anti-CD3ɛ and crosslinking antibodies. Dashed green arrows highlight the differences of calcium flux between DMSO-treated (red) and CS-treated (blue) thymocytes.

Supplementary Figure 6 CS does not induce apoptosis in DP thymocytes of OT-1 Rag2−/− β2m-null mice.

β2m-null mice were subjected to lethal dose radiation, then reconstituted with OT-I TCR transgenic Rag2−/− bone marrow. Six weeks after bone marrow transfer, DMSO control (left) or cholesterol sulfate (right) was injected intrathymically into the mice. After 2 days, Annexin V staining of DP thymocytes was analyzed by flow cytometry, as in Figure 5.

Statistical analysis for the percentage of Annexin V positive populations in DP thymocytes. Data are from one experiment representative of three independent experiments. Unpaired t tests were performed resulting in the p value shown on the right (mean with SEM, n=5).

Supplementary Figure 7 Thymocytes and splenocytes of Sult2b1−/− mice have normal percentages of CD4+ and CD8+ cells.

(a) Lipids were extracted from total thymoctyes of WT and Sult2b1 KO mice. CS and cholesterol were quantified by small molecule mass spectrometry. The ratio of CS/ cholesterol is shown.

(b) Thymocytes and splenocytes were isolated from either WT (left) or Sult2b1 KO mice (right). CD4 and CD8 surface markers were analyzed by flow cytometry. Representative data of three independent experiments are shown.

Supplementary Figure 8 Effects of SULT2B1 deficiency on thymocytes recognizing the HY self-antigen.

(a) FACS plots of CD8 SP thymocytes gated on H-Y Uty Db tetramer+ populations from wild-type (WT) and Sult2b1 knock-out (KO) mice after magnetic bead enrichment. Representative data of three independent experiments are shown.

(b) The total number of the H-Y tetramer positive CD8 SP cells. Data were collected from three independent experiments. Unpaired t tests were performed (mean with SD, n=10 in male mice group, and n=8 in female mice group, P values were shown on the right).

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Wang, F., Beck-García, K., Zorzin, C. et al. Inhibition of T cell receptor signaling by cholesterol sulfate, a naturally occurring derivative of membrane cholesterol. Nat Immunol 17, 844–850 (2016). https://doi.org/10.1038/ni.3462

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