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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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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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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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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DOI: https://doi.org/10.1038/ni.3462
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