Abstract

CD28 provides an essential costimulatory signal for T cell activation, and its function is critical in antitumor immunity. However, the molecular mechanism of CD28 transmembrane signaling remains elusive. Here we show that the conformation and signaling of CD28 are regulated by two counteractive charged factors, acidic phospholipids and Ca2+ ions. NMR spectroscopy analyses showed that acidic phospholipids can sequester CD28 signaling motifs within the membrane, thereby limiting CD28 basal signaling. T cell receptor (TCR) activation induced an increase in the local Ca2+ concentration around CD28, and Ca2+ directly disrupted CD28-lipid interaction, leading to opening and signaling of CD28. We observed that the TCR, Ca2+, and CD28 together form a dual-positive-feedback circuit that substantially amplifies T cell signaling and thus increases antigen sensitivity. This work unravels a new regulatory mechanism for CD28 signaling and thus contributes to the understanding of the dependence of costimulation signaling on TCR signaling and the high sensitivity of T cells.

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Acknowledgements

The NMR spectroscopy and imaging experiments were performed at the National Center for Protein Science Shanghai, Core Facility for Molecular Biology of SIBCB, and Core Facility for Cell Biology of SIBCB. We thank Z. Liu (NMR section, NCPSS) for help in setting up filter NOESY experiments. We thank Y. Yu (Integrated Laser Microscopy section, NCPSS) for help in setting up imaging experiments. We thank K. Wucherpfennig and E. Gagnon for helpful discussions. The CD28-deficient cell line was a generous gift from O. Acuto (Oxford University, Oxford, UK). This work was supported by NSFC (grants 31370860, 31425009, 31530022, and 31621003 to C.X.; grants 31470734 and 31670751 to H.L.), CAS (Strategic Priority Research Program grant XDB08020100 to C.X.). MOST (grant 2014CB541903 to H.L.), and the Wellcome Trust and the Royal Society (Sir Henry Dale Fellowship, grant 098363 to O.D.). N.T acknowledges support from Systems Biology DTC supported by the Engineering and Physical Science Research Council (UK).

Author information

Author notes

    • Wei Yang
    • , Weiling Pan
    • , Shuokai Chen
    • , Nicola Trendel
    •  & Shutan Jiang

    These authors contributed equally to this work.

Affiliations

  1. State Key Laboratory of Molecular Biology, National Center for Protein Science Shanghai, Shanghai Science Research Center, CAS Center for Excellence in Molecular Cell Science, Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China.

    • Wei Yang
    • , Weiling Pan
    • , Shuokai Chen
    • , Shutan Jiang
    • , Manman Xue
    • , Wei Wu
    • , Zeli Peng
    • , Hua Li
    •  & Chenqi Xu
  2. Department of Pathology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, China.

    • Wei Yang
  3. Sir William Dunn School of Pathology, University of Oxford, Oxford, UK.

    • Nicola Trendel
    •  & Omer Dushek
  4. Institute of Image Processing & Pattern Recognition, Shanghai Jiaotong University, Shanghai, China.

    • Feng Xiao
    •  & Hongbin Shen
  5. State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China.

    • Xiaoxi Li
    • , Hongbin Ji
    • , Xiaolong Liu
    •  & Hai Jiang
  6. School of Life Science and Technology, ShanghaiTech University, Shanghai, China.

    • Haopeng Wang
    •  & Chenqi Xu

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Contributions

C.X. conceived the project and wrote the manuscript. C.X., H.L., O.D., and H.S. designed the experiments. W.P. and H.L. performed the NMR experiments. W.Y., W.P., and S.J. performed the biochemical and immunological experiments. S.C. and W.Y. performed the imaging experiments. N.T. and O.D. performed the mathematical modeling. F.X., W.W., and S.C. performed the bioinformatics analysis. H.W., X. Liu and H. Ji provided extensive discussion. Z.P. and M.X. helped with the protein purification. X. Li and H. Jiang generated BBN-14 cells. All authors contributed to revision of the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Omer Dushek or Hua Li or Chenqi Xu.

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    Single-pass transmembrane proteins in immune cells that contain intracellular juxtamembrane polybasic regions (length of 10 amino acids; pI ≥ 11).

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https://doi.org/10.1038/nsmb.3489