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Dynamic regulation of CD28 conformation and signaling by charged lipids and ions

Nature Structural & Molecular Biology volume 24, pages 1081–1092 (2017)Cite this article

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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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Figure 1: The CD28 cytoplasmic domain inserts into the membrane bilayer.
Figure 2: The NMR structure of membrane-bound CD28 cytoplasmic domain.
Figure 3: The CD28-proximal Ca2+ concentration increases after TCR stimulation.
Figure 4: Ca2+ induces an open conformation of CD28CD.
Figure 5: TCR-induced Ca2+-influx-enhanced CD28CD opening and signaling.
Figure 6: A mathematical model of immune receptor conformational dynamics showing the importance of Ca2+ feedback to the antigen 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).

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Author notes

  1. Wei Yang, Weiling Pan, Shuokai Chen, Nicola Trendel and Shutan Jiang: These authors contributed equally to this work.

Authors and 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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  1. Wei Yang
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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.

Corresponding authors

Correspondence to Omer Dushek, Hua Li or Chenqi Xu.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 The pI-value distribution of single-pass membrane proteins at the plasma membrane in immune cells and in total cells.

The PI values were measured for the first 10 amino acids in the cytoplasmic domains of human single-pass transmembrane proteins that localized at the plasma membrane. Source data are available in Source Data 3.

Source data

Supplementary Figure 2 CD28 cytoplasmic domain specifically binds to acidic phospholipids.

(a) Alignment of CD28CD from different species. Most residues are highly conserved, especially the two important YxxM and PYAP signaling motifs (marked in red) and basic residues (marked in green). The residue conservation is visualized by the sequence logo of CD28CD generated from the Skylign web server http://www.skylign.org.

(b) Net charges of human and mouse CD28 cytoplasmic domains at pH 7.0.

(c) The protein sequences of mouse CD28CD and three mutants used in (d-g). Two polybasic regions (PBR) are marked in red, and mutant sites are marked in blue.

(d-g) Binding of CD28CD WT and mutants to acidic phospholipids was measured by the equilibrium-based microdialysis assay.

(d) A cartoon illustration of the equilibrium-based microdialysis assay. See Online Methods for details.

(e) Binding of CD28CD WT to different acidic phospholipids measured by the microdialysis assay. LUVs in the experimental chamber were composed of either 100% of the acidic phospholipids POPG, POPS, PI, PA, or a lipid mixture (40% POPC, 20% POPS, 20% POPE, 10% POPG and 10% PI). LUVs in the control chamber were composed of 100% of the zwitterionic lipid POPC. CD28CD-Alexa 488 was used at a concentration of 10 nM and LUVs at 5 mM. Two to three independent samples were measured for each condition and the results are plotted as the percentage of the fluorescence intensity in the experimental and control chambers.

(f-g) Binding of CD28CD WT and mutants to acidic phospholipids measured by the equilibrium-based microdialysis assay. Two to three independent samples were measured for each condition and the results are plotted as the percentage of the fluorescence intensity in the experimental and control chambers (F), and are further converted to the binding efficiencies of the mutants to POPG (G). Data were analyzed by unpaired t-test. **P < 0.01; ***P < 0.001; ns: no significant difference.

Data are representative of three (e) or seven (f, g) independent experiments. The center value and error bar in e-g denote mean and range. Source data for e-g are available in Source Data 4.

Source data

Supplementary Figure 3 Measurement of membrane binding of CD28CD by the aromatic fluorescence emission (AFE) assay.

(a) Incubation of 2 μM CD28CD with 0.2 mM acidic POPG LUVs led to significant increase of AFE value but such an increase was not observed in that with 0.2 mM zwitterionic POPC LUVs.

(b-c) Titration of acidic POPG LUVs with the indicated concentrations into 2 μM CD28CD sample led to the gradient increase of AFE value (at 310nm).

For each condition, three independent samples were measured in one experiment. Data are representative of three independent experiments. Unpaired t-test was used for comparing each two groups. One-Way ANOVA was used to test whether POPG treatment could cause significant change of the AFE value, P < 0.0001 (C).

The center value and error bar in c denote mean and s.e.m.. Source data are available in Source Data 5.

Source data

Supplementary Figure 4 Membrane binding of Notch1 juxtamembrane polybasic region.

(a) Sequence information and charge property of human Notch1 juxtamembrane domain.

(b) Incubation of 2 μM Notch1_JM with 0.1 mM acidic POPG LUVs led to significant increase of AFE value but such an increase was not observed in that with 0.1 mM zwitterionic POPC LUVs.

(c-d) Titration of acidic POPG LUVs with the indicated concentrations into 2 μM Notch1_JM sample led to the gradient increase of AFE value (at approximate 350nm). Three independent samples were measured for each condition. Unpaired t-test was used for the statistical analysis of each two groups. One-Way ANOVA was used to test whether POPG treatment could cause significant change of the AFE value, P < 0.0001.

(e-f) The de-quenching FRET was used to measure the interaction of Notch1 juxtamembrane polybasic region with the plasma membrane in live Jurkat T cells. Representative cells are shown in e. Data in f are representative of three independent experiments. Bars in e represent 2 μm. N = 31, 32, 35 for the three conditions (from left to right). Each dot represents the FRET value from one individual cell. One-Way ANOVA was used to analyze difference among three groups, the p-values of these three panels from left to right were <0.0001, 0.2074 and 0.1555. Unpaired t-test was used to analyze difference between each two groups.

The center value and error bar in d,f denote mean and s.e.m.. Source data for b-d, and f are available in Source Data 6.

Source data

Supplementary Figure 5 Intrinsic dynamics of membrane-bound CD28CD.

15N backbone spin relaxation measurements of CD28CD in POPG bicelles. (a) 15N longitudinal relaxation rates (R1), (b) 15N transverse relaxation rates (R2) and (c) the heteronuclear 1H-15N NOEs. Disappeared or overlapped residues are marked by asterisks. Prolines that lack backbone amides are marked by P.

Supplementary Figure 6 Disruption of CD28CD-membrane binding promoted the protein’s basal signaling.

(a) The de-quenching FRET was used to measure the membrane binding of CD28 WT and CD28 linker mutant (n = 21, 38 (from left to right), each dot represents the FRET value from one individual cell).

(b-e) TFP-T2A-HA-CD28 WT or linker mutant construct was transduced into mouse CD28−/− T cells by retrovirus, respectively. After protein translation of the fusion construct, TFP and HA-CD28 were separated due to the self-cleaving property of T2A. Basal CD28 signaling was measured in transduced cells without stimulation. IL-2 production was measured in transduced cells under TCR and/or CD28 stimulation.

(b-c) The cells without stimulation were lysed and the immunoprecipitation assay was performed to detect basal phosphorylation of CD28 and its interactions with signaling proteins. The bands were quantitated by ImageJ. The pCD28/HA, P85/HA, Lck/HA and Grb2/HA ratios were obtained and further normalized to the value of WT condition of each strip. Average results of 4 independent samples are shown in panel C.

(d-e) The cells were stimulated for 4 hours with plated-bound α-CD3 (0.5 μg/ml) alone or α-CD3 (0.5 μg/ml) + α-CD28 (2 μg/ml), and IL-2 production was measured by intracellular staining and flow cytometric analysis. CD4+ TFP+ cells were gated for the analysis of IL-2 level. TFP levels of CD28 WT cells and CD28 linker cells were matched, reflecting the comparable expression level of CD28 WT and CD28 linker. Percentage of IL-2 positive cells and median fluorescence intensity (MFI) of all cells are shown (n = 3).

Data are representative of three (a, c) or four (e) independent experiments, and were analyzed by unpaired t-test. The center value and error bar in a, c and e denote mean and s.e.m.. The original gel image of b can be found in Supplementary Data Set 2. Source data for a, c and e are available in Source Data 7.

Source data

Supplementary Figure 7 Ionomycin-induced Ca2+ influx enhances the openness of CD28.

The de-quenching FRET method was used to measure the effect of Ca2+ on CD28CD-membrane binding in live Jurkat T cells. Ionomycin (1 μM) was used to trigger Ca2+ influx in T cells. Ca2+ influx led to the significant decrease of the FRET value of CD28-TFP (a), indicating the dissociation of CD28CD from the membrane. In contrast, the FRET values of the 3aa-TFP and 50aa-TFP control constructs were insensitive to Ca2+ influx (b, c). The FRET efficiency was measured and plotted as mean +/- s.e.m. (n = 12, 15 in (A), n = 17, 16 in (B), n = 22, 19 in (C) (from left to right), each dot represents the FRET value from one individual cell). The surface mTFP level and R18 level were comparable.

Data are representative of two independent experiments. All the data were analyzed by unpaired t-test except the left sub-panels in (a) and (c) where Mann-Whitney test were used because the FERT efficiency data sets of the CD28 group and the 50aa-TFP group do not fit the normal distribution. The center value and error bar denote mean and s.e.m.. Source data are available in Source Data 8.

Source data

Supplementary Figure 8 Ca2+ influx enhances the openness and signaling of CD28.

(a-b) To further demonstrate the effect of Ca2+ on CD28CD-membrane binding, we use different concentrations of α-CD3 to generate different levels of Ca2+ influx. We also used BAPTA pre-treatment to chelate the intracellular Ca2+ (a). The de-quenching FRET was used to detect the effect of TCR-induced Ca2+ influx on the CD28CD-membrane interaction. Different doses of α-CD3 induced different levels of Ca2+ influx (a). Treatment of 10 μM BAPTA led to partial chelation of intracellular Ca2+. N = 64, 28, 22, 36 (from left to right) for each condition. Each dot represents the FRET value from one individual cell. Mouse IgG (3 μg/ml) was used as the isotype control antibody for α-CD3.

(c) To exam the effect of Ca2+ influx on CD28 signaling in primary T cell, the mouse CTL were generated and stimulated with α-CD3 (0.5 μg/ml) + α-CD28 (2 μg/ml) with indicated times in the Ca2+/Mg2+-free Ringer’s buffer containing 1 mM Ca2+ or not at 37 oC. After stimulation, cells were lysed for immunoprecipitation and immunoblotting. The bands were quantitated by ImageJ. The pCD28/CD28, P85/CD28, Lck/CD28 and Grb2/CD28 ratios were obtained and further normalized to the value of no stimulation condition of each strip.

Data are representative of three independent experiments. In (b), for the left sub-panel, One-Way ANOVA was used to analyze difference among four groups (P<0.0001) and unpaired t-test was used to analyze difference between two groups. For the middle and right sub-panels, the R18 level of the α-CD3 (3μg/ml) group and the mTFP level of the isotype control group do not fit the normal distribution. To compare each two groups, Mann Whitney test was used for data sets that do not fit the normal distribution and unpaired t-test was used for the rest. The center value and error bar in b denote mean and s.e.m.. The original gel image of b can be found in Supplementary Data Set 2. Source data for b are available in Source Data 9.

Source data

Supplementary Figure 9 CD28 ligation caused the dissociation of CD28 cytoplasmic domain from the membrane but was insufficient to induce CD28 phosphorylation.

(a) The de-quenching FRET method was used to measure whether CD28 ligation could induce CD28 cytoplasmic domain to dissociate from the membrane in live Jurkat T cells. The stimulating buffer was Ca2+/Mg2+-free Ringer’s buffer. Compared with mock treatment (no antibody), addition of 5 μg/ml α-CD28 induced dissociation of CD28CD from the membrane. To test whether this induction was dependent on the phosphorylation of CD28, 100 μM PP2 was pre-incubated with T cells for 10 min to inhibit Src family kinase and then the cells were stimulated by 5 μg/ml a-CD28. The result indicated CD28 phosphorylation was not required for the dissociation of CD28CD from the membrane. N = 22, 11, 12 for each condition (from left to right). Each dot represents the FRET value from one individual cell. Data are representative of three independent experiments. Unpaired t-test was used to analyze difference between two groups.

(b) CD28-deficient Jurkat T cells expressing HA-mCD28 were stimulated by 1 μg/ml a-CD3 alone, 2 μg/ml a-CD28 alone or 2 μg/ml a-CD28 and 1 μg/ml a-CD3 in normal Ringer’s buffer (containing 1 mM Ca2+) for the indicated time at 37 oC. Either CD3 ligation alone or CD28 ligation alone could not trigger CD28 phosphorylation, but CD28 ligation and TCR ligation together could trigger CD28 phosphorylation. The bands were quantitated by Image J. The pCD28 band intensity was divided by the corresponding CD28 band intensity to obtain the pCD28/CD28 ratio, which were further normalized to the value of 0 min of α-CD3 condition.

The center value and error bar in a denote mean and s.e.m.. The original gel image of b can be found in Supplementary Data Set 2. Source data for a are available in Source Data 10.

Source data

Supplementary Figure 10 Low surface expression of CD28 ligands in tumor cells.

CD80 and CD86 surface levels were detected on CT26 murine colon carcinoma cell line, EL4 murine lymphoma cell line, Eμ-Myc p19Afr−/− mouse B cell lymphoma cell line and BBN-14 murine bladder carcinoma cell line. Activated dendritic cell (activated by 100 ng/ml LPS at 37°C for 24 h) was used as a positive control.

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Supplementary Text and Figures

Supplementary Figures 1–10, Supplementary Table 1 and Supplementary Note 1 (PDF 2880 kb)

Life Sciences Reporting Summary (PDF 200 kb)

Supplementary Data Set 1

Single-pass transmembrane proteins in immune cells that contain intracellular juxtamembrane polybasic regions (length of 10 amino acids; pI ≥ 11). (XLSX 22 kb)

Supplementary Data Set 2

Original gel images for Figure 5e and Supplementary Figures 6b, 8c, and 9b. (PDF 35585 kb)

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Yang, W., Pan, W., Chen, S. et al. Dynamic regulation of CD28 conformation and signaling by charged lipids and ions. Nat Struct Mol Biol 24, 1081–1092 (2017). https://doi.org/10.1038/nsmb.3489

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