Letter | Published:

FBXO38 mediates PD-1 ubiquitination and regulates anti-tumour immunity of T cells

Naturevolume 564pages130135 (2018) | Download Citation

Abstract

Dysfunctional T cells in the tumour microenvironment have abnormally high expression of PD-1 and antibody inhibitors against PD-1 or its ligand (PD-L1) have become commonly used drugs to treat various types of cancer1,2,3,4. The clinical success of these inhibitors highlights the need to study the mechanisms by which PD-1 is regulated. Here we report a mechanism of PD-1 degradation and the importance of this mechanism in anti-tumour immunity in preclinical models. We show that surface PD-1 undergoes internalization, subsequent ubiquitination and proteasome degradation in activated T cells. FBXO38 is an E3 ligase of PD-1 that mediates Lys48-linked poly-ubiquitination and subsequent proteasome degradation. Conditional knockout of Fbxo38 in T cells did not affect T cell receptor and CD28 signalling, but led to faster tumour progression in mice owing to higher levels of PD-1 in tumour-infiltrating T cells. Anti-PD-1 therapy normalized the effect of FBXO38 deficiency on tumour growth in mice, which suggests that PD-1 is the primary target of FBXO38 in T cells. In human tumour tissues and a mouse cancer model, transcriptional levels of FBXO38 and Fbxo38, respectively, were downregulated in tumour-infiltrating T cells. However, IL-2 therapy rescued Fbxo38 transcription and therefore downregulated PD-1 levels in PD-1+ T cells in mice. These data indicate that FBXO38 regulates PD-1 expression and highlight an alternative method to block the PD-1 pathway.

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Data availability

The processed RNA-sequencing data are shown in Supplementary Data 1. Raw data will be provided upon request. Source Data for Figs. 1, 3, 4 and Extended Data Figs. 19 are included in the online version of the paper. Gel source data can be found in Supplementary Fig. 1.

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References

  1. 1.

    Ishida, Y., Agata, Y., Shibahara, K. & Honjo, T. Induced expression of PD-1, a novel member of the immunoglobulin gene superfamily, upon programmed cell death. EMBO J. 11, 3887–3895 (1992).

  2. 2.

    Dong, H. et al. Tumor-associated B7-H1 promotes T-cell apoptosis: a potential mechanism of immune evasion. Nat. Med. 8, 793–800 (2002).

  3. 3.

    Iwai, Y. et al. Involvement of PD-L1 on tumor cells in the escape from host immune system and tumor immunotherapy by PD-L1 blockade. Proc. Natl Acad. Sci. USA 99, 12293–12297 (2002).

  4. 4.

    Sharma, P. & Allison, J. P. The future of immune checkpoint therapy. Science 348, 56–61 (2015).

  5. 5.

    Oestreich, K. J., Yoon, H., Ahmed, R. & Boss, J. M. NFATc1 regulates PD-1 expression upon T cell activation. J. Immunol. 181, 4832–4839 (2008).

  6. 6.

    Kao, C. et al. Transcription factor T-bet represses expression of the inhibitory receptor PD-1 and sustains virus-specific CD8+ T cell responses during chronic infection. Nat. Immunol. 12, 663–671 (2011).

  7. 7.

    Xiao, G., Deng, A., Liu, H., Ge, G. & Liu, X. Activator protein 1 suppresses antitumor T-cell function via the induction of programmed death 1. Proc. Natl Acad. Sci. USA 109, 15419–15424 (2012).

  8. 8.

    Staron, M. M. et al. The transcription factor FoxO1 sustains expression of the inhibitory receptor PD-1 and survival of antiviral CD8+ T cells during chronic infection. Immunity 41, 802–814 (2014).

  9. 9.

    Mathieu, M., Cotta-Grand, N., Daudelin, J.-F., Thébault, P. & Labrecque, N. Notch signaling regulates PD-1 expression during CD8+ T-cell activation. Immunol. Cell Biol. 91, 82–88 (2013).

  10. 10.

    Youngblood, B. et al. Chronic virus infection enforces demethylation of the locus that encodes PD-1 in antigen-specific CD8+ T cells. Immunity 35, 400–412 (2011).

  11. 11.

    Stephen, T. L. et al. SATB1 expression governs epigenetic repression of PD-1 in tumor-reactive T cells. Immunity 46, 51–64 (2017).

  12. 12.

    Burr, M. L. et al. CMTM6 maintains the expression of PD-L1 and regulates anti-tumour immunity. Nature 549, 101–105 (2017).

  13. 13.

    Li, C.-W. et al. Glycosylation and stabilization of programmed death ligand-1 suppresses T-cell activity. Nat. Commun. 7, 12632 (2016).

  14. 14.

    Zhao, J. et al. F-box protein FBXL19-mediated ubiquitination and degradation of the receptor for IL-33 limits pulmonary inflammation. Nat. Immunol. 13, 651–658 (2012).

  15. 15.

    Skaar, J. R., Pagan, J. K. & Pagano, M. SCF ubiquitin ligase-targeted therapies. Nat. Rev. Drug Discov. 13, 889–903 (2014).

  16. 16.

    Komander, D. & Rape, M. The ubiquitin code. Annu. Rev. Biochem. 81, 203–229 (2012).

  17. 17.

    Singer, M. et al. A distinct gene module for dysfunction uncoupled from activation in tumor-infiltrating T cells. Cell 166, 1500–1511 (2016).

  18. 18.

    Paley, M. A. et al. Progenitor and terminal subsets of CD8+ T cells cooperate to contain chronic viral infection. Science 338, 1220–1225 (2012).

  19. 19.

    Im, S. J. et al. Defining CD8+ T cells that provide the proliferative burst after PD-1 therapy. Nature 537, 417–421 (2016).

  20. 20.

    Wei, S. C. et al. Distinct cellular mechanisms underlie anti-CTLA-4 and anti-PD-1 checkpoint blockade. Cell 170, 1120–1133 (2017).

  21. 21.

    Mitra, S. & Leonard, W. J. Biology of IL-2 and its therapeutic modulation: mechanisms and strategies. J. Leukoc. Biol. 103, 643–655 (2018).

  22. 22.

    Thommen, D. S. & Schumacher, T. N. T cell dysfunction in cancer. Cancer Cell 33, 547–562 (2018).

  23. 23.

    Krieg, C., Létourneau, S., Pantaleo, G. & Boyman, O. Improved IL-2 immunotherapy by selective stimulation of IL-2 receptors on lymphocytes and endothelial cells. Proc. Natl Acad. Sci. USA 107, 11906–11911 (2010).

  24. 24.

    Zhu, E. F. et al. Synergistic innate and adaptive immune response to combination immunotherapy with anti-tumor antigen antibodies and extended serum half-life IL-2. Cancer Cell 27, 489–501 (2015).

  25. 25.

    Rosenberg, S. A. IL-2: the first effective immunotherapy for human cancer. J. Immunol. 192, 5451–5458 (2014).

  26. 26.

    West, E. E. et al. PD-L1 blockade synergizes with IL-2 therapy in reinvigorating exhausted T cells. J. Clin. Invest. 123, 2604–2615 (2013).

  27. 27.

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

  28. 28.

    Zhou, P. et al. In vivo discovery of immunotherapy targets in the tumour microenvironment. Nature 506, 52–57 (2014).

  29. 29.

    Gao, Y. et al. Inflammation negatively regulates FOXP3 and regulatory T-cell function via DBC1. Proc. Natl Acad. Sci. USA 112, E3246–E3254 (2015).

  30. 30.

    Yang, X. P. et al. Opposing regulation of the locus encoding IL-17 through direct, reciprocal actions of STAT3 and STAT5. Nat. Immunol. 12, 247–254 (2011).

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Acknowledgements

We thank C. Li for biochemical support; Y. Gao and B. Li for providing FOXP3-expressing Jurkat cells; A. Bietz for proofreading; X. Shi, C. Yan and J. Zhang for discussion; Genome Tagging Project (GTP) Center, Animal facility and Cell Biology facility of SIBCB for technical support. C.X. is funded by NSFC grants (31530022, 31425009, 31621003), CAS grants (Strategic Priority Research Program XDB08020100, XDB29000000; Facility-based Open Research Program; QYZDB-SSW-SMC048), STSMC 16JC1404800 and the Ten Thousand Talent Program ‘National Program for Support of Top-notch Young Professionals’ of China.

Reviewer information

Nature thanks R. Deshaies, W. Ouyang, K. Paukan and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information

Author notes

  1. These authors contributed equally: Xiangbo Meng, Xiwei Liu

Affiliations

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

    • Xiangbo Meng
    • , Xiwei Liu
    • , Xingdong Guo
    • , Shutan Jiang
    • , Yibing Bai
    • , Manman Xue
    • , Ronggui Hu
    •  & Chenqi Xu
  2. State Key Laboratory of Ophthalmology, Sun Yat-sen University, Guangzhou, China

    • Tingting Chen
    •  & Lai Wei
  3. Department of Liver Surgery and Transplantation, Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai, China

    • Zhiqiang Hu
    •  & Xiaowu Huang
  4. Key Laboratory of Carcinogenesis and Cancer Invasion, Ministry of Education, Fudan University, Shanghai, China

    • Zhiqiang Hu
    •  & Xiaowu Huang
  5. State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China

    • Haifeng Liu
    •  & Xiaolong Liu
  6. Department of Immunology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

    • Shao-cong Sun
  7. Sun Yat-sen University Cancer Center, Guangzhou, China

    • Penghui Zhou
  8. Department of Pathology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, China

    • Wei Yang
  9. Department of Pathology, Nanfang Hospital, Southern Medical University, Guangzhou, China

    • Wei Yang
  10. School of Life Science and Technology, ShanghaiTech University, Shanghai, China

    • Chenqi Xu

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Contributions

C.X. conceived the project. Xiaolong Liu, S.S. and R.H. contributed to the design of the project and extensive discussions. X.M., Xiwei Liu and H.L. performed the animal experiments. X.M., Xiwei Liu and X.G. performed ex vivo T cell experiments and biochemistry experiments. X.M. analysed the mass spectrometry samples. S.J., T.C. and L.W. performed chromatin-immunoprecipitation and quantitative PCR with reverse transcription experiments. W.Y. provided human colorectal carcinoma cDNA samples. X.H. and Z.H. provided human hepatocellular carcinoma samples. Y.B. purified the GST–PD-1 protein. P.Z. provided technical assistance. C.X., X.M. and Xiwei Liu. wrote the manuscript and the other authors revised it. X.M. made the figures. M.X. helped to proofread the manuscript.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Chenqi Xu.

Extended data figures and tables

  1. Extended Data Fig. 1 Analysis of surface PD-1 dynamics.

    a, PD-1 lysine residues are conserved across different species. Amino acid sequences of PD-1 cytoplasmic domains from different species. Lysine residues are highlighted in red. The Lys210 and Lys233 (numbered according to the human sequence) are highly conserved. b, Experimental approach to study surface-labelled PD-1 internalization and ubiquitination. c, Confocal imaging of PD-1 internalization. Cells were stimulated with PHA (150 ng ml−1) for three days to induce PD-1 expression. Surface PD-1 molecules were labelled by non-conjugated anti-PD-1 and cells were incubated for 3 h at 37 °C. Cell samples were collected before and after 37 °C incubation and subjected to fixation and permeabilization. An Alexa-647-labelled secondary antibody was used to image PD-1 molecules. d, Change in cell-surface expression of PD-1 after inhibition of lysosome activity by bafilomycin A1 (BFA). n = 3 biologically independent samples. e, Expression of p62 was used as an indicator of lysosome inhibition. Experiments were independently repeated three times (ce). **P < 0.01, unpaired Student’s t-test. For gel source data, see Supplementary Fig. 1. Source data

  2. Extended Data Fig. 2 FBXO38 specifically regulates PD-1 protein level.

    a, Generation of FBXO38-HA knock-in and FBXO38 knockout Jurkat T cells by CRISPR–Cas9 technology. The knock-in and knockout efficiencies were confirmed by immunoblotting. b, Co-immunoprecipitation of Myc–FBXO47 and HA–PD-1 in HEK293FT cells. WCL, whole-cell lysate. cf, Effect of FBXO38 and FBXO47 overexpression on surface PD-1 levels in Jurkat T cells. Cells were transduced by Myc-FBXO38-IRES-GFP, Myc-FBXO47-IRES-GFP or vector control using lentiviruses. Transduced cells were sorted by GFP expression. c, FBXO38 or FBXO47 protein levels. d, Surface TCR levels in unstimulated Jurkat cells were measured by flow cytometry. e, f, Cell-surface levels of PD-1 in Jurkat cells stimulated with anti-CD3 (5 μg ml−1) for 24 h. n = 3 biologically independent samples. gm, Effect of FBXO38 knockdown on PD-1 expression levels in Jurkat T cells. Cells were transduced with constructs containing control shRNA or FBXO38 shRNA (knockdown) as well as a GFP marker. Transduced cells were sorted by GFP expression. g, h, FBXO38 expression and transcriptional level in control and knockdown cells. i, Surface TCR levels in unstimulated Jurkat cells. j, Total PD-1 levels in control and knockdown cells stimulated with PHA (150 ng ml−1) for 72 h. km, Surface PD-1 levels in control and knockdown cells stimulated with PHA (50, 75 or 100 ng ml−1) for the indicated number of hours. n = 3 biologically independent samples. np, Effect of Fbxo38 overexpression on PD-1 surface levels in activated OT-1 CD8+ T cells. Activated OT-1 T cells were transduced by Myc-FBXO38-IRES-GFP or vector control using a retrovirus. PD-1 levels in transduced CD8+ T cells (GFP positive) were measured 24 and 48 h after transduction. n, Transduction efficiency in activated OT-1 T cells. o, p, PD-1 surface levels in vector control and Fbxo38-overexpressing (FBXO38) OT-1 T cells. n = 3 biologically independent samples. Data are representative of two (j) or three (ai, kp) independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, unpaired Student’s t-test except the 72-h group of PD-1 MFI in m, in which an unpaired Student’s t-test with Welch’s correction was used. For gel source data, see Supplementary Fig. 1. Source data

  3. Extended Data Fig. 3 Fbxo38 knockout does not affect thymocyte development and peripheral T cell homeostasis.

    a, Protein levels of FBXO38 in wild-type or Fbxo38CKO CD8+ T cells. bi, Flow cytometry analysis of thymocytes, peripheral T cells isolated from spleen and lymph nodes of wild-type and Fbxo38CKO mice (8 weeks old, n = 6 mice). bi, Left, representative flow cytometry profiles. Right, statistical analyses. b, Percentages of CD4CD8 double-negative (DN), CD4+CD8+ double-positive (DP), CD4+ single-positive (CD4SP) and CD8+ single-positive (CD8SP) cells out of the total number of thymocytes from wild-type or Fbxo38CKO mice. c, Percentages of CD44+ single-positive (DN1), CD44+CD25+ double-positive (DN2), CD25+ single-positive (DN3) and CD44CD25double-negative (DN4) cells in CD4CD8 cells from b. d, e, Percentages of CD4+ and CD8+ T cells in spleen and lymph nodes of wild-type and Fbxo38CKO mice. fi, Percentages of naive (CD44lowCD62Lhigh), central memory (CD44highCD62Lhigh; CM) and effector/effector memory (CD44highCD62Llow, effector/EM) CD4+ and CD8+ T cells in spleen (f, h) and lymph nodes (g, i) of wild-type and Fbxo38CKO mice. a, Data are representative of three independent experiments. bi, Data are a combination of three independent experiments and analysed by unpaired Student’s t-test (b, d, g, h), unpaired Student’s t-test with Welch’s correction (e) or Mann–Whitney U-test (c, f, i). For gel source data, see Supplementary Fig. 1. Source data

  4. Extended Data Fig. 4 Fbxo38 knockout does not affect CD8+ T cell activation, proliferation and apoptosis.

    a, TCR and CD28 surface levels in unstimulated wild-type or Fbxo38CKO CD8+ T cells measured by anti-CD3ε and anti-CD28 staining and flow cytometry. b, c, CD44 and LAG-3 surface levels in wild-type or Fbxo38CKO CD8+ T cells stimulated with anti-CD3/CD28 (2 μg ml−1) for 96 h. n = 3 biologically independent samples. d, Cytokine production of wild-type or Fbxo38CKO CD8+ T cells stimulated with anti-CD3/CD28 (2 μg ml−1) for the indicated time. n = 3 biologically independent samples. NS, not stimulated; S, stimulated. e, RNA-sequencing analysis of naive and activated mouse CD8+ T cells isolated from three paired wild-type and Fbxo38CKO mice. Naive CD8+ T cells were stimulated with anti-CD3/CD28 (2 μg ml−1) for 96 h. Full information for the RNA-sequencing data are available in Supplementary Data 1. f, g, Naive CD8+ T cells isolated from wild-type or Fbxo38CKO mice were stimulated with anti-CD3/CD28 (2 μg ml−1) for the indicated time. f, Proliferation of wild-type or Fbxo38CKO CD8+ T cells measured by Celltracker APC dilution. n = 3 biologically independent samples. Cells were loaded with Celltracker (0.5 μM) for 20 min at 37 °C before stimulation. g, Apoptosis of wild-type or Fbxo38CKO CD8+ T cells measured by annexin V and propidium iodide (PI) staining. n = 3 biologically independent samples. Data are representative of three (a, c, d, f, g) or four (b) independent experiments. Data were analysed by unpaired Student’s t-test (bd, f, g). Source data

  5. Extended Data Fig. 5 Regulation of PD-1 surface levels by FBXO38.

    a, b, PD-1 surface levels in activated CD8+ T cells of Fbxo38flox/flox (wild-type) or Fbxo38CKO (CKO) mice at different time points. Naive CD8+ T cells were stimulated with anti-CD3/CD28 (2 μg ml−1 or 5 μg ml−1). n = 3 biologically independent samples. cf, Assessment of the role of FBXO38 in p97-mediated degradation of PD-1 and TCR at the endoplasmic reticulum. c, d, Naive CD8+ T cells from wild-type or Fbxo38CKO mice were stimulated with anti-CD3/CD28 (2 μg ml−1) for 96 h and incubated with the p97 inhibitor CB-5083 (0.5 or 1 μM) for 12 h. n = 3 biologically independent samples. e, f, Naive CD8+ T cells from wild-type or Fbxo38CKO mice were incubated with the p97 inhibitor CB-5083 (0.5 or 1 μM) for 12 h. n = 3 biologically independent samples. Data are representative of two independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001,  unpaired Student’s t-test (ac, e, f) or unpaired Student’s t-test with Welch’s correction (d). Source data

  6. Extended Data Fig. 6 Functional roles of FBXO38 in MC38 colorectal carcinoma model.

    a, Gating strategy of tumour-infiltrating leukocytes. Cells were first gated by FSC/SSC to exclude debris, followed by gating FSC-A and FSC-H to eliminate non-singlet cells. CD8+ cells were gated for CD44 and PD-1 expression analysis. Conventional CD4+ (CD25) cells and Treg cells (CD4+FOXP3+) were gated for PD-1 expression. This gating strategy is applicable to c–g and Fig. 3e–o. bg, MC38 tumour progression and phenotype of tumour-infiltrating T cells were assessed in wild-type and Fbxo38CKO mice. b, Tumour size was recorded every two days from day 7. n = 10 mice. cg, Tumour-infiltrating T cells were isolated and analysed on day 16. n = 6 mice. PD-1 levels of CD8+, conventional CD4 (CD4+CD25) and Treg (CD4+FOXP3+) cells were assessed in ce. Total number of CD8+ T cells, total number of CD4+ T cells, the CD8+/CD4+ cell ratio and Treg cell population in the tumour microenvironment were also assessed in f, g. bg, Data are representative of two independent experiments. **P < 0.01, unpaired Student’s t-test (c, d (right), e, f (left, middle)), unpaired Student’s t-test with Welch’s correction (d (left), f (right), g) or two-way ANOVA (b). Source data

  7. Extended Data Fig. 7 Fbxo38 knockdown does not affect CD8+ T cell activation.

    a, b, Fbxo38 knockdown efficiency. c, TCR and CD28 surface levels of control and Fbxo38-knockdown cells. dg, Control and Fbxo38-knockdown CD8+ T cells were sorted and stimulated with anti-CD3/CD28 to measure TCR signalling and cytokine production. d, Phosphorylation of TCR signalling proteins in control and Fbxo38-knockdown CD8+ T cells after stimulation for indicated times with soluble anti-CD3/CD28 (5 μg ml−1). e, CD69 surface levels in control and Fbxo38-knockdown CD8+ T cells after stimulation for 5 h with anti-CD3/CD28 (5 μg ml−1). n = 3 biologically independent samples. f, g, IFNγ secretion in control and Fbxo38-knockdown CD8+ T cells after stimulation for 24 h (f) or 48 h (g) with anti-CD3/CD28 (5 μg ml−1). n = 3 biologically independent samples. ag, Data are representative of at least three independent experiments. eg, Data were analysed by unpaired Student’s t-test. For gel source data, see Supplementary Fig. 1. Source data

  8. Extended Data Fig. 8 Fbxo38 knockdown impairs anti-tumour function of CD8+ T cells due to higher PD-1 expression.

    a, PD-1 surface levels of control or Fbxo38-knockdown CD8+ T cells. Cells were stimulated with anti-CD3/CD28 (5 μg ml−1) for 48 h. n = 3 biologically independent samples. b, PD-L1 expression in the EL-4 cell line. c, Killing ability of Fbxo38-knockdown OT-1 CD8+ T cells. OT-1 CD8+ T cells were transduced with control or Fbxo38 shRNA constructs. Transduced cells were sorted by GFP expression and stimulated with OVA-pulsed splenocytes for three days. Sorted cells were stimulated with OVA-pulsed EL-4 cells and a lactate dehydrogenase (LDH) assay was used to measure cytotoxicity. n = 4 biologically independent samples. The ratio of cytotoxic T lymphocytes (CTL) and EL-4 cells was 2:1. Anti-PD-1 antibody (J43, 1 μg ml−1) was used to block PD-1 signalling during the killing process. dm, Adoptive transfer therapy of control or Fbxo38-knockdown OT-1 cells against B16F10-OVA melanoma. d, Experimental approach of the adoptive T cell transfer therapy. Melanoma-bearing mice received T cell therapy alone (ek) or received T cell therapy followed by anti-PD-1 therapy (l, m). e, Gating strategy for transferred OT-1 CD8+ cells in the lymph node. Cells isolated form lymph nodes were stained by anti-CD8–PE–Cy7 and anti-PD-1–APC and analysed by flow cytometry. Transferred CD8+ T cells were gated by GFP (FITC) expression. fi, Transferred CD8+ T cells were analysed in the draining and non-draining lymph nodes on day 18. n = 5 (shRNA1) or 6 (control and shRNA2) mice. jm, Tumour size and survival curves. n = 16 mice for the PBS group and controls treated with anti-PD-1 in l, m; n = 15 mice for other groups in jm. Data are representative of three (ac) or two (fm) independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, unpaired Student’s t-test (a, c, h, i), Mann–Whitney U-test (f, g), two-way ANOVA (j, l) or log-rank (Mantel–Cox) test (k, m). Source data

  9. Extended Data Fig. 9 Effect of cytokines on regulating FBXO38 and PD-1 levels.

    af, Naive CD8+ T cells were freshly isolated from the spleen and stimulated under different conditions. n = 3 biologically independent samples in all experiments. a, b, Cells were stimulated with anti-CD3 (2 μg ml−1) alone without additional cytokines for 0–4 days. a, Fbxo38 transcriptional levels were measured by RT–qPCR. b, FBXO38 expression levels on day 4 were measured by immunoblotting. c, d, Cells were stimulated with anti-CD3/CD28 (2 μg ml−1) in the presence or absence of 10 ng ml−1 IL-10 or IL-15 for 0–4 days. e, f, Cells were stimulated with anti-CD3/CD28 (2 μg ml−1) in the presence of 0–100 ng ml−1 interleukins (IL-10 or IL-15) or interferons (IFNα, IFNβ or IFNγ) for four days. Naive cells were used as a control. g, The genetic regions to which STAT5 binds on the Fbxo38 gene loci are shown. This figure was reconstructed using the previously published GEO dataset GSM652878 and viewed with UCSC genome browser (genome version mm9)30. hn, Naive CD8+ T cells were freshly isolated from spleen and stimulated with anti-CD3/CD28 (2 μg ml−1) in the presence of 0.5, 1, 10 or 100 ng ml−1 IL-2 for 0–4 days. ik, Percentages of PD-1+ and CD44+ of total CD8+ T cells and surface PD-1 MFI of CD8+ PD-1+ cells were assessed. l, m, Fbxo38 and Pdcd1 transcriptional levels of total CD8+ T cells were assessed by RT–qPCR and normalized to those of naive cells. n, FBXO38 expression was measured by immunoblotting. n = 3 biologically independent samples. Data are representative of four (a, c), three (d, e) or two (b, f, hn) independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, unpaired Student’s t-test with Welch’s correction (j (48 h, 0.5 versus 1), k (72 h, 0.5 versus 100, 96 h, 0.5 versus 1), l (48 h, 0.5 versus 1 and 0.5 versus 10, 96 h, 0.5 versus 10, 0.5 versus 100)), or  unpaired Student’s t-test. For gel source data, see Supplementary Fig. 1. Source data

Supplementary information

  1. Supplementary Figure 1

    This file contains gel source data

  2. Reporting Summary

  3. Supplementary Table 1

    A list of PD-1 interacting proteins identified by GST pull-down coupled with mass spectrometry

  4. Supplementary Data 1

    RNA-seq information of WT and CKO CD8+ T cells at naïve or activated state

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