Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

The ubiquitin-specific protease USP8 is critical for the development and homeostasis of T cells

Nature Immunology volume 16, pages 950–960 (2015)Cite this article

Subjects

Abstract

The modification of proteins by ubiquitin has a major role in cells of the immune system and is counteracted by various deubiquitinating enzymes (DUBs) with poorly defined functions. Here we identified the ubiquitin-specific protease USP8 as a regulatory component of the T cell antigen receptor (TCR) signalosome that interacted with the adaptor Gads and the regulatory molecule 14-3-3β. Caspase-dependent processing of USP8 occurred after stimulation of the TCR. T cell–specific deletion of USP8 in mice revealed that USP8 was essential for thymocyte maturation and upregulation of the gene encoding the cytokine receptor IL-7Rα mediated by the transcription factor Foxo1. Mice with T cell–specific USP8 deficiency developed colitis that was promoted by disturbed T cell homeostasis, a predominance of CD8+ γδ T cells in the intestine and impaired regulatory T cell function. Collectively, our data reveal an unexpected role for USP8 as an immunomodulatory DUB in T cells.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: USP8 interacts with Gads, 14-3-3β and the TCR signalosome.
Figure 2: Altered T cell development in Usp8f/fCd4-Cre mice.
Figure 3: Diminished thymocyte proliferation after depletion of USP8.
Figure 4: Signaling defects in Usp8f/fCd4-Cre thymocytes.
Figure 5: Usp8f/fCd4-Cre mice exhibit lymphopenia and hyperactivation of peripheral T cells.
Figure 6: T cell–specific deficiency in USP8 causes lethal colitis.
Figure 7: Intrinsic function of USP8 is critical for T cell homeostasis.
Figure 8: γδ T cells promote colitis in the absence of functional Treg cells.

Similar content being viewed by others

Accession codes

Primary accessions

Gene Expression Omnibus

References

  1. Jiang, X. & Chen, Z.J. The role of ubiquitylation in immune defence and pathogen evasion. Nat. Rev. Immunol. 12, 35–48 (2012).

    Article  CAS  Google Scholar 

  2. Sun, S.C. Deubiquitylation and regulation of the immune response. Nat. Rev. Immunol. 8, 501–511 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Niendorf, S. et al. Essential role of ubiquitin-specific protease 8 for receptor tyrosine kinase stability and endocytic trafficking in vivo. Mol. Cell. Biol. 27, 5029–5039 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Wright, M.H., Berlin, I. & Nash, P.D. Regulation of endocytic sorting by ESCRT-DUB-mediated deubiquitination. Cell Biochem. Biophys. 60, 39–46 (2011).

    Article  CAS  PubMed  Google Scholar 

  5. Kato, M., Miyazawa, K. & Kitamura, N. A deubiquitinating enzyme UBPY interacts with the Src homology 3 domain of Hrs-binding protein via a novel binding motif PX(V/I)(D/N)RXXKP. J. Biol. Chem. 275, 37481–37487 (2000).

    Article  CAS  PubMed  Google Scholar 

  6. Reincke, M. et al. Mutations in the deubiquitinase gene USP8 cause Cushing's disease. Nat. Genet. 47, 31–38 (2014).

    Article  PubMed  CAS  Google Scholar 

  7. Mizuno, E., Kitamura, N. & Komada, M. 14–3-3-dependent inhibition of the deubiquitinating activity of UBPY and its cancellation in the M phase. Exp. Cell Res. 313, 3624–3634 (2007).

    Article  CAS  PubMed  Google Scholar 

  8. Berry, D.M., Nash, P., Liu, S.K., Pawson, T. & McGlade, C.J. A high-affinity Arg-X-X-Lys SH3 binding motif confers specificity for the interaction between Gads and SLP-76 in T cell signaling. Curr. Biol. 12, 1336–1341 (2002).

    Article  CAS  PubMed  Google Scholar 

  9. Harkiolaki, M. et al. Structural basis for SH3 domain-mediated high-affinity binding between Mona/Gads and SLP-76. EMBO J. 22, 2571–2582 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Kaneko, T. et al. Structural insight into modest binding of a non-PXXP ligand to the signal transducing adaptor molecule-2 Src homology 3 domain. J. Biol. Chem. 278, 48162–48168 (2003).

    Article  CAS  PubMed  Google Scholar 

  11. Brownlie, R.J. & Zamoyska, R. T cell receptor signalling networks: branched, diversified and bounded. Nat. Rev. Immunol. 13, 257–269 (2013).

    Article  CAS  PubMed  Google Scholar 

  12. Heissmeyer, V. & Vogel, K.U. Molecular control of Tfh-cell differentiation by Roquin family proteins. Immunol. Rev. 253, 273–289 (2013).

    Article  PubMed  CAS  Google Scholar 

  13. Naik, E. et al. Regulation of proximal T cell receptor signaling and tolerance induction by deubiquitinase Usp9X. J. Exp. Med. 211, 1947–1955 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Zou, Q. et al. USP15 stabilizes MDM2 to mediate cancer-cell survival and inhibit antitumor T cell responses. Nat. Immunol. 15, 562–570 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Soares, L. et al. Two isoforms of otubain 1 regulate T cell anergy via GRAIL. Nat. Immunol. 5, 45–54 (2004).

    Article  CAS  PubMed  Google Scholar 

  16. Mackall, C.L., Fry, T.J. & Gress, R.E. Harnessing the biology of IL-7 for therapeutic application. Nat. Rev. Immunol. 11, 330–342 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Kerdiles, Y.M. et al. Foxo1 links homing and survival of naive T cells by regulating L-selectin, CCR7 and interleukin 7 receptor. Nat. Immunol. 10, 176–184 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Ouyang, W., Beckett, O., Flavell, R.A. & Li, M.O. An essential role of the Forkhead-box transcription factor Foxo1 in control of T cell homeostasis and tolerance. Immunity 30, 358–371 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Takada, K. & Jameson, S.C. Naive T cell homeostasis: from awareness of space to a sense of place. Nat. Rev. Immunol. 9, 823–832 (2009).

    Article  CAS  PubMed  Google Scholar 

  20. Ouyang, W. et al. Novel Foxo1-dependent transcriptional programs control Treg cell function. Nature 491, 554–559 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Obsil, T. & Obsilova, V. Structural basis of 14–3-3 protein functions. Semin. Cell Dev. Biol. 22, 663–672 (2011).

    Article  CAS  PubMed  Google Scholar 

  22. Tzivion, G., Dobson, M. & Ramakrishnan, G. FoxO transcription factors; Regulation by AKT and 14–3-3 proteins. Biochim. Biophys. Acta 1813, 1938–1945 (2011).

    Article  CAS  PubMed  Google Scholar 

  23. Surh, C.D. & Sprent, J. Homeostasis of naive and memory T cells. Immunity 29, 848–862 (2008).

    Article  CAS  PubMed  Google Scholar 

  24. Park, S.G. et al. T regulatory cells maintain intestinal homeostasis by suppressing γδ T cells. Immunity 33, 791–803 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  26. Lasserre, R. et al. Release of serine/threonine-phosphorylated adaptors from signaling microclusters down-regulates T cell activation. J. Cell Biol. 195, 839–853 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Yoder, J. et al. Requirement for the SLP-76 adaptor GADS in T cell development. Science 291, 1987–1991 (2001).

    Article  CAS  PubMed  Google Scholar 

  28. Vantourout, P. & Hayday, A. Six-of-the-best: unique contributions of γδ T cells to immunology. Nat. Rev. Immunol. 13, 88–100 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Ketscher, L. et al. Selective inactivation of USP18 isopeptidase activity in vivo enhances ISG15 conjugation and viral resistance. Proc. Natl. Acad. Sci. USA 112, 1577–1582 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. De, A., Dainichi, T., Rathinam, C.V. & Ghosh, S. The deubiquitinase activity of A20 is dispensable for NF-κB signaling. EMBO Rep. 15, 775–783 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Lu, T.T. et al. Dimerization and ubiquitin mediated recruitment of A20, a complex deubiquitinating enzyme. Immunity 38, 896–905 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Wang, X. et al. Attenuation of T cell receptor signaling by serine phosphorylation-mediated lysine 30 ubiquitination of SLP-76 protein. J. Biol. Chem. 287, 34091–34100 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. McCaughtry, T.M. et al. Conditional deletion of cytokine receptor chains reveals that IL-7 and IL-15 specify CD8 cytotoxic lineage fate in the thymus. J. Exp. Med. 209, 2263–2276 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Ouyang, W. et al. Foxo proteins cooperatively control the differentiation of Foxp3+ regulatory T cells. Nat. Immunol. 11, 618–627 (2010).

    Article  CAS  PubMed  Google Scholar 

  35. Nanno, M. et al. Exacerbating role of γδ T cells in chronic colitis of T-cell receptor α mutant mice. Gastroenterology 134, 481–490 (2008).

    Article  CAS  PubMed  Google Scholar 

  36. Gubbels Bupp, M.R. et al. T cells require Foxo1 to populate the peripheral lymphoid organs. Eur. J. Immunol. 39, 2991–2999 (2009).

    Article  CAS  PubMed  Google Scholar 

  37. Rubtsov, Y.P. et al. Regulatory T cell-derived interleukin-10 limits inflammation at environmental interfaces. Immunity 28, 546–558 (2008).

    Article  CAS  PubMed  Google Scholar 

  38. Fontenot, J.D., Rasmussen, J.P., Gavin, M.A. & Rudensky, A.Y. A function for interleukin 2 in Foxp3-expressing regulatory T cells. Nat. Immunol. 6, 1142–1151 (2005).

    Article  CAS  PubMed  Google Scholar 

  39. Förster, R., Davalos-Misslitz, A.C. & Rot, A. CCR7 and its ligands: balancing immunity and tolerance. Nat. Rev. Immunol. 8, 362–371 (2008).

    Article  PubMed  CAS  Google Scholar 

  40. Garcia, S., DiSanto, J. & Stockinger, B. Following the development of a CD4 T cell response in vivo: from activation to memory formation. Immunity 11, 163–171 (1999).

    Article  CAS  PubMed  Google Scholar 

  41. Lee, P.P. et al. A critical role for Dnmt1 and DNA methylation in T cell development, function, and survival. Immunity 15, 763–774 (2001).

    Article  CAS  PubMed  Google Scholar 

  42. Sledzińska, A. et al. TGF-β signalling is required for CD4+ T cell homeostasis but dispensable for regulatory T cell function. PLoS Biol. 11, e1001674 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  43. Kisielow, P., Bluthmann, H., Staerz, U.D., Steinmetz, M. & von Boehmer, H. Tolerance in T-cell-receptor transgenic mice involves deletion of nonmature CD4+8+ thymocytes. Nature 333, 742–746 (1988).

    Article  CAS  PubMed  Google Scholar 

  44. Orban, P.C., Chui, D. & Marth, J.D. Tissue- and site-specific DNA recombination in transgenic mice. Proc. Natl. Acad. Sci. USA 89, 6861–6865 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Lugassy, J. et al. Modulation of TCR responsiveness by the Grb2-family adaptor, Gads. Cell. Signal. 27, 125–134 (2015).

    Article  CAS  PubMed  Google Scholar 

  46. Larghi, P. et al. VAMP7 controls T cell activation by regulating the recruitment and phosphorylation of vesicular Lat at TCR-activation sites. Nat. Immunol. 14, 723–731 (2013).

    Article  CAS  PubMed  Google Scholar 

  47. Gnesutta, N. et al. Cloning and characterization of mouse UBPy, a deubiquitinating enzyme that interacts with the ras guanine nucleotide exchange factor CDC25(Mm)/Ras-GRF1. J. Biol. Chem. 276, 39448–39454 (2001).

    Article  CAS  PubMed  Google Scholar 

  48. Yeh, W.C. et al. Early lethality, functional NF-κB activation, and increased sensitivity to TNF-induced cell death in TRAF2-deficient mice. Immunity 7, 715–725 (1997).

    Article  CAS  PubMed  Google Scholar 

  49. Lahl, K. et al. Selective depletion of Foxp3+ regulatory T cells induces a scurfy-like disease. J. Exp. Med. 204, 57–63 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Klipper-Aurbach, Y. et al. Mathematical formulae for the prediction of the residual beta cell function during the first two years of disease in children and adolescents with insulin-dependent diabetes mellitus. Med. Hypotheses 45, 486–490 (1995).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank M. Oberle, C. Fix, T. el Gaz, A. Nikolaev and T. Bass for technical assistance; J. Wersing for cell sorting; S. Hemmers for support with generation of the Cd4-CreERT2 mice; A. Izcue for protocols; S. Feller (University of Halle) and E. Martegani (University of Milano-Bicocca) for anti-USP8 antiserum; and R. Beyaert (University of Ghent) for tagged ubiquitin-expression vectors. Supported by Deutsche Forschungsgemeinschaft (KN590/4-1 to K.-P.K.; support via EXC294 (the Center for Biological Signaling Studies) to W.W.S.).

Author information

Author notes

  1. Agnes Kisser, Sandra Niendorf & Andreas Schlosser

    Present address: Present addresses: Ludwig Boltzmann Institute for Health Technology Assessment, Vienna, Austria (A.K.), Department of Infectious Diseases, Robert Koch Institute, Berlin, Germany (S.N.), and Rudolf-Virchow-Zentrum für Experimentelle Biomedizin, Würzburg, Germany (A.S.).,

Authors and Affiliations

  1. Institute of Neuropathology, University of Freiburg, Freiburg, Germany

    Almut Dufner, Anja Basters, Anne Schönle, Marco Prinz & Klaus-Peter Knobeloch

  2. Leibniz Institute for Molecular Pharmacology, Berlin, Germany

    Agnes Kisser, Sandra Niendorf & Klaus-Peter Knobeloch

  3. Institute for Molecular Medicine, University Medical Center of the Johannes Gutenberg University of Mainz, Mainz, Germany

    Sonja Reissig & Ari Waisman

  4. Biotechnology Institute Thurgau at the University of Konstanz, Kreuzlingen, Switzerland

    Annette Aichem & Marcus Groettrup

  5. Center for Biological Systems Analysis, University of Freiburg, Freiburg, Germany

    Thorsten Kurz & Andreas Schlosser

  6. Ruth and Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel

    Deborah Yablonski

  7. Division of Immunology, Department of Biology, University of Konstanz, Konstanz, Germany

    Marcus Groettrup

  8. Institute for Medical Microbiology, Immunology, and Hygiene, Technische Universität München, Germany

    Thorsten Buch

  9. Institute of Laboratory Animal Sciences, University of Zurich, Zurich, Switzerland

    Thorsten Buch

  10. Department of Molecular Immunology, Faculty of Biology, and Center of Chronic Immunodeficiency, University of Freiburg, Freiburg, Germany

    Wolfgang W Schamel

  11. BIOSS Center for Biological Signaling Studies, University of Freiburg, Freiburg, Germany

    Wolfgang W Schamel & Marco Prinz

Authors
  1. Almut Dufner
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Agnes Kisser
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sandra Niendorf
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Anja Basters
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Sonja Reissig
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Anne Schönle
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Annette Aichem
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Thorsten Kurz
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Andreas Schlosser
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Deborah Yablonski
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Marcus Groettrup
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Thorsten Buch
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Ari Waisman
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Wolfgang W Schamel
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Marco Prinz
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Klaus-Peter Knobeloch
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

A.D. designed and performed experiments, analyzed the data and wrote the paper; A.K. designed and performed experiments; S.N. generated the Usp8f/f mice and designed and performed experiments; A.B. performed the yeast two-hybrid screen; S.R. and A.W. contributed to or carried out the endoscopic recording and histological analyses; A.Schö provided help with experiments; A.A. and M.G. contributed to the yeast two-hybrid screen and provided reagents; T.K. performed the gene-expression microarray analysis; A.Schl performed the identification of ubiquitination sites by mass spectrometry; D.Y. generated and provided mutant Jurkat cells; T.B. generated Cd4-CreERT2 mice; W.W.S. provided reagents and contributed to the calcium-influx experiment; M.P. contributed to the histological analyses; and K.-P.K. supervised the project and wrote the paper.

Corresponding authors

Correspondence to Almut Dufner or Klaus-Peter Knobeloch.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Identification of USP8-interacting proteins by yeast two-hybrid screening.

(a) Schematic representation of USP8 baits: Bait 1 (residues 143-485 of human USP8) encodes the N-terminal SH3BM. Bait 2 (residues 481-764) harbors the 14-3-3BM and the C-terminal SH3BM. (b) In the initial screen 4 independent clones for each 14-3-3β and Gads were identified. For validation NMY51 cells were co-transformed with vectors encoding bait 1 or 2 and either Gads or 14-3-3β. The interaction between the proteins was confirmed by yeast growth on agar plates lacking tryptophane, leucine and histidine (-TLH) and by β-Galactosidase assay (X-Gal). As a control, LaminC was coexpressed with Gads and 14-3-3β, respectively.

Supplementary Figure 2 USP8 modulates ubiquitination of Gads and 14-3-3β.

(a) HEK293T cells were transfected with an empty vector or expression vectors for FLAG-tagged USP8 and/or FLAG-tagged ubiquitin, HA-tagged Gads or HA-tagged 14-3-3β as indicated. IP was performed with anti-HA(3F10) and analyzed by immunoblot with indicated antibodies. *, ubiquitin modification. Results are representative of 2 independent experiments. (b) MS analysis of 14-3-3β ubiquitination sites. Flag-tagged USP8(C786A) was cotransfected with FLAG-ubiquitin and HA-tagged 14-3-3β in 293HEKT cells. An anti-HA-IP was performed and the isolated proteins were separated by SDS-PAGE. A protein band corresponding to the accumulated ubiquitin-modified 14-3-3β molecule was excised and analyzed as described in Methods. Briefly, proteins were digested in gel in separate experiments with trypsin and with thermolysin. Both digests were analyzed with CID on a Q-TOF instrument and with ETD on an ion trap instrument. The peptide MDKSELVQKAK with an N-terminal acetylation, an oxidized methionine and with a lysine linked to LRGG was generated in the thermolysin digest and identified by three CID spectra (Mascot scores 34, 35 and 43) and one ETD spectrum (Mascot score 35). The shown ETD spectrum allows unambiguous localization of the ubiquitination site to K9. The peptide VFYYEILNSPEKACS with a carbamidomethylated cysteine and a LRGG-modified lysine residue was also generated in the thermolysin digest and identified by one CID spectrum (Mascot score 59) and one ETD spectrum (Mascot score 59). The shown CID spectrum allowed unambiguous localization of the ubiquitination site to position K189.

Supplementary Figure 3 Deletion of Usp8 in thymocytes.

(a) Verification of Usp8 gene deletion in thymocytes of Usp8f/fCd4-Cre mice by Southern blot. Genomic DNA from sorted thymocytes derived from wild-type (WT) and Usp8f/f Cd4-Cre mice was digested with NcoI and analyzed3. Deletion is indicated by a 6kb band representing the deleted allele. The 5kb band represents the floxed allele. (b) USP8 protein expression in sorted thymocyte subsets of Usp8f/f (ctrl) and Usp8f/fCd4-Cre (ΔUsp8) mice was analyzed by immunoblot. (c) USP8 protein expression in sorted thymocyte subsets of Usp8f/f (ctrl) and Usp8f/fLck-Cre (ΔUsp8) mice was analzed by immunoblot. Results shown in (a-c) are representative of 3 (a) or 2 (b,c) independent experiments. (d) CD25 and CD44 expression on DN thymocytes for analysis of the early DN1 (CD44+CD25−), DN2 (CD44+CD25+), DN3 (CD44−CD25+) and DN4(CD44−CD25−) stages of thymocyte development. (e) Thymocyte development in Usp8f/fLck-Cre mice. Top, flow cytometric analysis of CD4 and CD8 expression on thymocytes derived from Usp8f/f and Usp8f/fLck-Cre mice. Bottom, TCRβ expression on thymocyte subsets of Usp8f/f and Usp8f/fLck-Cre mice. Results in (d) and (e) are representative of 3 independent experiments. (f) Thymocyte survival assay. Thymocytes derived from Usp8f/f and Usp8f/fCd4-Cre mice (Cd4-Cre:+) were stimulated as in Fig. 3a for 48 h and stained with propidium iodide (PI) and a fluorescent conjugate of Annexin V. The percentages of cells in early apoptosis (AnnexinV+PI−) and late apoptosis (AnnexinV+PI+) are indicated. Results in (f) are the means of 3 independent experiments ±SD.

Supplementary Figure 4 Normal TCR signaling in Usp8f/fCd4-Cre thymocytes.

(a,b) Expression of STAM2 and overall ubiquitination in CD4+ thymocytes. (a) Protein lysates from thymocytes were analyzed by immunoblot for USP8 and STAM2 expression. (b) Protein lysates from anti-CD4–purified thymocytes were analyzed by immunoblot for abundance of proteins harboring K48-linked and K63-linked ubiquitin chains. (c) Ligand-induced TCR downmodulation. Thymocytes were stained on ice with biotinylated hamster anti-TCRβ. Subsequently, the TCR was crosslinked with hamster-specific secondary antibodies at 37°C for the indicated times. Remaining surface TCR was labeled with streptavidin-PE and measured by flow cytometry in SP4 (left) and DP gates. The percentages of surface expression are presented as the mean fluorescence using the untreated controls as reference (n = 5). (d–i) Total thymocytes (d,e,i) or enriched DP thymocytes (f,g,h) were stimulated for the indicated times with anti-CD3 plus anti-CD28. Phosphorylation of Erk1/2, Jnk, p38, Akt (d) and IκBα (e), and actin protein abundance was monitored by immunoblot. (f) Total protein abundance of IκBα, 14-3-3β, Gads and actin was monitored by immunoblot. (g,h) NF-κB and AP-1 activation were determined by EMSA. (i) Tyrosine phosphorylation was detected by immunoblot. Total protein abundance corresponds to the actin as shown in (d). (j) Ca2+ flux in DP-enriched thymocytes loaded with indo-1-AM (5 μg/ml) and stimulated with anti-CD3 plus anti-CD28. The graph shows the ratio of bound to unbound indo-1-AM as a measure of Ca2+ influx. Results for a,b and d-j are representative of at least 3 independent experiments. SFo, surface fluorescence.

Supplementary Figure 5 Microarray analysis of gene expression in DP thymocytes upon depletion of Usp8.

(a) Microarray analysis of 2 groups of Usp8f/f and Usp8f/fCd4-Cre DP thymocyte populations (2 per group in each experiment, 8 mice in total), which resulted in a list of 74 genes with a significant >2-fold difference. Heatmap of the 74 relevant genes. (b) Top network extracted by analysis of the microarray gene expression data using the Ingenuity software.

Supplementary Figure 6 Consequences of Usp8 deletion in peripheral T cells.

(a) Representative flow cytometric analysis of lymphocyte subsets in MLNs derived from Usp8f/f, Usp8+/+Cd4-Cre and Usp8f/fCd4-Cre mice was performed as in Fig. 5a (n ≥ 6). (b) Usp8 gene deletion in peripheral T cells from Usp8f/fCd4-Cre mice. Southern blot analysis was performed using sorted CD4+ and CD8+ cells from spleen and MLNs derived from Usp8+/+(WT) and Usp8f/fCd4-Cre mice. (c) USP8 and Foxo1 protein expression in sorted effector T cells derived from Usp8f/f and Usp8f/fCd4-Cre mice was determined by immunoblot. (d) IL-7Rα expression on effector (CD44hiCD62Llo) and naïve (CD44loCD62Lhi) T cells. CD3+CD4+ splenocytes from Usp8f/f and Usp8f/fCd4-Cre mice were analyzed for CD44, CD62L and IL-7Rα expression by flow cytometry. (n = 5; p = 0.006; paired, 2-sided t-test). (e) T cell homeostasis in Usp8f/fLck-Cre mice. Percentages of B220+, CD3+, CD4+ and CD8+ lymphocytes and the distribution of naïve and effector T cells within the CD3+CD4+ population were determined in spleen (n = 3). Bottom, percentage of IL-7Rα+ naïve T cells. Results shown are the means of 4 independent experiments ±SD. *, P < 0.01 (unpaired, 2-sided t-test). (f) Representative flow cytometry analysis of CCR7 expression on CD4+ splenocytes derived from Usp8f/f and Usp8f/fCd4-Cre mice (n = 3).

Supplementary Figure 7 Proximal TCR signaling is not affected by OHT-induced deletion of Usp8.

(a–d) CD4+ T cells were enriched from spleens of Usp8f/+Cd4-CreERT2 (ctrl) and Usp8f/fCd4-CreERT2 (iKO) mice, expanded and treated with OHT for 48h as in Fig. 7c. Subsequently, cells were starved for 2h and restimulated with anti-CD3 and anti-CD28 for the times indicated. (a) Immunoblot analysis of USP8, Lat, phospho-Lat, phospho-PLC-γ1, phospho-Erk1/2 and actin expression. (b) Cells were pretreated with MG132 and chloroquine to inhibit proteasomal and lysosomal degradation, respectively. Immunoblot analysis of USP8, Gads, 14-3-3β, phospho-IκBα, and actin expression. (c) Cell lysates were analyzed for actin and USP8 expression. In parallel, p38- and PKB-phosphorylation was monitored using phospho-specific antibodies. (d) Immunoblot analysis of USP8, proteins modified by K48-linked ubiquitination, tyrosine-phosphorylated proteins, SLP-76, SLP-76 phosphorylated at S376 (14-3-3BM), phospho-Jnk, Foxo1, GRAIL and actin. The results shown (a-d) are representative of at least 3 independent experiments.

Supplementary Figure 8 TCR signalosome formation upon OHT-induced deletion of Usp8 and binding of USP8 to the CD3-CD28 cluster in Jurkat-derived USP8-deficient cells.

(a) CD4+ T cells were enriched from spleens of Usp8f/+Cd4-CreERT2 (ctrl) and Usp8f/fCd4-CreERT2 (iKO) mice, expanded and treated with OHT for 48h as in Fig. 7c. Subsequently, TCR signalosomes were purified after restimulation with Dynabeads® Mouse T-Activator CD3/CD28. Total lysate (input) and components of the signaling complex were analyzed by immunoblot using antibodies directed against USP8, tyrosine-phosphorylated proteins, CD3ε and the indicated signaling components. (b) Jurkat-derived Gads-KO (dG32Gads−/−), Lat-KO (J.Cam2.5), and SLP-76-KO (J14) cells were stimulated with Dynabeads® Human T-Activator CD3/CD28 prior to purification of TCR signalosomes. Antibodies directed against USP8 and TCR-proximal signaling components were used to analyze the input and purified signalosome components as indicated. Results (a,b) are representative of at least 3 independent experiments.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8, Supplementary Table 1 (PDF 1523 kb)

41590_2015_BFni3230_MOESM19_ESM.mov

Appearance of healthy colon visualized by in vivo miniendoscopic analysis of Usp8f/f mouse. Movies are representative for endoscopic examination of 3 mice. (MOV 3765 kb)

41590_2015_BFni3230_MOESM20_ESM.mov

Endoscopic recording of Usp8f/fCd4-Cre colon revealing typical signs of colitis like enhanced granularity, loss of the vascular pattern of the mucosa and a reduction in translucency. Movies are representative for endoscopic examination of 5 mice (MOV 4062 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Dufner, A., Kisser, A., Niendorf, S. et al. The ubiquitin-specific protease USP8 is critical for the development and homeostasis of T cells. Nat Immunol 16, 950–960 (2015). https://doi.org/10.1038/ni.3230

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ni.3230

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing