Arginine methylation controls the strength of γc-family cytokine signaling in T cell maintenance

Abstract

The methylation of arginine residues in proteins is a post-translational modification that contributes to a wide range of biological processes. Many cytokines involved in T cell development and activation utilize the common cytokine receptor γ-chain (γc) and the kinase JAK3 for signal transduction, but the regulatory mechanism that underlies the expression of these factors remains unclear. Here we found that the arginine methyltransferase PRMT5 was essential for the maintenance of invariant natural killer T cells (iNKT cells), CD4+ T cells and CD8+ T cells. T cell–specific deletion of Prmt5 led to a marked reduction in signaling via γc-family cytokines and a substantial loss of thymic iNKT cells, as well as a decreased number of peripheral CD4+ T cells and CD8+ T cells. PRMT5 induced the symmetric dimethylation of Sm proteins that promoted the splicing of pre-mRNA encoding γc and JAK3, and this critically contributed to the expression of γc and JAK3. Thus, arginine methylation regulates strength of signaling via γc-family cytokines by facilitating the expression of signal-transducing components.

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Fig. 1: PRMT5 expression and symmetric arginine dimethylation in T cells.
Fig. 2: T cell–specific deletion of PRMT5 causes a decrease in iNKT cells, CD4+ T cells and CD8+ T cells.
Fig. 3: Cell-autonomous function for PRMT5 in the regulation of iNKT cells, CD4+ T cells and CD8+ T cells.
Fig. 4: PRMT5 is required for γc expression in iNKT cells.
Fig. 5: Impaired survival and proliferation of PRMT5-deficient peripheral T cells.
Fig. 6: PRMT5 regulates signaling via γc-family cytokines by promoting the expression of γc and JAK3.
Fig. 7: PRMT5 facilitates splicing of pre-mRNA from Il2rg and Jak3.

Data availability

The data that support the findings of this study are available from the corresponding author upon request. The RNA-seq data have been deposited in GEO with accession code GSE118135.

References

  1. 1.

    Bedford, M. T. & Clarke, S. G. Protein arginine methylation in mammals: who, what, and why. Mol. Cell 33, 1–13 (2009).

    CAS  Article  Google Scholar 

  2. 2.

    Blanchet, F., Schurter, B. T. & Acuto, O. Protein arginine methylation in lymphocyte signaling. Curr. Opin. Immunol. 18, 321–328 (2006).

    CAS  Article  Google Scholar 

  3. 3.

    Pawlak, M. R., Scherer, C. A., Chen, J., Roshon, M. J. & Ruley, H. E. Arginine N-methyltransferase 1 is required for early postimplantation mouse development, but cells deficient in the enzyme are viable. Mol. Cell. Biol. 20, 4859–4869 (2000).

    CAS  Article  Google Scholar 

  4. 4.

    Tee, W.-W. et al. Prmt5 is essential for early mouse development and acts in the cytoplasm to maintain ES cell pluripotency. Genes Dev. 24, 2772–2777 (2010).

    CAS  Article  Google Scholar 

  5. 5.

    Yang, Y. & Bedford, M. T. Protein arginine methyltransferases and cancer. Nat. Rev. Cancer 13, 37–50 (2013).

    CAS  Article  Google Scholar 

  6. 6.

    Kim, S. et al. PRMT5 protects genomic integrity during global DNA demethylation in primordial germ cells and preimplantation embryos. Mol. Cell 56, 564–579 (2014).

    CAS  Article  Google Scholar 

  7. 7.

    Bezzi, M. et al. Regulation of constitutive and alternative splicing by PRMT5 reveals a role for Mdm4 pre-mRNA in sensing defects in the spliceosomal machinery. Genes Dev. 27, 1903–1916 (2013).

    CAS  Article  Google Scholar 

  8. 8.

    Zhang, T. et al. Prmt5 is a regulator of muscle stem cell expansion in adult mice. Nat. Commun. 6, 7140 (2015).

    CAS  Article  Google Scholar 

  9. 9.

    Liu, F. et al. Arginine methyltransferase PRMT5 is essential for sustaining normal adult hematopoiesis. J. Clin. Invest. 125, 3532–3544 (2015).

    Article  Google Scholar 

  10. 10.

    Blanc, R. S., Vogel, G., Chen, T., Crist, C. & Richard, S. PRMT7 preserves satellite cell regenerative capacity. Cell Rep. 14, 1528–1539 (2016).

    CAS  Article  Google Scholar 

  11. 11.

    Kim, J. D. et al. PRMT8 as a phospholipase regulates Purkinje cell dendritic arborization and motor coordination. Sci. Adv. 1, e1500615 (2015).

    Article  Google Scholar 

  12. 12.

    Mowen, K. A., Schurter, B. T., Fathman, J. W., David, M. & Glimcher, L. H. Arginine methylation of NIP45 modulates cytokine gene expression in effector T lymphocytes. Mol. Cell 15, 559–571 (2004).

    CAS  Article  Google Scholar 

  13. 13.

    Richard, S., Morel, M. & Cléroux, P. Arginine methylation regulates IL-2 gene expression: a role for protein arginine methyltransferase 5 (PRMT5). Biochem. J. 388, 379–386 (2005).

    CAS  Article  Google Scholar 

  14. 14.

    Blanchet, F., Cardona, A., Letimier, F. A., Hershfield, M. S. & Acuto, O. CD28 costimulatory signal induces protein arginine methylation in T cells. J. Exp. Med. 202, 371–377 (2005).

    CAS  Article  Google Scholar 

  15. 15.

    Geoghegan, V., Guo, A., Trudgian, D., Thomas, B. & Acuto, O. Comprehensive identification of arginine methylation in primary T cells reveals regulatory roles in cell signalling. Nat. Commun. 6, 6758 (2015).

    CAS  Article  Google Scholar 

  16. 16.

    Webb, L. M. et al. PRMT5-selective inhibitors suppress inflammatory T cell responses and experimental autoimmune encephalomyelitis. J. Immunol. 198, 1439–1451 (2017).

    CAS  Article  Google Scholar 

  17. 17.

    Takeshita, T. et al. Cloning of the γ chain of the human IL-2 receptor. Science 257, 379–382 (1992).

    CAS  Article  Google Scholar 

  18. 18.

    Noguchi, M. et al. Interleukin-2 receptor γ chain mutation results in X-linked severe combined immunodeficiency in humans. Cell 73, 147–157 (1993).

    CAS  Article  Google Scholar 

  19. 19.

    Boyman, O. & Sprent, J. The role of interleukin-2 during homeostasis and activation of the immune system. Nat. Rev. Immunol. 12, 180–190 (2012).

    CAS  Article  Google Scholar 

  20. 20.

    Lantz, O., Sharara, L. I., Tilloy, F., Andersson, A. & DiSanto, J. P. Lineage relationships and differentiation of natural killer (NK) T cells: intrathymic selection and interleukin (IL)-4 production in the absence of NKR-P1 and Ly49 molecules. J. Exp. Med. 185, 1395–1401 (1997).

    CAS  Article  Google Scholar 

  21. 21.

    Leonard, W. J. Cytokines and immunodeficiency diseases. Nat. Rev. Immunol. 1, 200–208 (2001).

    CAS  Article  Google Scholar 

  22. 22.

    Fernandez, D. R. et al. Activation of mammalian target of rapamycin controls the loss of TCRζ in lupus T cells through HRES-1/Rab4-regulated lysosomal degradation. J. Immunol. 182, 2063–2073 (2009).

    CAS  Article  Google Scholar 

  23. 23.

    Gapin, L., Matsuda, J. L., Surh, C. D. & Kronenberg, M. NKT cells derive from double-positive thymocytes that are positively selected by CD1d. Nat. Immunol. 2, 971–978 (2001).

    CAS  Article  Google Scholar 

  24. 24.

    Godfrey, D. I., Stankovic, S. & Baxter, A. G. Raising the NKT cell family. Nat. Immunol. 11, 197–206 (2010).

    CAS  Article  Google Scholar 

  25. 25.

    Lee, Y. J., Holzapfel, K. L., Zhu, J., Jameson, S. C. & Hogquist, K. A. Steady-state production of IL-4 modulates immunity in mouse strains and is determined by lineage diversity of iNKT cells. Nat. Immunol. 14, 1146–1154 (2013).

    CAS  Article  Google Scholar 

  26. 26.

    Savage, A. K. et al. The transcription factor PLZF directs the effector program of the NKT cell lineage. Immunity 29, 391–403 (2008).

    CAS  Article  Google Scholar 

  27. 27.

    Lazarevic, V. et al. The gene encoding early growth response 2, a target of the transcription factor NFAT, is required for the development and maturation of natural killer T cells. Nat. Immunol. 10, 306–313 (2009).

    CAS  Article  Google Scholar 

  28. 28.

    Egawa, T. et al. Genetic evidence supporting selection of the Vα14i NKT cell lineage from double-positive thymocyte precursors. Immunity 22, 705–716 (2005).

    CAS  Article  Google Scholar 

  29. 29.

    Matsuda, J. L. et al. Homeostasis of Vα14i NKT cells. Nat. Immunol. 3, 966–974 (2002).

    CAS  Article  Google Scholar 

  30. 30.

    Tani-ichi, S. et al. Interleukin-7 receptor controls development and maturation of late stages of thymocyte subpopulations. Proc. Natl Acad. Sci. U.S.A. 110, 612–617 (2013).

    CAS  Article  Google Scholar 

  31. 31.

    Gordy, L. E. et al. IL-15 regulates homeostasis and terminal maturation of NKT cells. J. Immunol. 187, 6335–6345 (2011).

    CAS  Article  Google Scholar 

  32. 32.

    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).

    CAS  Article  Google Scholar 

  33. 33.

    Onishi, M. et al. Identification and characterization of a constitutively active STAT5 mutant that promotes cell proliferation. Mol. Cell. Biol. 18, 3871–3879 (1998).

    CAS  Article  Google Scholar 

  34. 34.

    Friesen, W. J. et al. The methylosome, a 20S complex containing JBP1 and pICln, produces dimethylarginine-modified Sm proteins. Mol. Cell. Biol. 21, 8289–8300 (2001).

    CAS  Article  Google Scholar 

  35. 35.

    Meister, G. et al. Methylation of Sm proteins by a complex containing PRMT5 and the putative U snRNP assembly factor pICln. Curr. Biol. 11, 1990–1994 (2001).

    CAS  Article  Google Scholar 

  36. 36.

    Friesen, W. J., Massenet, S., Paushkin, S., Wyce, A. & Dreyfuss, G. SMN, the product of the spinal muscular atrophy gene, binds preferentially to dimethylarginine-containing protein targets. Mol. Cell 7, 1111–1117 (2001).

    CAS  Article  Google Scholar 

  37. 37.

    Hong, C. et al. Activated T cells secrete an alternatively spliced form of common γ-chain that inhibits cytokine signaling and exacerbates inflammation. Immunity 40, 910–923 (2014).

    CAS  Article  Google Scholar 

  38. 38.

    Rochman, Y., Spolski, R. & Leonard, W. J. New insights into the regulation of T cells by γc family cytokines. Nat. Rev. Immunol. 9, 480–490 (2009).

    CAS  Article  Google Scholar 

  39. 39.

    Yoshimura, A., Naka, T. & Kubo, M. SOCS proteins, cytokine signalling and immune regulation. Nat. Rev. Immunol. 7, 454–465 (2007).

    CAS  Article  Google Scholar 

  40. 40.

    Davari, K. et al. Rapid genome-wide recruitment of RNA polymerase II drives transcription, splicing, and translation events during T cell responses. Cell Rep. 19, 643–654 (2017).

    CAS  Article  Google Scholar 

  41. 41.

    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).

    CAS  Article  Google Scholar 

  42. 42.

    Cheng, D., Côté, J., Shaaban, S. & Bedford, M. T. The arginine methyltransferase CARM1 regulates the coupling of transcription and mRNA processing. Mol. Cell 25, 71–83 (2007).

    Article  Google Scholar 

  43. 43.

    Yang, Y. et al. PRMT9 is a type II methyltransferase that methylates the splicing factor SAP145. Nat. Commun. 6, 6428 (2015).

    CAS  Article  Google Scholar 

  44. 44.

    Brahms, H. et al. The C-terminal RG dipeptide repeats of the spliceosomal Sm proteins D1 and D3 contain symmetrical dimethylarginines, which form a major B-cell epitope for anti-Sm autoantibodies. J. Biol. Chem. 275, 17122–17129 (2000).

    CAS  Article  Google Scholar 

  45. 45.

    Koh, C. M. et al. MYC regulates the core pre-mRNA splicing machinery as an essential step in lymphomagenesis. Nature 523, 96–100 (2015).

    CAS  Article  Google Scholar 

  46. 46.

    Nakashima, T. et al. Evidence for osteocyte regulation of bone homeostasis through RANKL expression. Nat. Med. 17, 1231–1234 (2011).

    CAS  Article  Google Scholar 

  47. 47.

    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).

    CAS  Article  Google Scholar 

  48. 48.

    Miyao, T. et al. Plasticity of Foxp3+ T cells reflects promiscuous Foxp3 expression in conventional T cells but not reprogramming of regulatory T cells. Immunity 36, 262–275 (2012).

    CAS  Article  Google Scholar 

  49. 49.

    Kawano, T. et al. CD1d-restricted and TCR-mediated activation of vα14 NKT cells by glycosylceramides. Science 278, 1626–1629 (1997).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank S. Hori (University of Tokyo) for pMSCV-Foxp3-IG; H. Kawamoto (Kyoto University) and T. Ikawa (Tokyo University of Science) for Tst4-DLL4 stromal cells; and C. Tsuda, T. Ono, K. Hamada, K. Kaneki, T. Suda, M. Tsukasaki, A. Suematsu, S. Sawa, N. Komatsu and Y. Nakayama for discussions and technical assistance. This work was supported in part by a grant for the ERATO Takayanagi Osteonetwork Project from the Japan Science and Technology Agency, and Grants-in-Aid for Specially Promoted Research (15H05703), Scientific Research (B) (16H05202 and 18H02919), Young Scientists (A) (15H05653), Young Scientists (B) (16K19102) and JSPS Research Fellow (15J05761) from the Japan Society for the Promotion of Science (JSPS). M.I. was supported by a JSPS Research Fellowship for Young scientists (15J05761).

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Contributions

M.I. performed most of the experiments, interpreted the results and prepared the manuscript; K.O. provided advice on project planning and data interpretation and contributed to experiments of Il2rg splicing and manuscript preparation; A.T., T. Nitta and T.N.-K. contributed to data interpretation and discussions; R.M. performed retroviral transduction into thymocytes and contributed to data interpretation; T.K. provided a retroviral vector and advice on data analysis; T. Nakashima generated genetically modified mice and provided advice on data analysis; and H.T. directed the project and wrote the manuscript.

Corresponding author

Correspondence to Hiroshi Takayanagi.

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The Department of Osteoimmunology (of The University of Tokyo) is an endowed department supported by an unrestricted grant from Chugai Pharmaceutical Co., AYUMI Pharmaceutical Corporation and Noevir Co.

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Integrated supplementary information

Supplementary Figure 1 PRMT5 expression in T-cell subsets in the thymus and spleen.

(a) Prmt5 mRNA expression in CD4SP, CD8SP and iNKT cells in the thymus, and Treg, naïve CD4+ T and naïve CD8+ T cells in the spleen and lymph nodes, and activated CD4+ T and activated CD8+ T cells of Foxp3hCD2 knock-in mice (n = 3). Treg cells were isolated as CD4+CD25+hCD2+ T cells. T cells stimulated with anti-CD3/CD28 in the presence of IL-2 for 2 days were used as activated T cells. The level of mRNA expression was normalized with that of Actb mRNA expression. (b) Protein expression level of PRMT5 in thymocytes, naïve T cells and activated T cells. n = 2-4. (c) Prmt5 mRNA expression in CD4SP, CD8SP, iNKT and Treg cells in the thymus of Foxp3hCD2 knock-in mice (n = 3). Statistical analysis was carried out using One-way ANOVA with Tukey’s multiple-comparison test. *P < 0.05; **P < 0.01. Data are mean ± s.e.m. and representative of more than 2 independent experiments.

Supplementary Figure 2 Conditional gene-targeting of Prmt5.

(a) The targeting strategy to generate Prmt5 conditional mutant mice. The genomic structure of the wild-type Prmt5 gene is indicated. Exon 2 and 3 are flanked by loxP sequences; the Neo cassette is flanked by frt sequences. The modified Prmt5 locus after homologous recombination (Prmt5floxNeo allele) as well as the deleted Prmt5 gene after Cre-mediated excision of exons 2 and 3 (Prmt5Δ allele) are shown. The arrows below the diagram of the wild-type allele indicate the positions of the primers (P1, P2 and P3) used for the PCR genotyping. (b) The expression of Prmt5 mRNA in DN, DP, CD4SP and CD8SP cells of control (n = 3) and Prmt5flox/Δ Cd4-Cre (DN, n = 4; DP and CD4SP, n = 5; and CD8SP, n = 3) mice. (c) The number of TCRβ+ cells in the lymph nodes (control, n = 3; Prmt5flox/Δ Cd4-Cre, n = 6), liver (n = 7) and bone marrow (control, n = 3; Prmt5flox/Δ Cd4-Cre, n = 4). Statistical analysis was carried out using two-tailed Student’s t-test. NS, not significant (P > 0.05); *P < 0.05; ***P < 0.005. Data are mean ± s.e.m.

Supplementary Figure 3 Analysis of thymic iNKT cell development and the activation state of peripheral CD4+ and CD8+ T cells of Prmt5flox/Δ Cd4-Cre mice.

(a) The frequency of CD45.1+ cells and CD45.2+ cells in DP, CD4SP, CD8SP and iNKT cells in the thymus (n = 4), and splenic B cells (n = 3) of BM chimeric mice. Each line indicates individual mice. Prmt5flox/Δ Cd4-Cre BM cells were able to develop into DP cells, SP cells and B cells similarly to control BM cells. (b) CD1d expression on DP cells of control (n = 5) and Prmt5flox/Δ Cd4-Cre (n = 3) mice. The bar graphs show the mean fluorescence intensity (MFI) of CD1d. (c) The frequencies of CD62L+CD44, CD62LCD44+ and CD62L+CD44+ cells among CD4+ T and CD8+ T cells in the spleen of control and Prmt5flox/Δ Cd4-Cre mice. (d) The frequencies of CD44+ cells in CD4+ T and CD8+ T cells of control (n = 8) and Prmt5flox/Δ Cd4-Cre (n = 10) mice. (e) Va14-Ja18 transcripts in pre-selected (CD69loTCRβlo) DP cells of control and Prmt5flox/Δ Cd4-Cre mice (n = 3). Statistical analysis was carried out using two-tailed Student’s t-test. NS, not significant (P > 0.05); *P < 0.05; ***P < 0.005. Data are mean ± s.e.m. and representative of more than 2 independent experiments.

Supplementary Figure 4 Carm1 and Prmt9 expression in thymic Treg cells and effects of retroviral transduction of Foxp3 on Prmt5 expression.

(a) The expression of Carm1 and Prmt9 was measured in iNKT and Treg cells in the thymus, and Treg and naïve CD4+ T cells in the spleen and lymph nodes of Foxp3hCD2 knock-in mice (n = 4). Treg cells were isolated as CD4+CD25+hCD2+ T cells. (b, c) Effects of retroviral expression of Foxp3 on Prmt5 expression in thymocytes (n = 6) (a) and peripheral CD4+ T cells (n = 3) (b). Statistical analysis was carried out using One-way ANOVA with Tukey’s multiple-comparison test (a) and two-tailed Student’s t-test (b,c). *P < 0.05; **P < 0.01; ***P < 0.005. Data are mean ± s.e.m. and representative of 2 independent experiments.

Supplementary Figure 5 Retroviral expression of a constitutively active form of STAT5 rescued impaired expression of CD25 in PRMT5-deficient T cells.

Effects of retroviral expression of a constitutively active form of STAT5 (STAT5A1*6) on CD25 expression in CD4+ and CD8+ T cells (n = 3). Empty: empty vector. Statistical analysis was carried out using two-tailed Student’s t-test. NS, not significant (P > 0.05); *P < 0.05; **P < 0.01. Data are mean ± s.e.m.

Supplementary Figure 6 Effects of the disruption of the Prmt5 gene on the splicing of Il2rg and Jak3 pre-mRNA.

(a) A procedure to analyze genes, the splicing of which was affected in PRMT5-deficient stage 1 iNKT cells. Ninety-five genes were arranged in the order of fold change in intron ratio and the top 30 genes were listed. The Il2rg and Jak3 genes ranked 16th and 22nd, respectively. (b) Schematic diagram of γc mRNA splice isoforms. The arrows indicate a primer set specifically targeting membrane γc or soluble γc. (c) mRNA expression of soluble γc in control (n = 4-9) and PRMT5-deficient (n = 4-9) T cells. (d) Subcloning and sequencing of the PCR products amplified by primers targeting exon 1 and exon 8 of the Il2rg gene in activated T cells of control and PRMT5-deficient mice (n = 1). Transcripts retaining intron 3, introns 3 and 5, and introns 3, 5 and 6, as well as a transcript skipping exon 6 were detected in PRMT5-deficient T cells. (e) Effects of retroviral expression of γc and JAK3 on surface expression of human γc in 7AADCD4+ T cells of control (n = 5) and Prmt5flox/Δ Cd4-Cre mice (n = 4). Empty: empty vector. The number shows the mean fluorescence intensity (MFI) of human γc. (f) The ratio of the unspliced mRNA to spliced mRNA in each intron of the Il2rg gene in naïve CD4+ T (control, n = 4; Prmt5flox/Δ Cd4-Cre, n = 5) and CD8+ T cells (control, n = 6; Prmt5flox/Δ Cd4-Cre, n = 7). (g) mRNA expression of soluble γc in naïve CD4+ T (control, n = 4; Prmt5flox/Δ Cd4-Cre, n = 5) and CD8+ T cells (control, n = 6; Prmt5flox/Δ Cd4-Cre, n = 7) Statistical analysis was carried out using two-tailed Student’s t-test. NS, not significant (P > 0.05); *P < 0.05; **P < 0.01; ***P < 0.005. Data are mean ± s.e.m. and representative of 2 independent experiments.

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Inoue, M., Okamoto, K., Terashima, A. et al. Arginine methylation controls the strength of γc-family cytokine signaling in T cell maintenance. Nat Immunol 19, 1265–1276 (2018). https://doi.org/10.1038/s41590-018-0222-z

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