||The novel role of signal transducer and activator of transcription 5B (STAT5B) in mediating T cell differentiation and regulatory functions
||Institute of Clinical Medicine
Human STAT5B mutation
Induced regulatory T cells (iTreg)
Growth hormone insensitivity syndrome (GHIS)
轉錄訊息傳遞及活化子蛋白5B (STAT5B)是生長激素和共γ鏈家族細胞激素(例如:介白素2 (IL-2) , 7,15等) 的必要轉錄因子，IL-2-STAT5B路徑在誘導型調節性T細胞 (iTreg) 分化扮演了很重要的角色，iTreg的功能是維持免疫平衡和控制發炎疾病的一種細胞。近年來，STAT5B突變患者的臨床、生化和遺傳學的研究迅速發展，人類STAT5B突變會導致”非典型”生長激素不敏感症候群、免疫失調、自體免疫、慢性肺部疾病等臨床症狀。然而，STAT5B突變是如何導致免疫失調症狀和自體免疫疾病的機制仍未被清楚研究，此外，目前尚未有治療方法同時改善STAT5B突變病患的生長遲緩和免疫缺陷症狀。
我們團隊診斷出一位男性病童 (以下簡稱為病童C)，其臨床症狀包含:出生後生長遲緩、嚴重皮膚發炎、高γ免疫球蛋白血症和淋巴細胞增生症。我們利用全外顯子定序鑑定出病童C帶有異形合子的STAT5B點突變 (DNA核甘酸1281位點鳥嘌呤至腺嘌呤變異；第371個胺基酸的丙氨酸至蘇氨酸變異)，然而這個突變和病童的免疫失調症狀之間的關係仍未知，因此本篇研究探討異形合子STAT5BA371T 突變在免疫失調中所扮演的致病機轉。我們已經確認這個突變STAT5B蛋白質可正常表達且IL-2刺激後的磷酸化表現正常，且在T細胞刺激前CD4+CD25+ T細胞比例是正常的，但是，病童C的周邊血單核細胞在刺激後的CD4+CD25high T細胞族群表現較少。已知CD25是STAT5B的目標下游基因，且較少的CD25表現將導致對IL-2不敏感，因此造成分化iTreg的功能異常。
為了更進一步研究STAT5BA371T 突變在T細胞分化和基因調控中所扮演的角色，我們建立了一個轉染野生型或突變STAT5B的T細胞株。冷光報導基因分析結果顯示，STAT5BA371T 突變在T細胞活化後會顯著地降低CD25轉錄活性且具有顯性抑制突變的效應，這個實驗結果代表這個點突變會去減弱STAT5B轉錄因子下游的轉錄功能，但是我們發現到點突變仍保有百分之七十的CD25啟動子轉錄活性。我們接著利用RNA定序去鑑定TCR刺激後STAT5BA371T下游基因的表現變化，GSEA分析結果發現和組織駐留Treg相關的基因群在STAT5BA371T細胞株相較於野生型有顯著集合的現象。
Signal transducer and activator of transcription 5B (STAT5B) is an essential transcription factor of growth hormone and common γ-chain family cytokines, including interleukin (IL)-2, IL-7, and IL-15. IL-2-STAT5B pathway plays an important role in differentiation of induced regulatory T (iTreg) cells. The iTreg cells are crucial for maintaining immune tolerance and controlling inflammatory diseases. In recent years, the clinical, biological, and genetic characteristics of patients with STAT5B mutations have rapidly expanded. Human STAT5B mutations can cause “non-classical” growth hormone insensitivity syndrome (GHIS), immune dysregulation, autoimmunity, and chronic lung disease. However, the mechanisms of how STAT5B mutations lead to the immune dysregulation and autoimmune syndrome are still unclear. In addition, therapeutic options to reverse both post-natal growth retardation and immunodeficiency at present are lacking at present.
We recently encountered a male patient (designated patient C), who suffered from post-natal growth retardation, severe skin inflammation, hypergammaglobulinemia and lymphoproliferative syndrome. A novel heterozygous STAT5B missense mutation (c.1281G to A；p.Ala371Thr) was identified in this patient through whole exome sequencing (WES). However, the relationship between the mutation and immune dysregulation syndrome is still unknown. Therefore, we investigated the role of heterozygous STAT5BA371T variant in the pathogenesis of immune dysregulation syndrome in this study. We confirmed that the mutant STAT5B protein is expressed and tyrosine phosphorylation in response to IL-2 stimulation is normal. We found normal proportion of CD4+CD25+ T cells in the peripheral blood mononuclear cell (PBMC) of the patient before T cell receptor (TCR) stimulation. Furthermore, significant decrease in CD4+CD25high T cells was found in the patient’s PBMC after T cell activation. Given that CD25 is one of the STAT5B target genes in T cells, lower CD25 expression implicates that novel STAT5BA371T variant is a hypomorphic mutation. Lower CD25 expression in turn leads to lower sensitivity to IL-2 hence ineffective induction of iTreg cells.
To investigate the role of this STAT5BA371T mutation in T cell differentiation and gene regulation, we have established a human T cell lines model which was transfected with wild type (WT) or mutant STAT5B genes. Dual luciferase reporter assay indicated the STAT5BA371T mutation showed dominate negative effect and significantly diminish transcription activity of CD25 after TCR activation. The data indicated that the missense mutation partially compromised STAT5B in its downstream transcriptional function with 70% of the transcription activation activity on CD25 promoter conserved. We then performed RNA sequencing (RNA-seq) to identify the changes in expression of STAT5BA371T target genes after TCR stimulation. Gene set enrichment analysis (GSEA) showed that genes associated with tissue resident Treg were enriched in the STAT5BA371T compared to WT cell line.
In conclusion, our data showed that a dominate-negative heterozygous STAT5BA371T variant may cause the immune dysregulation syndrome by deterring iTreg differentiation, suggesting a critical role of STAT5B in iTreg differentiation. This novel mutation may provide a direction into a better understanding of the complicated roles of STAT5B in regulating the human immune system.
誌 謝 V
Chapter 1 Introduction 1
1.1 The role of Treg cells in autoimmune diseases 2
1.2 IL-2-STAT5 signaling pathway in T cells 3
1.3 STAT5B can regulate distinct genes to maintain immune tolerance 5
1.4 Primary immune deficiency 6
1.5 Growth hormone insensitivity syndrome (GHIS) 6
1.6 Somatic gain-of-function human STAT5B mutations 7
1.7 Germline loss-of-function human STAT5B mutation 8
The aim of this study 11
Chapter 2 Material and methods 12
2.1 Reagents 13
2.2 PBMC and cell line culture 13
2.3 Whole Exome Sequencing 13
2.4 Sanger sequencing and exon PCR 14
2.5 Plasmids and site specific mutagenesis 14
2.6 Electroporation of Jurkat cells 15
2.7 Cell treatment and immunoblotting 16
2.8 ELISA 17
2.9 Flow cytometry 17
2.10 Dual reporter assay 18
2.11 Real-time quantitative PCR 18
2.12 RNA sequencing 19
2.13 Gene set enrichment analysis 19
2.14 Statistical analysis 19
Chapter 3 Results 20
3.1 Patient and disease-causing gene identification 21
3.2 The STAT5B protein expression and IL-2 induced STAT5 phosphorylation were normal in STAT5B mutant patient 22
3.3 The populations of CD4+CD25high cells were significant decrease in patient C after TCR activation 23
3.4 Establishment of human T cell line constructed with STAT5B p.Ala371Thr 24
3.5 PMA+ionomycin induced CD25 expression were impaired in STAT5BA371T-expressing Jurkat cells 25
3.6 STAT5BA371T mutation caused significantly decrease CD25 transcription activity in vitro 26
3.7 Clarify the FOXP3 expression and iTreg differentiation in STAT5BA371T-Jurkat cells 27
3.8 Identification of potential STAT5BA371T target genes in Jurkat cells by RNA-seq 28
Chapter 4 Discussion 29
4.1 Future works of our study 32
4.2 Promising therapeutic approaches for STAT5B mutant patients 34
Chapter 5 Figures and tables 36
Table 1. Exon PCR and cloning primers 37
Table 2. RT-qPCR primers 37
Table 3. Endocrine features of patient C. 37
Table 4. Immunological evaluation of patient C. 38
Table 5. Serum immunoglobulins level of patient C. 38
Chart 1. Growth curve for height and weight of patient C. 39
Figure 1. 40
Figure 1. Clinical features of the STAT5B mutation patient. 41
Figure 2. 42
Figure 2. Novel heterozygous STAT5B mutation in patient C. 44
Figure 3. 45
Figure 3. Expression and IL-2-induced phosphorylation of STAT5B protein were normal in patient’s PBMCs. 48
Figure 4. 49
Figure 4. Less abundant CD4+CD25high cells in the patient when compared with healthy controls after TCR activation. 51
Figure 5. 52
Figure 5. The novel STAT5B point mutation didn’t interfere protein expression and IL-2 induced phosphorylation in transfected-Jurkat cells. 54
Figure 6. 55
Figure 6. Lower CD25 levels were found in activated STAT5BA371T Jurkat cells. 56
Figure 7. 57
Figure 7. The STAT5B-driven transcription activity is impaired in STAT5BA371T-expressing Jurkat cells after stimulation. 58
Figure 8. 59
Supplemental Figure 1. 60
Supplemental Figure 1. Overexpressed STAT5BWT or STAT5BA371T no significantly changed in FOXP3 mRNA expression in Jurkat cells. 61
Supplemental Figure 2. 62
Supplemental Figure 2. RNA-seq analysis of STAT5BA371T Jurkat cells. 65
Chapter 6 References 66
1. Nutsch, K., et al., Rapid and Efficient Generation of Regulatory T Cells to Commensal Antigens in the Periphery. Cell Rep, 2016. 17(1): p. 206-220.
2. Sakaguchi, S., et al., Regulatory T Cells and Human Disease. Annu Rev Immunol, 2020. 38: p. 541-566.
3. Gitelman, S.E. and J.A. Bluestone, Regulatory T cell therapy for type 1 diabetes: May the force be with you. J Autoimmun, 2016. 71: p. 78-87.
4. Hull, C.M., M. Peakman, and T.I.M. Tree, Regulatory T cell dysfunction in type 1 diabetes: what's broken and how can we fix it? Diabetologia, 2017. 60(10): p. 1839-1850.
5. Meyer, A., et al., Regulatory T cell frequencies in patients with rheumatoid arthritis are increased by conventional and biological DMARDs but not by JAK inhibitors. Ann Rheum Dis, 2019.
6. Li, W., et al., The Regulatory T Cell in Active Systemic Lupus Erythematosus Patients: A Systemic Review and Meta-Analysis. Frontiers in immunology, 2019. 10: p. 159-159.
7. Sharabi, A., et al., Regulatory T cells in the treatment of disease. Nat Rev Drug Discov, 2018. 17(11): p. 823-844.
8. Lourenço, E.V. and A. La Cava, Natural regulatory T cells in autoimmunity. Autoimmunity, 2011. 44(1): p. 33-42.
9. Malek, T.R., The biology of interleukin-2. Annu Rev Immunol, 2008. 26: p. 453-79.
10. Knochelmann, H.M., et al., When worlds collide: Th17 and Treg cells in cancer and autoimmunity. Cell Mol Immunol, 2018. 15(5): p. 458-469.
11. Abbas, A.K., et al., Revisiting IL-2: Biology and therapeutic prospects. Sci Immunol, 2018. 3(25).
12. Shi, H., et al., Hippo Kinases Mst1 and Mst2 Sense and Amplify IL-2R-STAT5 Signaling in Regulatory T Cells to Establish Stable Regulatory Activity. Immunity, 2018. 49(5): p. 899-914.e6.
13. Yu, A., et al., Selective IL-2 responsiveness of regulatory T cells through multiple intrinsic mechanisms supports the use of low-dose IL-2 therapy in type 1 diabetes. Diabetes, 2015. 64(6): p. 2172-83.
14. Klatzmann, D. and A.K. Abbas, The promise of low-dose interleukin-2 therapy for autoimmune and inflammatory diseases. Nat Rev Immunol, 2015. 15(5): p. 283-94.
15. Trotta, E., et al., A human anti-IL-2 antibody that potentiates regulatory T cells by a structure-based mechanism. Nat Med, 2018. 24(7): p. 1005-1014.
16. Romano, M., et al., Past, Present, and Future of Regulatory T Cell Therapy in Transplantation and Autoimmunity. Front Immunol, 2019. 10: p. 43.
17. Villarino, A.V., et al., Subset- and tissue-defined STAT5 thresholds control homeostasis and function of innate lymphoid cells. J Exp Med, 2017. 214(10): p. 2999-3014.
18. Villarino, A., et al., Signal transducer and activator of transcription 5 (STAT5) paralog dose governs T cell effector and regulatory functions. Elife, 2016. 5.
19. Kanai, T., J. Jenks, and K.C. Nadeau, The STAT5b Pathway Defect and Autoimmunity. Frontiers in immunology, 2012. 3: p. 234-234.
20. Ross, S.H. and D.A. Cantrell, Signaling and Function of Interleukin-2 in T Lymphocytes. Annu Rev Immunol, 2018. 36: p. 411-433.
21. Kanai, T., et al., Identification of STAT5A and STAT5B Target Genes in Human T Cells. PLOS ONE, 2014. 9(1): p. e86790.
22. Liao, W., J.-X. Lin, and W.J. Leonard, Interleukin-2 at the crossroads of effector responses, tolerance, and immunotherapy. Immunity, 2013. 38(1): p. 13-25.
23. Malek, T.R. and I. Castro, Interleukin-2 receptor signaling: at the interface between tolerance and immunity. Immunity, 2010. 33(2): p. 153-165.
24. Lin, J.X., et al., Critical Role of STAT5 transcription factor tetramerization for cytokine responses and normal immune function. Immunity, 2012. 36(4): p. 586-99.
25. Lin, J.X., et al., Critical functions for STAT5 tetramers in the maturation and survival of natural killer cells. Nat Commun, 2017. 8(1): p. 1320.
26. Yanai, H., Autoimmune Polyendocrine Syndromes. N Engl J Med, 2018. 378(26): p. 2542-3.
27. Torgerson, T.R. and H.D. Ochs, Immune dysregulation, polyendocrinopathy, enteropathy, X-linked: forkhead box protein 3 mutations and lack of regulatory T cells. J Allergy Clin Immunol, 2007. 120(4): p. 744-50; quiz 751-2.
28. Storr, H.L., et al., Nonclassical GH Insensitivity: Characterization of Mild Abnormalities of GH Action. Endocr Rev, 2019. 40(2): p. 476-505.
29. Goudy, K., et al., Human IL2RA null mutation mediates immunodeficiency with lymphoproliferation and autoimmunity. Clin Immunol, 2013. 146(3): p. 248-61.
30. Janecka, A., M. Kolodziej-Rzepa, and B. Biesaga, Clinical and Molecular Features of Laron Syndrome, A Genetic Disorder Protecting from Cancer. In Vivo, 2016. 30(4): p. 375-81.
31. Suwinski, P., et al., Advancing Personalized Medicine Through the Application of Whole Exome Sequencing and Big Data Analytics. Frontiers in genetics, 2019. 10: p. 49-49.
32. Kucuk, C., et al., Activating mutations of STAT5B and STAT3 in lymphomas derived from gammadelta-T or NK cells. Nat Commun, 2015. 6: p. 6025.
33. Pham, H.T.T., et al., STAT5BN642H is a driver mutation for T cell neoplasia. J Clin Invest, 2018. 128(1): p. 387-401.
34. Ito, M., et al., A novel JAK inhibitor, peficitinib, demonstrates potent efficacy in a rat adjuvant-induced arthritis model. J Pharmacol Sci, 2017. 133(1): p. 25-33.
35. Bezrodnik, L., et al., Long-term follow-up of STAT5B deficiency in three argentinian patients: clinical and immunological features. J Clin Immunol, 2015. 35(3): p. 264-72.
36. Kofoed, E.M., et al., Growth hormone insensitivity associated with a STAT5b mutation. N Engl J Med, 2003. 349(12): p. 1139-47.
37. Hwa, V., STAT5B deficiency: Impacts on human growth and immunity. Growth hormone & IGF research : official journal of the Growth Hormone Research Society and the International IGF Research Society, 2016. 28: p. 16-20.
38. Pugliese-Pires, P.N., et al., A novel STAT5B mutation causing GH insensitivity syndrome associated with hyperprolactinemia and immune dysfunction in two male siblings. Eur J Endocrinol, 2010. 163(2): p. 349-55.
39. Scaglia, P.A., et al., A novel missense mutation in the SH2 domain of the STAT5B gene results in a transcriptionally inactive STAT5b associated with severe IGF-I deficiency, immune dysfunction, and lack of pulmonary disease. J Clin Endocrinol Metab, 2012. 97(5): p. E830-9.
40. Swigris, J.J., et al., Lymphoid interstitial pneumonia: a narrative review. Chest, 2002. 122(6): p. 2150-64.
41. Laron, Z., Laron syndrome (primary growth hormone resistance or insensitivity): the personal experience 1958-2003. J Clin Endocrinol Metab, 2004. 89(3): p. 1031-44.
42. Klammt, J., et al., Dominant-negative STAT5B mutations cause growth hormone insensitivity with short stature and mild immune dysregulation. Nat Commun, 2018. 9(1): p. 2105.
43. Majri, S.S., et al., STAT5B: A Differential Regulator of the Life and Death of CD4(+) Effector Memory T Cells. J Immunol, 2018. 200(1): p. 110-118.
44. Matsubara, Y., et al., Transcription activator-like effector nuclease-mediated transduction of exogenous gene into IL2RG locus. Scientific reports, 2014. 4: p. 5043-5043.
45. Subramanian, A., et al., Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A, 2005. 102(43): p. 15545-50.
46. Schmidt, A., et al., Comparative Analysis of Protocols to Induce Human CD4+Foxp3+ Regulatory T Cells by Combinations of IL-2, TGF-beta, Retinoic Acid, Rapamycin and Butyrate. PLoS One, 2016. 11(2): p. e0148474.
47. Zhao, C., et al., Induced regulatory T-cells (iTregs) generated by activation with anti-CD3/CD28 antibodies differ from those generated by the physiological-like activation with antigen/APC. Cell Immunol, 2014. 290(2): p. 179-84.
48. Simpson, H.M., et al., STAT5 inhibition induces TRAIL/DR4 dependent apoptosis in peripheral T-cell lymphoma. Oncotarget, 2018. 9(24): p. 16792-16806.
49. Iwashima, M., et al., Genetic evidence for Shc requirement in TCR-induced c-Rel nuclear translocation and IL-2 expression. Proc Natl Acad Sci U S A, 2002. 99(7): p. 4544-9.
50. Ilnicka, A., et al., Regulation of FOXP3 expression in myeloid cells in response to all-trans-retinoic acid, interleukin 2 and transforming growth factor β. Dev Comp Immunol, 2019. 96: p. 18-26.
51. Kearney, C.J., K.L. Randall, and J. Oliaro, DOCK8 regulates signal transduction events to control immunity. Cell Mol Immunol, 2017. 14(5): p. 406-411.
52. Biggs, C.M., S. Keles, and T.A. Chatila, DOCK8 deficiency: Insights into pathophysiology, clinical features and management. Clin Immunol, 2017. 181: p. 75-82.
53. Jenks, J.A., et al., Differentiating the roles of STAT5B and STAT5A in human CD4+ T cells. Clinical immunology (Orlando, Fla.), 2013. 148(2): p. 227-236.
54. Ramírez, L., et al., A novel heterozygous STAT5B variant in a patient with short stature and partial growth hormone insensitivity (GHI). Growth Horm IGF Res, 2020. 50: p. 61-70.
55. Darrigues, J., J.P.M. van Meerwijk, and P. Romagnoli, Age-Dependent Changes in Regulatory T Lymphocyte Development and Function: A Mini-Review. Gerontology, 2018. 64(1): p. 28-35.
56. Garg, S.K., et al., Aging is associated with increased regulatory T-cell function. Aging cell, 2014. 13(3): p. 441-448.
57. Holland, S.M., et al., STAT3 mutations in the hyper-IgE syndrome. N Engl J Med, 2007. 357(16): p. 1608-19.
58. Renner, E.D., et al., Novel signal transducer and activator of transcription 3 (STAT3) mutations, reduced T(H)17 cell numbers, and variably defective STAT3 phosphorylation in hyper-IgE syndrome. The Journal of allergy and clinical immunology, 2008. 122(1): p. 181-187.
59. Minegishi, Y., et al., Dominant-negative mutations in the DNA-binding domain of STAT3 cause hyper-IgE syndrome. Nature, 2007. 448(7157): p. 1058-62.
60. Perl, A., Activation of mTOR (mechanistic target of rapamycin) in rheumatic diseases. Nat Rev Rheumatol, 2016. 12(3): p. 169-82.
61. Lai, Z.W., et al., Sirolimus in patients with clinically active systemic lupus erythematosus resistant to, or intolerant of, conventional medications: a single-arm, open-label, phase 1/2 trial. Lancet, 2018. 391(10126): p. 1186-1196.
62. Spolski, R., P. Li, and W.J. Leonard, Biology and regulation of IL-2: from molecular mechanisms to human therapy. Nat Rev Immunol, 2018. 18(10): p. 648-659.
63. Spangler, J.B., et al., Antibodies to Interleukin-2 Elicit Selective T Cell Subset Potentiation through Distinct Conformational Mechanisms. Immunity, 2015. 42(5): p. 815-25.