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系統識別號 U0026-2308202023563600
論文名稱(中文) STAT5B在T細胞分化和調節功能中所扮演的新穎角色
論文名稱(英文) The novel role of signal transducer and activator of transcription 5B (STAT5B) in mediating T cell differentiation and regulatory functions
校院名稱 成功大學
系所名稱(中) 臨床醫學研究所
系所名稱(英) Institute of Clinical Medicine
學年度 108
學期 2
出版年 109
研究生(中文) 謝皓
研究生(英文) Hao Hsieh
學號 S96074019
學位類別 碩士
語文別 英文
論文頁數 73頁
口試委員 指導教授-謝奇璋
指導教授-陳芃潔
口試委員-許育祥
口試委員-蔡曜聲
中文關鍵字 人類轉錄訊息傳遞及活化子蛋白5B突變  介白素2  誘導型調節性T細胞  生長激素不敏感症候群 
英文關鍵字 Human STAT5B mutation  Interleukin-2 (IL-2)  Induced regulatory T cells (iTreg)  Growth hormone insensitivity syndrome (GHIS) 
學科別分類
中文摘要 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。
總結來說,我們的實驗結果顯示顯性抑制突變的異形合子STAT5BA371T透過阻止iTreg分化而導致免疫失調症狀,代表STAT5B在iTreg分化中扮演了重要的角色,這個新穎的點突變使我們更加深入理解STAT5B轉錄因子在調節人類免疫系統中所扮演的複雜角色。
英文摘要 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.
論文目次 Abstract I
中文摘要: III
誌 謝 V
Abbreviations VI
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
References: 67
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