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The Molecular Mechanism of Th9 Cell Differentiation is Regulated by Batf3 and Id1

  • 이우호 (Wooho Lee, 서강대학교 일반대학원)

초록

Th9 cells, relatively recently discovered among CD4 T cells, predominantly secrete IL- 9 cytokines and play a role in triggering allergic diseases, autoimmune disorders, and suppressing cancer. In this study, transcription factors associated with Th9 cell differentiation were investigated as a fundamental approach to treating Th9 cell-related diseases. The first transcription factor studied is Batf3, which has been reported to be upregulated by OX40 signaling, inducing Th9 cell differentiation. Batf3 overexpression experiments revealed an increase in IL-9, a cytokine important for the differentiation and function of Th9 cells. Furthermore, it revealed the molecular mechanism by which Batf3 increases Il9 promoter activity to induce Th9 cell differentiation. The second transcription factor studied is Id1, showing specific and higher expression in Th9 cells compared to other CD4 T cells. Id1-deficient CD4 T cells had no issues differentiating into other CD4 T cell subsets but were unable to differentiate into Th9 cells. Tcf3 and Tcf4, which bind to Id1, were identified, and it was revealed that these factors decrease Il9 promoter activity, thereby inhibiting Th9 cell differentiation. Additionally, when asthma was induced in mice with Id1-deficient CD4 T cells, a significant reduction in disease symptoms was observed. These two studies suggest that Batf3 and Id1 are crucial transcription factors in Th9 cell differentiation.

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초록

Th9 세포는 CD4 T 세포 중에서 비교적 최근에 발견된 세포이다. 주로 IL-9 사이토카인을 분비하며 알레르기 질환, 자가면역 질환을 일으키고 암을 억제하는 역할을 한다. 본 연구에서는 Th9 세포 관련 질병의 치료에 대한 근본적인 접근을 위해 Th9 세포 분화와 관련된 전사 인자들을 연구했다. 첫 번째로 연구한 전사 인자는 Batf3 로 Th9 세포분화를 돕는 OX40 신호에 의해 증가한다는 보고가 있었다. Batf3 과발현 실험을 통해 Th9 세포의 분화와 기능에 중요한 사이토카인 IL-9 이 증가하는 것을 밝혀냈다. 또한, Batf3 가 Il9 프로모터 활성을 증가시켜 Th9 세포 분화를 돕는 분자기전을 밝혀냈다. 두 번째로 연구한 전사 인자는 Id1 으로 Th9 세포에서 다른 CD4 T 세포에 비해 특이적으로 높게 발현되는 것이 관찰되었다. Id1 유전자가 결핍된 CD4 T 세포의 경우 다른 CD4 T 세포로의 분화에는 문제가 없지만 Th9 세포로는 분화가 이루어지지 않는 것을 밝혀냈다. Id1 과 결합하는 Tcf3 와 Tcf4 를 찾아냈으며, 두 인자가 Il9 의 프로모터 활성을 감소시켜 Th9 세포 분화를 억제하는 분자기전을 밝혀냈다. 또한 Id1 이 결핍된 CD4 T 세포를 가진 쥐에게 천식을 유도하자 질병의 증상이 눈에 띄게 감소하였다. 이 두 연구는 Batf3 와 Id1 이 Th9 세포 분화에 중요한 전사인자임을 시사한다.

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목차

I. 초록 1
II. General Abstract 2
III. General Introduction 3

Chapter 1. BATF3 is sufficient for the induction of Il9 expression and can
compensate for BATF during Th9 cell differentiation 6
1.1 Abstract 7
1.2 Introduction 8
1.3 Materials and Methods . 11
1.3.1. Mice 11
1.3.2. Intracellular Staining (ICS) 11
1.3.3. Purification of CD4+ T cells and stimulation in vitro 11
1.3.4. Retroviral transduction 12
1.3.5. RNA isolation and qRT-PCR . 13
1.3.6. ChIP assay 14
1.3.7. Immunoblot analysis 14
1.3.8. ELISA 15
1.3.9. Co-IP 16
1.3.10. Transient reporter assay 16
1.3.11. A doptive transfer model for assessment of airway inflammation 17
1.3.12. Analysis of BAL fluid . 17
1.3.13. RNA isolation and histology of lung tissue 18
1.3.14. Analysis of serum IgE 18
1.4 Results 20
1.4.1. OX40 signaling increases the expression of Batf3 in Th9 cells. 20
1.4.2. Overexpression of Batf3 increases the production of IL-9 in Th9 cells23
1.4.3. The BATF3–IRF4 complex increases Il9 promoter activity 27
1.4.4 BATF and BATF3 compensate for each other in Th9 cells 31
1.4.5. BATF3 induces the expression of IL-9 in Batf KO Th9 cells . 36
1.4.6 Batf3-overexpressing Th9 cells induce airway inflammation, even in the absence of BATF 39
1.5 Discussion . 42
1.6 Publication Information . 45

Chapter 2. Transcription Factor Id1 Plays an Essential Role in Th9 Cell
Differentiation by Inhibiting Tcf3 and Tcf4 . 46
2.1 Abstract 47

2.2 Introduction 48
2.3 Materials and Methods . 51
2.3.1. Mice 51
2.3.2. Isolation and Differentiation of CD4 T Cells 51
2.3.3. Flow Cytometry 53
2.3.4. ELISA 54
2.3.5. RNA Isolation and Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) 54
2.3.6. Retroviral Transduction . 55
2.3.7. Co-IP Analysis 55
2.3.8. ChIP . 56
2.3.9. RNA-seq . 56
2.3.10. Mass spectrometry . 57
2.3.11. Transient Reporter Assay . 57
2.3.12. Animal Model of Asthma . 58
2.3.13. Adoptive Transfer Model of Asthma 58
2.3.14. Analysis of Asthma-Induced Mice . 59
2.3.15. Statistical Analysis 59
2.4. Results . 64
2.4.1. Id1 is an Essential Positive Regulator of Th9 Cell Differentiation . 64
2.4.2. Differentiation of Th9 Cells is Dependent on Id1 Level and Both Id1 and Id3 are Positive Regulators of Th9 Differentiation . 69
2.4.3. Tcf3 and Tcf4, Binding Partners of Id1, Act as Negative Regulators of Th9 Cell Differentiation . 74
2.4.4. Tcf3 and Tcf4 Inhibit Il9 Promoter Activity by Blocking Positive Transcription Factors 79
2.4.5. Both Tcf3 and Tcf4 play crucial roles in inhibiting Th9 cell differentiation. 83
2.4.6. Id1-Deficient Th9 Cells Show Increased Expression of Genes Related to Type 1 Immune Responses 86
2.4.7. Id1-Deficient Th9 Cells Ameliorate Airway Inflammation in an Animal Model of Asthma . 91
2.4.8. Id1-Deficient Th9 Cells Ameliorate Airway Inflammation in an Adoptive Transfer Model of Asthma . 95
2.5. Discussion 100
2.6 Publication Information . 104
IV. Concluding Remarks 105
V. References 108

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