Title Page
Contents
Ⅰ. 초록 13
Ⅱ. General Abstract 14
Ⅲ. General Introduction 15
Chapter 1. BATF3 is sufficient for the induction of Il9 expression and can compensate for BATF during Th9 cell differentiation 18
1.1. Abstract 19
1.2. Introduction 20
1.3. Materials and Methods 23
1.3.1. Mice 23
1.3.2. Intracellular Staining (ICS) 23
1.3.3. Purification of CD4+ T cells and stimulation in vitro 23
1.3.4. Retroviral transduction 24
1.3.5. RNA isolation and qRT-PCR 25
1.3.6. CHIP assay 26
1.3.7. Immunoblot analysis 26
1.3.8. ELISA 27
1.3.9. Co-IP 28
1.3.10. Transient reporter assay 28
1.3.11. A doptive transfer model for assessment of airway inflammation 29
1.3.12. Analysis of BAL fluid 29
1.3.13. RNA isolation and histology of lung tissue 30
1.3.14. Analysis of serum IgE 30
1.4. Results 32
1.4.1. OX40 signaling increases the expression of Batf3 in Th9 cells 32
1.4.2. Overexpression of Batf3 increases the production of IL-9 in Th9 cells 35
1.4.3. The BATF3-IRF4 complex increases Il9 promoter activity 39
1.4.4. BATF and BATF3 compensate for each other in Th9 cells 43
1.4.5. BATF3 induces the expression of IL-9 in BarfKO Th9 cells 48
1.4.6. Batf3-overexpressing Th9 cells induce airway inflammation, even in the absence of BATF 51
1.5. Discussion 54
1.6. Publication Information 57
Chapter 2. Transcription Factor Id1 Plays an Essential Role in Th9 Cell Differentiation by Inhibiting Tcf3 and Tcf4 58
2.1. Abstract 59
2.2. Introduction 60
2.3. Materials and Methods 63
2.3.1. Mice 63
2.3.2. Isolation and Differentiation of CD4 T Cells 63
2.3.3. Flow Cytometry 65
2.3.4. ELISA 66
2.3.5. RNA Isolation and Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) 66
2.3.6. Retroviral Transduction 67
2.3.7. Co-IP Analysis 67
2.3.8. ChIP 68
2.3.9. RNA-seq 68
2.3.10. Mass spectrometry 69
2.3.11. Transient Reporter Assay 69
2.3.12. Animal Model of Asthma 70
2.3.13. Adoptive Transfer Model of Asthma 70
2.3.14. Analysis of Asthma-Induced Mice 71
2.3.15. Statistical Analysis 71
2.4. Results 76
2.4.1. Id1 is an Essential Positive Regulator of Th9 Cell Differentiation 76
2.4.2. Differentiation of Th9 Cells is Dependent on Id1 Level and Both Id1 and Id3 are Positive Regulators of Th9 Differentiation 81
2.4.3. Tcf3 and Tcf4, Binding Partners of Id1, Act as Negative Regulators of Th9 Cell Differentiation 86
2.4.4. Tcf3 and Tcf4 Inhibit Il9 Promoter Activity by Blocking Positive Transcription Factors 91
2.4.5. Both Tcf3 and Tcf4 play crucial roles in inhibiting Th9 cell differentiation. 95
2.4.6. Id1-Deficient Th9 Cells Show Increased Expression of Genes Related to Type 1 Immune Responses 98
2.4.7. Id1-Deficient Th9 Cells Ameliorate Airway Inflammation in an Animal Model of Asthma 103
2.4.8. Id1-Deficient Th9 Cells Ameliorate Airway Inflammation in an Adoptive Transfer Model of Asthma 107
2.5. Discussion 112
2.6. Publication Information 116
Ⅳ. Concluding Remarks 117
Ⅴ. References 120
Chapter 1. BATF3 is sufficient for the induction of Il9 expression and can compensate for BATF during Th9 cell differentiation 11
Figure 1-1. Batf3 expression in Th9 cells increases significantly upon exposure to OX40L. 34
Figure 1-2. Increased expression of IL-9 in Batf3-overexpressing Th9 cells. 38
Figure 1-3. BATF interacts physically with IRF4 and increases Il9 promoter activity by binding to the CNS1 within the Il9 locus. 42
Figure 1-4. BATF and BATF3 play redundant roles and compensate for each other. 47
Figure 1-5. BATF3 can compensate for the loss of BATF and induce Il9 expression. 50
Figure 1-6. BatfKO Th9 cells induce airway inflammation upon overexpression of Batf3. 53
Chapter 2. Transcription Factor Id1 Plays an Essential Role in Th9 Cell Differentiation by Inhibiting Tcf3 and Tcf4 11
Figure 2-1. Id1 deficiency inhibits differentiation of Th9 cells. 80
Figure 2-2. The differentiation of Th9 cells is dependent on the expression level of Id1. 85
Figure 2-3. Tcf3 and Tcf4 act as negative regulators of Th9 cell differentiation. 90
Figure 2-4. Tcf3 and Tcf4 bind to Il9 promoter and inhibit transcription. 94
Figure 2-5. Both Tcf3 and Tcf4 are important for blockade of Th9 cell differentiation. 97
Figure 2-6. Global gene expression analyses. 102
Figure 2-7. Id1 cKO mice show ameliorated airway inflammation. 106
Figure 2-8. Id1-deficient Th9 cells ameliorate airway inflammation in a cell-intrinsic manner. 111