Title Page
ABSTRACT
국문 초록
PREFACE
Contents
NOMENCLATURE(OR LIST OF SYMBOLS) 19
CHAPTER 1. INTRODUCTION 22
1.1. Monocyte-lineage cells in tumor microenvironment 22
1.2. Immune exhaustion 22
1.3. CD244 (2B4, SLAMF4) 23
1.4. Role of CD244 in monocyte-lineage cells in the tumor 23
CHAPTER 2. MATERIALS AND METHODS 25
2.1. Study design 25
2.2. Mice 25
2.3. Cell lines and tumor models 26
2.4. Cell isolation 26
2.5. Bone marrow-derived macrophages (BMDM) and tumor-infiltrating monocytes differentiation 26
2.6. BMDM adaptive transfer 27
2.7. Flow cytometry 27
2.8. Quantitative PCR 30
2.9. ELISPOT 31
2.10. Antigen presentation and phagocytosis assay 31
2.11. Fluorescence microscopy 31
2.12. scRNA-seq data analysis 32
2.13. Bulk RNA-seq data analysis 33
2.14. Statistical analyses 33
CHAPTER 3. RESULTS 34
3.1. Absence of CD244 signaling in monocyte-lineage cells suppresses melanoma tumorigenesis 34
3.1.1. CD244 expression significantly upregulated in monocytes and macrophages after tumor induction 34
3.1.2. Monocyte-lineage specific CD244-deletion abrogates melanoma tumor growth 36
3.2. Targeted CD244 deletion in monocyte-lineage cells enhances antigen-specific anti-tumor immunity of T cells 38
3.2.1. Targeted CD244 deletion in monocyte-lineage cells enhances IFN-γ and granzyme-b secretion of T cells 38
3.2.2. Targeted CD244 deletion in monocyte-lineage cells increases antigen-specific CD8 T cells 40
3.3. CD244 suppresses maturation of monocyte-lineage cells 42
3.3.1. CD244 regulates proportion of Ly6Chigh and Ly6Clow macrophages in tumor microenvironment[이미지참조] 42
3.3.2. CD244 signaling suppresses maturation of bone marrow monocyte-lineage cells into Ly6Clow macrophages[이미지참조] 44
3.3.3. CD244 suppresses maturation into Ly6Clow macrophages in tumor microenvironment[이미지참조] 47
3.4. CD244 suppresses anti-tumorigenic functions of macrophages 49
3.4.1. CD244 suppresses anti-tumorigenic cytokine secretion of macrophages 49
3.4.2. CD244 suppresses antigen presentation, phagocytosis in macrophages 51
3.5. ER stress increases the expression of CD244 on immunosuppressive monocytes, which ultimately inhibits autophagy 55
3.5.1. Public mouse scRNA-seq reveals 4 sub-clusters of monocyte-lineage cells in tumor microenvironment 55
3.5.2. CD244 high immunosuppressive monocytes show increased ER stress pathway 58
3.5.3. ER stress induces CD244 expression and suppresses macrophage differentiation 60
3.5.4. Deletion of CD244 increases autophagy in monocyte-lineage cells 62
3.6. CD244-deficient macrophages increase memory T cells and potentiate anti-PD-L1 therapy 66
3.6.1. Anti-PD-L1 therapy increases activation and memory of T cells without affecting exhaustion in monocyte-lineage specific CD244-deficient mice 66
3.6.2. Combination therapy of anti-PD-L1 antibody and CD244-deficient macrophages synergistically suppresses tumor growth 69
3.7. Monocyte/macrophage CD244 expression is negatively correlated with patient survival 71
3.7.1. CD244 regulates monocyte-lineage cells of patients same as in mice 71
3.7.2. CD244-negative monocyte-lineage cells increase T cell activation in patients 74
3.7.3. CD244-negative monocyte-lineage cells increase survival of melanoma patients 77
3.7.4. CD244 exhibits regulatory effects on monocyte-lineage cells in patients with other cancers similar to those observed in melanoma 79
CHAPTER 4. DISCUSSION 81
CHAPTER 5. CONCLUSTION 85
REFERENCES 86
Table 1. Flow cytometry antibody list 28
Table 2. Quantitative PCR primer list 30
Table 3. CD244 low and high patients classification 76
Figure 1. CD244 expression upregulated in tumor infiltrating monocyte-lineage cells 35
Figure 2. Specific deletion of CD244 in monocyte-lineage cells suppresses melanoma tumorigenesis 37
Figure 3. CD244 deletion in monocyte-lineage cells increases anti-tumor activity of T cells 39
Figure 4. CD244 deletion in monocyte-lineage cells increases antigen specific activation of CD8 T cells 41
Figure 5. CD244 deletion in monocyte-lineage cells alters proportion of Ly6Chigh and Ly6Clow macrophages in tumor microenvironment[이미지참조] 43
Figure 6. CD244 deletion in bone marrow monocytes increases differentiation into Ly6Clow macrophages[이미지참조] 45
Figure 7. CD244 deletion in tumor-infiltrating monocytes increases differentiation into Ly6Clow macrophages[이미지참조] 48
Figure 8. CD244 deletion in monocyte-lineage cells increases anti-tumorigenic cytokine secretion of macrophages 50
Figure 9. CD244 deletion in monocyte-lineage cells increases antigen presentation and phagocytosis of Ly6Clow macrophages[이미지참조] 53
Figure 10. Identification of monocyte-lineage cell sub-populations in public mouse scRNA-seq data 56
Figure 11. CD244 expression upregulated in immunosuppressive monocytes and positively correlates with ER stress pathway 59
Figure 12. ER stress regulators modulate both CD244 expression and maturation into Ly6Clow macrophages[이미지참조] 61
Figure 13. CD244 suppresses autophagy and consequently hinders macrophage differentiation 64
Figure 14. Anti-PD-L1 antibody alters T cell proportion and phenotype in LysMcreCD244fl/fl mice[이미지참조] 67
Figure 15. Anti-PD-L1 therapy synergistically eradicates melanoma and lung cancer, when combined with CD244-deficient BMDMs 70
Figure 16. CD244 suppresses anti-tumorigenic functions of monocyte-lineage cells in human melanoma patients 72
Figure 17. CD244-negative monocyte-lineage cells increases CD8 T cell activation in melanoma patients 75
Figure 18. Patients with CD244-negative monocyte-lineage cells showed significantly improved survival 78
Figure 19. CD244 exerts regulatory effects on monocyte-lineage cells in patients with other cancers, resembling the effects observed in melanoma 80