CD244 in the pathogenesis of experimental autoimmune encephalomyelitis : Implication of natural killer cells in autoimmune disease

목차 (Table of Contents)

• Abstract 1
• Table of Content 2
• List of Figures 5
• List of Tables 8
• List of Abbreviations 9
• 1. Introduction 12
• 1.1. Central nervous system (CNS) autoimmune disease 12
• 1.2. Experimental autoimmune encephalomyelitis (EAE) as a model for multiple sclerosis 12
• 1.3. Role of natural killer (NK) cells in experimental autoimmune encephalomyelitis (EAE) 14
• 1.4. CD244 (SLAM4) 16
• 2. Aim 19
• 3. Materials and Methods 20
• 3.1. Mice 20
• 3.2. Induction and clinical evaluation of EAE 20
• 3.3. Histology 21
• 3.3.1. Luxol fast blue and hematoxylin/eosin staining 21
• 3.3.2. Immunohistochemistry and immunofluorescence staining 21
• 3.4. Western blot analysis 22
• 3.5. Isolation of mononuclear cells from spleen and CNS 23
• 3.5.1. Isolation of mononuclear cells from spleen 23
• 3.5.2. Isolation of CNS-infiltrating mononuclear cells 23
• 3.6. Flow cytometry 24
• 3.7. Proliferation assay 25
• 3.7.1. 3H-thymidine incorporation assay 25
• 3.7.2. Carboxyfluorescein succinimidyl ester (CFSE) labeling assay 25
• 3.8. Cytometric bead array (CBA) 26
• 3.9. Real-time polymerase chain reaction (Real-time PCR) 26
• 3.10. Depletion of NK1.1+ cells by mAb 27
• 3.11. NK cytotoxicity assay 28
• 3.11.1. Isolation of CD4+ T cells and NK cells 28
• 3.11.2. Cytotoxicity assay 28
• 3.12. Statistical Analysis 28
• 4. Results 29
• 4.1. CD244 expression on NK cells in the periphery is strongly reduced during the early and peak phase of EAE. 29
• 4.2. Expressions of CD48, CD244 ligand, increase on T cell subsets during EAE. 32
• 4.3. Expression patterns of CD244 and CD48 in the CNS change more dramatically than those in the periphery. 35
• 4.4. CD244 is mostly expressed on NK cells and macrophages in CNS-infiltrating cells. 38
• 4.5. Experimental autoimmune encephalomyelitis attenuated and delayed in CD 244 KO mice. 41
• 4.6. CNS inflammatory lesions of CD244 KO mice contain fewer T cells and more NK cells. 44
• 4.7. CD244 KO mice show impaired recall proliferation of autoreactive T cells 49
• 4.8. CD244 KO mice have reduced mRNA expression of Th1/Th17 type cytokines and transcriptional factor in the CNS inflammatory lesion. 51
• 4.9. CD244 KO mice produce less Th1 and Th17 effector cytokines from CNSinfiltrating cells. 54
• 4.10. CD244 deficiency does not affect the expression of NKG2A on NK cells. 59
• 4.11. An antibody depletion of NK cells in CD244 KO mice promotes the development of EAE. 60
• 4.12. CNS-infiltrating NK cells have less cytotoxicity along with less expression of SLAM-associated protein (SAP) in EAE-induced mice. 63
• 4.13. CNS-infiltrating NK cells efficiently kill autoreactive T cells via up-regulating SAP expression. 67
• 4.14. Target cells killing by NK cells are protected by the CD48 blockade. 72
• 5. Discussion 75
• References 82
• Abstract in Korean 93
• Acknowledgements 95