Title Page

Contents

ABSTRACT 11

Ⅰ. INTRODUCTION 13

1. History of natural killer cells 13

2. Characterization of NK cells 14

3. Development of NK cells 14

4. NK cells and Cancer 16

4.1. NK cell receptors and their ligands 16

4.2. Mechanisms of NK cells activity 19

4.3. NK cell functions and immune modulation 19

5. NK cell based immunotherapy 21

6. Objective and strategies 21

Ⅱ. MATERIALS AND METHODS 23

1. Cell lines 23

2. Ex vivo expansion of NK cells 23

3. Flow cytometry 24

4. CFSE proliferation assay 26

5. Western blot analysis 26

6. CD107a degranulation assay 27

7. Cytotoxicity assay 27

8. Cytokine bead assay (CBA) 29

9. In vivo tumor model 29

10. Statistical analysis 30

Ⅲ. RESULTS 31

1. Effect of γ-globulin on lymphocytes 31

2. Co-stimulatory effect of γ-globulin and IL-18 35

3. Optimization of NK cell expansion 37

4. Characterization of ex vivo expanded NK cells 44

4.1. Cell composition of expanded cells 44

4.2. Expression of activating and inhibitory receptors 47

4.3. Expression of intracellular cytokines 50

4.4. Functional activity of NK cells 52

4.5. Cytokine secretion 54

5. Evaluation of the cryopreservation of NK cells 56

6. in vitro anti-cancer effect 59

7. in vivo study 62

Ⅳ. DISCUSSION 64

Ⅴ. CONCLUSION 66

REFERENCES 67

ABSTRACT IN KOREAN 73

Table 1. List of antibodies used for flow cytometry 25

Table 2. List of antibodies used for the blocking assay 28

Table 3. Cell composition in expanded cells[내용없음] 9

Table 4. Relative MFI of NK cell receptors (CD3⁻CD56⁺ gated) 49

Fig. 1. Comparison of activity by γ-globulin concentration. 33

Fig. 2. Comparison of proliferation by γ-globulin concentration. 34

Fig. 3. Synergistic effect of IL-18 and γ-globulin on NK cells. 36

Fig. 4. Evaluation of changes in NK cells during the culture period. 39

Fig. 5. Analysis of cellular senescence markers by culture period. 40

Fig. 6. Optimization of NK cell expansion. 41

Fig. 7. Comparison in static and dynamic culture conditions. 42

Fig. 8. Scale-up process evaluation of NK cells using a bioreactor. 43

Fig. 9. Characterization of ex vivo expanded NK cells. 46

Fig. 10. Characterization of NK ligands in ex vivo expanded NK cells. 48

Fig. 11. Intracellular cytokine expression. 51

Fig. 12. Functional activity of NK cells. 53

Fig. 13. Measurement of secreted cytokines. 55

Fig. 14. Evaluation of in vitro equivalence of fresh and frozen NK cells. 57

Fig. 15. Evaluation of in vivo equivalence of fresh and frozen NK cells. 58

Fig. 16. Expression of NK cell ligands on glioblastoma. 60

Fig. 17. in vitro anti-tumor effect against glioblastoma. 61

Fig. 18. in vivo anti-tumor efficacy in glioblastoma mouse orthotopic model. 63