Potential Tumor-Promoting Effects of Ectopic Cd137 Expression on Hodgkin Lymphoma
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POTENTIAL TUMOR-PROMOTING EFFECTS OF ECTOPIC CD137 EXPRESSION ON HODGKIN LYMPHOMA HO WENG TONG (B. Sci (Biomedical Sci.), UPM, Malaysia) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHYSIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2013 i Acknowledgement I would like to take this opportunity to address my appreciation to my thesis supervisor, Associate Professor Herbert Schwarz, who had provided me with exceptional guidance and advice throughout my candidature. Besides that, he also gave me a lot of supports and this study would not be possible without his truly contributions. I would also like to thank Dr. Shao Zhe, Dr Jiang Dongsheng and Dr Shaqireen for guiding me through basic laboratory technique when I first joined the group, Dr. Gan Shu Uin, Dr. Angela Moh and Ms Tan Teng Ee for assisting me in the generation of transfected and knock down cell lines, and Ms Lee Shu Ying from the confocal unit, NUHS, for providing the necessary facility for my confocal imaging. In addition, I would also like to show my appreciation to Ms Pang Wan Lu who worked closely with me in this Hodgkin lymphoma project. Last but not least, I would also like to express my pleasure to work with all the members from Herbert Schwarz' laboratory, especially Zulkarnain, Qianqiao, Liang Kai and Andy who gave me a lot of support both technically and morally. ii TABLE OF CONTENTS DECLARATION i ACKNOWLEDGEMENT ii TABLE OF CONTENTS iii ABSTRACT vii LIST OF TABLES ix LIST OF FIGURES x LIST OF ABBREVIATION xii 1. INTRODUCTION 1 1.1 Hodgkin lymphoma 2 1.1.1 Etiology and pathophysiology 3 1.1.2 Immunosuppressive microenvironment 6 1.1.3 Association of HL with members of tumor necrosis 8 factor receptor family 1.2 CD137 and CD137L 1.2.1 Expression and characteristic of CD137 and 12 CD137L 1.2.2 Targeting CD137 for immunotherapy 16 1.2.3 CD137 and CD137L in malignant diseases 18 1.3 Trogocytosis 19 1.4 Research objectives 22 iii 2. MATERIALS AND METHODS 24 2.1 Cells and cell culture 24 2.1.1 Cell lines 24 2.1.2 CD137 overexpressing cell lines 24 2.1.3 PBMC isolation 25 2.1.4 T cells, B cells and Monocytes purification 26 2.1.5 Storage of cell lines and primary cells 27 2.2 Antibodies and reagents 27 2.3 Flow cytometry 28 2.4 Enzyme linked immunosorbent assay (ELISA) 29 2.5 Western Blot 29 2.5.1 Protein purification 29 2.5.2 Electrophoresis and electroblotting 30 2.5.3 Antibody probing and visualization 30 2.6 Co-immunoprecipitation 31 2.7 Confocal microscopy 31 2.8 Endocytosis inhibition assay 32 2.9 Cell viability assay 32 2.10 Trogocytosis assay 33 2.11 Reserve-transcriptase polymerase chain reaction 33 2.11.1 RNA extraction 33 2.11.2 Reverse transcription 34 2.11.3 Polymerase chain reaction (PCR) 35 2.12 Light microscopic examination 36 2.13 Antibody treatment 36 iv 2.13.1 CD137 and CD137L neutralization 36 2.13.2 Agonistic CD137 stimulation 36 2.14 Statistics 37 3. RESULT 38 3.1 Screening and generation of RS cell lines 38 3.2 Ectopically expressed CD137 reduces the T cell 42 stimulatory capacity of HRS cells 3.2.1 Co-culturing HRS cells with PBMC and T cells 42 3.2.2 Ectopically expressed CD137 down-regulates 46 CD137L expression on RS cells 3.2.3 Induction of IFN-γ release is due to CD137L 48 upregulation after CD137 silencing 3.2.4 Summary 51 3.3 Mechanism of CD137L disappearance 52 3.3.1 CD137 neutralization does not affect CD137L 53 mRNA expression 3.3.2 CD137L protein expression increases after CD137 54 neutralization 3.3.3 CD137-CD137L co-localization in RS cells 56 3.3.4 CD137 and CD137L are co-internalized via 61 endocytosis 3.3.5 Trogocytosis mediates CD137 transfer which 65 causes CD137L downregulation 3.3.6 Summary 74 3.4 Transfer of CD137 to surrounding monocytes and B cells 75 3.4.1 Ectopically expressed CD137 induces CD137L 76 downregulation in monocytes and B cells v 3.4.2 CD137 overexpressing cell lines abrogate IFN-γ 83 release from PBMC 3.4.3 Summary 84 3.5 Potential of CD137 and CD137L signaling on HRS cells 85 3.5.1 Agonistic anti-CD137 antibody 85 3.5.2 CD137 stimulation causes morphological changes in 87 HRS cells 3.5.3 Involvement of the cytoplasmic domain of CD137L in 89 HRS cell signaling 3.5.4 Summary 91 4. DISCUSSION 92 4.1 Ectopically expressed CD137 abrogates T cell activity 92 4.2 CD137 trogocytosis 99 4.2.1 CD137 and CD137L transfer 101 4.2.2 Aggregation of CD137 and CD137L in the cytoplasm 103 4.3 CD137 and CD137L induce signaling into HRS cells 104 4.4 Limitations 106 4.5 Future works 107 4.6 Conclusion 110 REFERENCES 111 APPENDIX I ACRYLAMIDE GEL CASTING 125 APPENDIX II MEDIA AND BUFFERS 126 APPENDIX III ANTIBODIES LIST 134 APPENDIX IV CD137L DOWNREGULATION ON 136 PRIMARY MONOCYTES AND B CELLS vi ABSTRACT Hodgkin lymphoma (HL) is a hematological malignancy. The malignant cells in HL, the Hodgkin and Reed Sternberg (HRS) cells, comprise only a minority of the entire tumor mass while infiltrating inflammatory cells constitute the vast majority of the tumor mass. HRS cells were reported to express CD137 ectopically but the function(s) of CD137 in the pathogenesis of HL remained unidentified. CD137 is a potent co-stimulatory molecule expressed by activated T cells. Upon ligation by CD137 ligand (CD137L) which is mainly expressed by antigen presenting cells (APC), CD137 signalling enhances T cell activity. This study shows that ectopic CD137 expression on HRS cells reduces IFN-γ release from T cells by downregulating CD137L expression on HRS cells. The IFN-γ suppression due to ectopic CD137 expression can be reversed by neutralizing CD137 with antibodies or by knocking down CD137 with siRNA. The downregulation of CD137L is not due to a reduction of de novo synthesis of CD137L but due to an increased CD137L turnover. When CD137 binds to CD137L, the CD137-CD137L complex will be internalized, and hence the surface CD137L levels available for T cell co-stimulation are reduced. Ectopic CD137 also gets transferred from HRS cells to surrounding cells including B cells and monocytes and leads to the downregulation of CD137L on monocytes. The CD137L downregulation on monocytes results in a lower T cell co-stimulation and a reduction of IFN-γ release from T cells. In addition, this study finds that ectopic CD137 on HRS cells might induce signalling into HRS cells but the benefits that HRS cells may gain from this signalling are yet to be identified. In conclusion, this study provides new vii insights into the immune escape mechanism of HL, and opens new research areas for the development of novel therapeutic approaches. viii LIST OF TABLES Table 1. Characteristic of Hodgkin lymphoma subtype 4 Table 2. RT-PCR reaction mix 34 Table 3. Standard PCR reaction mix 35 Table 4. Primers set for CD137 and cyclophilin PCR 35 Table 5. PCR thermal cycling for CD137L amplification 36 Table 6. Changes in CD137L expression on monocytes and B 82 cells after co-culturing with HRS cell lines ix LIST OF FIGURES Figure 1. CD137 expression on Hodgkin Lymphoma 11 Figure 2. CD137 and CD137L bidirectional signaling 15 Figure 3. PBMC isolation with density gradient centrifugation. 26 Figure 4. Expression of CD137 on Reed Sternberg cell lines 40 Figure 5. CD137 silencing of KM-H2 cells 41 Figure 6. CD137 expression on L428, L540 and L1236 cells after 41 CD137 transfection Figure 7. CD137 neutralization on KM-H2 cells induces higher 44 IFN-γ release from PBMC Figure 8. Impaired CD137 expression on KM-H2 cells leads to 45 higher IFN-g realease from PBMC and T cells Figure 9. CD137 and CD137L expression in KM-H2 cells after 47 CD137 neutralization Figure 10. CD137L is upregulated in KM-H2 cells after CD137 47 silencing Figure 11. CD137L neutralizing antibodies abrogate the 49 induction of IFN-γ release from PBMC during co- culture with KM-H2-CD137 - cells. Figure 12. CD137L neutralizing antibodies abrogate the 50 induction of IFN-γ release from T cells during co- culture with KM-H2-CD137 - cells. Figure 13. CD137 neutralization does not affect mRNA expression 53 level of CD137L in KM-H2 cells Figure 14. Total CD137L protein in KM-H2 cells was upregulated 55 after CD137 neutralization Figure 15. Upregulation of CD137L protein in KM-H2 cells after 55 CD137 neutralization Figure 16. CD137L co-immunoprecipitates with CD137 in KM-H2 58 cells. x Figure 17. CD137 neutralization reduces CD137 and CD137L 58 interaction in KM-H2 cells. Figure 18. Colocalization of CD137 and CD137L in KMH2 cells 60 Figure 19. Toxicity titration of MDC on KM-H2 cells 63 Figure 20. Inhibition of endocytosis increases CD137 and CD137L 64 expression on KM-H2 cells Figure 21. CD137 and CD137L induce trogocytosis between donor 67 and recipient cells Figure 22. The presence of CD137 and CD137L induce L-428 and L- 68 1236 cell aggregation with KMS-11 cells 70 Figure 23. Downregulation of CD137L on KMS-11 cells following CD137L dependent transfer of CD137 from L-428 cells to KMS-11 cells Figure 24. Downregulation of CD137L on KMS-11 cells following 71 CD137L dependent transfer of CD137 from L-1236 cells to KMS-11 cells Figure 25. CD137L transfer from KMS-11-CD137L cells to L-428- 72 CD137 cells Figure 26.