Potential tumor promoting effects of ectopic CD137 expression on hodgkin lymphoma

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 INTRODUCTION 1.1 Hodgkin lymphoma 1.1.1 Etiology and pathophysiology 1.1.2 Immunosuppressive microenvironment 1.1.3 Association of HL with members of tumor necrosis factor receptor family 1.2 CD137 and CD137L 1.2.1 Expression and characteristic of CD137 and CD137L 12 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 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 RESULT 38 3.1 Screening and generation of RS cell lines 38 3.2 Ectopically expressed CD137 reduces the T cell stimulatory capacity of HRS cells 42 3.2.1 Co-culturing HRS cells with PBMC and T cells 42 3.2.2 Ectopically expressed CD137 down-regulates CD137L expression on RS cells 46 3.2.3 Induction of IFN-γ release is due to CD137L upregulation after CD137 silencing 48 3.2.4 Summary 51 Mechanism of CD137L disappearance 52 3.3.1 CD137 neutralization does not affect CD137L mRNA expression 53 3.3.2 CD137L protein expression increases after CD137 neutralization 54 3.3.3 CD137-CD137L co-localization in RS cells 56 3.3.4 3.3 CD137 and CD137L are co-internalized via 61 endocytosis 3.3.5 65 3.3.6 3.4 Trogocytosis mediates CD137 transfer which causes CD137L downregulation Summary 74 Transfer of CD137 to surrounding monocytes and B cells 75 3.4.1 76 Ectopically expressed CD137 induces CD137L downregulation in monocytes and B cells v 3.4.2 CD137 overexpressing cell lines abrogate IFN-γ release from PBMC 83 3.4.3 Summary 84 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 HRS cells 87 3.5.3 Involvement of the cytoplasmic domain of CD137L in HRS cell signaling 89 3.5.4 3.5 Summary 91 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 110 111 REFERENCES APPENDIX I ACRYLAMIDE GEL CASTING 125 APPENDIX II MEDIA AND BUFFERS 126 APPENDIX III ANTIBODIES LIST 134 APPENDIX IV CD137L DOWNREGULATION ON PRIMARY MONOCYTES AND B CELLS 136 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 Characteristic of Hodgkin lymphoma subtype Table RT-PCR reaction mix 34 Table Standard PCR reaction mix 35 Table Primers set for CD137 and cyclophilin PCR 35 Table PCR thermal cycling for CD137L amplification 36 Table Changes in CD137L expression on monocytes and B cells after co-culturing with HRS cell lines 82 ix antibodies against human 4-1BB: distinct epitopes of human 4-1BB on lung tumor cells and immune cells Tissue Antigens 70:470-479 Zhang, X., C.J Voskens, M Sallin, A Maniar, C.L Montes, Y Zhang, W Lin, G Li, E Burch, M Tan, R Hertzano, A.I Chapoval, K Tamada, B.R Gastman, D.H Schulze, and S.E Strome 2010 CD137 promotes proliferation and survival of human B cells Journal of Immunology 184:787-795 Zhang, Y., and B Zhang 2008 TRAIL resistance of breast cancer cells is associated with constitutive endocytosis of death receptors and Molecular Cancer Research : MCR 6:1861-1871 Zhang, Y., T Yoshida, and B Zhang 2009 TRAIL induces endocytosis of its death receptors in MDA-MB-231 breast cancer cells Cancer Biology & therapy 8:917-922 Zhao, S., H Zhang, Y Xing, and Y Natkunam 2012 CD137 Ligand Is Expressed in Primary and Secondary Lymphoid Follicles and in B-cell Lymphomas: Diagnostic and Therapeutic Implications The American Journal of Surgical Pathology Zhou, Z., S Kim, J Hurtado, Z.H Lee, K.K Kim, K.E Pollok, and B.S Kwon 1995 Characterization of human homologue of 4-1BB and its ligand Immunology Letters 45:67-73 Zhu, B.Q., S.W Ju, and Y.Q Shu 2009 CD137 enhances cytotoxicity of CD3(+)CD56(+) cells and their capacities to induce CD4(+) Th1 responses Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie 63:509-516 124 APPENDIX I ACRYLAMIDE GEL CASTING For making of 10 ml of resolving gel: Reagent (ml) 8% gel 10% gel 12% gel 15% gel MilliQ water 4.6 4.0 3.3 2.3 30% Acrylamide mix (Bio-rad) 2.7 3.3 4.0 5.0 1.5M Tris (pH 8.8) 2.5 2.5 2.5 2.5 10% SDS (1st Base) 0.1 0.1 0.1 0.1 10% APS (Sigma-Aldrich) 0.1 0.1 0.1 0.1 TEMED (Sigma-Aldrich) 0.006 0.004 0.004 0.004 For making of ml of stacking gel: Reagent (ml) MilliQ water 1.4 30% Acrylamide mix (Bio-rad) 0.33 1.5M Tris (pH 6.8) 0.25 10% SDS (1st Base) 0.02 10% APS (Sigma-Aldrich) 0.02 TEMED (Sigma-Aldrich) 0.002 125 APPENDIX II MEDIA AND BUFFERS Cell culture RPMI with 10% FBS For preparing of L: Item Quantity Source RPMI-1640 16.4 g Sigma-Aldrich L-glutamine (200 mM) 10 ml Gibco Sodium bicarbonate 2g Sigma-Aldrich FBS 100 ml Biowest MilliQ water Top up to L - Adjusted to pH 7.0 and filtered with 0.22 µm filter unit (Nalgene) RPMI, 10% FBS with Penicillin and streptomycin For preparing of 100 ml: Item Quantity Source RPMI with 10% FBS 99 ml - Penicillin (10,0000 U) /Streptomycin(10,000 µg/ml) ml Gibco Selective medium (5 µg/ml Blasticidin) For preparing of 100 ml: Item Quantity Source RPMI with 10% FBS 99.5 ml - Blasticidin (1 mg/ml) 500 µl Sigma-Aldrich 126 Cell line freezing medium (20% FBS, 10% DMSO) For preparing of 100 ml: Item Quantity Source RPMI with 10% FBS 77.8 ml - FBS 12.2 ml Biowest DMSO 10 ml Sigma-Aldrich Primary cell freezing medium, Part A (40% FBS) For preparing of 100 ml: Item Quantity Source RPMI with 10% FBS 66.7 ml - FBS 33.3 ml Biowest Primary cell freezing medium, Part B (40% FBS, 20% DMSO) For preparing of 100 ml: Item Quantity Source RPMI with 10% FBS 44.4 ml - FBS 35.6 ml Biowest DMSO 20 ml Sigma-Aldrich 127 PBMC isolation and purification 10 x Phosphate buffer saline (PBS) For preparing of L: Item Quantity Source Sodium chloride (NaCl) 80 g Sigma-Aldrich Potassium chloride (KCl) Disodium hydrogen phosphate (Na2HPO4) Monopotassium phosphate (KH2PO4) 2g Sigma-Aldrich 14.4 g Sigma-Aldrich 2.4 g Sigma-Aldrich MilliQ water Top up to L - Item Quantity Source 10 x PBS 100 ml - MilliQ water Top up to L - Item Quantity Source x PBS 99.6 ml - EDTA (0.5 M) 400 µl 1st Base Adjusted to pH 7.4 x PBS For preparing of L: Sterilized with autoclaving PBS with mM EDTA For preparing of 100 ml: Sterilized with autoclaving 128 RBC lysis buffer For preparing of L: Item Quantity Source 8.29 g Sigma-Aldrich 0.84 g Sigma-Aldrich EDTA (0.5 M) 23 ml 1st Base MilliQ water Top up to L - Ammonium chloride (NH4Cl) Sodium bicarbonate (NaHCO3) Filtered with 0.22 µm filter unit (Nalgene) MASC buffer For preparing of L: Item Quantity Source 10 x PBS 100 ml - Bovine serum albumin (BSA) 5g Science Werke EDTA (0.5 M) ml 1st Base MilliQ water Top up to L - Stirred until dissolve and filtered with 0.22 µm filter unit (Nalgene) 129 Flow cytometry FACS buffer For preparing of 500 ml: Item Quantity Source 10 x PBS 50 ml - FBS 2.5 ml Biowest 20% Sodium azide 500 µl Sigma-Aldrich MilliQ water Top up to 500 ml - ELISA PBS with 0.05% Tween-20 (PBST) For preparing of L: Item Quantity Source 10 x PBS 100 ml - Tween-20 500 µl NUMI store, NUS MilliQ water Top up to L - Stirred until dissolve 130 Co-Immunoprecipitation and Western blot RIPA buffer For preparing of 100 ml: Item Quantity Source Tris 606 mg 1st Base NaCl 876 mg NUMI store, NUS SDS (10% w/v) ml 1st Base Sodium deoxycholate 0.5 µg Sigma-Aldrich Triton-X ml Bio-Rad PMSF (100mM) ml* Sigma-Aldrich MilliQ water Top up to 100 ml - Stirred until dissolve and adjusted to pH 7.4 *PMSF was added prior to use Non-denaturing lysis buffer (PBS with % Triton-X) For preparing of 100 ml: Item Quantity Source x PBS 98 ml - Triton-X ml Bio-Rad PMSF (100mM) ml* Sigma-Aldrich Stirred until dissolve *PMSF was added prior to use 131 x Tris-Glycine SDS Running Buffer For preparing of L: Item Quantity Source Tris 15.1 g 1st Base Glycine 72 g 1st Base SDS (10% w/v) 50 ml 1st Base MilliQ water Top up to L - Stirred until dissolve x Tris-Glycine SDS Running Buffer For preparing of L: Item Quantity Source x Tris-Glycine Buffer 200 ml - MilliQ water Top up to L - Item Quantity Source Tris 3.02 g 1st Base Glycine 14.4 g 1st Base Absolute Ethanol 200 ml NUMI store, NUS MilliQ water Top up to L - Transfer buffer For preparing of L: Stirred until dissolve and adjusted to pH 8.0 132 Western blot blocking buffer For preparing of 100 ml: Item Quantity Source Skim milk powder 5g Anlene PBST Top up to 100 ml - Stirred until dissolve Confocal microscopy Fixing buffer (4% PFA in PBS) For preparing of 100 ml: Item Quantity Source x PBS 100 ml - Paraformaldehyde (PFA) 4g Sigma-Aldrich M sodium hydroxide (NaOH) was added dropwise until PFA was fully dissolved Then adjusted to pH 7.2 with hydrochloric acid (HCl) Permeabilization buffer (0.2% Triton-X in PBS) For preparing of 100 ml: Item Quantity Source x PBS 100 ml - Triton-X 200 µl Bio-Rad Stirred until dissolve 133 APPENDIX III ANTIBODIES LIST Antigen Company 4B4-1-PE BD Pharmigen Flow cytometry staining 1:10 4-6 BBK-2 Lab Vision CD137 neutralization 1:200 7-9, 14, 15, 17, 20 Flow cytometry staining 1:200 9, 20, 23-28 Co-immunoprecipitation µg/1 mg of protein 16, 17 Confocal staining CD137 Clone Usage Dilution Figure 1:100 18 JG1.6a CD137 stimulation 1:200 31, 32 5F4-PE Biolegend Flow cytometry staining 1:10 9, 10, 20 Confocal staining CD137L Novus Biologicals 1:10 18 CD137L neutralization 1:100 10, 11 5F4 Biolegend 134 134 41B436 Adipogen CD137L neutralization 1:200 10, 11 Western blot 1:2000 14-17, 29 Flow cytometry staining 1:200 23-26 1:100 10, 11 C65-485 BD Pharmigen CD137L neutralization EPR1172Y Origene Confocal staining CD11b ICRF44 eBioscience Flow cytometry staining 1:10 27 CD20 2H7 eBioscience Flow cytometry staining 1:10 28 33 135 135 APPENDIX IV CD137L DOWNREGULATION ON PRIMARY MONOCYTES AND B CELLS Experiment A Monocytes HDLM2 KMH2 70% 41% 70% 55% CD137L B B cells HDLM2 KMH2 14% 13% 14% 15% CD137L PBMC alone Co-culture CD137L expression on monocytes and B cells following co-culture with HRS cell lines x 105 cells/ml of KM-H2 or HDLM-2 cells were co-cultured with 106 of PBMC suboptimally activated with ng/ml of anti-CD3 (Clone Okt3) 24 h later, the cells were harvested and stained for CD137L (clone 41B436), CD11b (for monocytes) and CD20 (for B cells), and analyzed with flow cytometry The histograms shown are gated on (A) CD11b+ or (B) CD20+ population only Open histograms show the PBMC alone condition, while grey histogram show PBMC co-culture with HRS cells condition The percentages of positively stained cells are shown in the histograms 136 Experiment A Monocytes PBMC alone 17% HDLM2 KMH2 12% 13% CD137L B cells B PBMC alone 17% HDLM2 KMH2 19% 39% CD137L CD137L expression on monocytes and B cells following co-culture with HRS cell lines x 105 cells/ml of KM-H2 or HDLM-2 cells were co-cultured with 106 of PBMC suboptimally activated with ng/ml of anti-CD3 (Clone Okt3) 24 h later, the cells were harvested and stained for CD137L (clone 41B436), CD11b (for monocytes) and CD20 (for B cells), and analyzed with flow cytometry The histograms shown are gated on (A) CD11b+ or (B) CD20+ population only Open histograms show the isotype staining of CD137L The percentages of positively stained cells are shown in the histograms 137 Experiment Monocytes A PBMC alone 4% HDLM2 KMH2 16% 25% CD137L B cells B PBMC alone 6% HDLM2 KMH2 8% 25% CD137L CD137L expression on monocytes and B cells following co-culture with HRS cell lines x 105 cells/ml of KM-H2 or HDLM-2 cells were co-cultured with 106 of PBMC suboptimally activated with ng/ml of anti-CD3 (Clone Okt3) 24 h later, the cells were harvested and stained for CD137L (clone 41B436), CD11b (for monocytes) and CD20 (for B cells), and analyzed with flow cytometry The histograms shown are gated on (A) CD11b+ or (B) CD20+ population only Open histograms show the isotype staining of CD137L The percentages of positively stained cells are shown in the histograms 138 ... LIST OF FIGURES Figure CD137 expression on Hodgkin Lymphoma 11 Figure CD137 and CD137L bidirectional signaling 15 Figure PBMC isolation with density gradient centrifugation 26 Figure Expression of. .. the effects of ectopic expression of CD137 on the survival of HRS cells In the next section of Introduction, the biology of CD137 and its involvement in disease state will be discussed Figure CD137. .. 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