CD137 ligand induced apoptosis in peripheral blood mononuclear cells (PBMCs)

CD137-INDUCED CELL DEATH IN PERIPHERAL BLOOD MONONUCLEAR CELLS NURULHUDA BINTE MUSTAFA (B.Sc(Hons.), NUS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY OF SCIENCE DEPARTMENT OF PHYSIOLOGY, MEDICINE NATIONAL UNIVERSITY OF SINGAPORE 2010 i ACKNOWLEDGEMENTS Oh humble graduate student how thy toils make thee weary! Be not for the Grace of God and thy teachers, family and friends, thou wouldst have surely perish. To Professor Shazib Pervaiz for the rigorous development of a scientific mind through your intellectual contributions and critical insights, for challenging me to rise to the occasion, for your understanding and patience, for nurturing an amazing lab which prizes friendship and love as much as Science, it will be difficult to find a better atmosphere to mature in, a profound Thank You. To Associate Professor Herbert Schwarz for always being there to assist and propel me in my research, for the unique broadening of my scientific perspective which comes when working with a co-supervisor in a different field of research and for your quiet encouragement, you have been an invaluable pillar of support in my progress and I am most grateful. To Dr. Jayshree, Ms. Kartini, Dr. Andrea and Dr. Alan, Thank You very much for your guidance especially at the initial stages as a naive UROPS student and throughout the course of my work, for the endless cups of coffee over science, enjoyable chats and selfless assistance beyond research. To each and every member of the ROS and Tumour Biology lab and the CD137 Immunotherapy Lab whom I have worked closely with, you guys know who you are. We brave not only the trials of scientific progress together, but also really care for each other’s personal life, you guys are like family. Special mention to the class of 2005, Inthrani, Sinong, Chew Hooi, Zhi Xiong and Greg for inspiring each other to continue marching down this difficult road and Shaqireen for being my sparring partner in CD137 research. To mom and dad, you have been unbelievable. For motivating me to always strive for excellence, for your precious prayers, unflagging love and support, and most importantly for your steadfast faith in me, words of gratitude are completely inadequate for all you have given me. To my siblings Adibah, Nabil and Hazi, thank you for inspiring me in ways that you not know. Finally to my husband, you alone truly know the rainbow of emotions and challenges that I have experienced in the course of my scientific research and in writing this thesis. For being willing to make any sacrifice so that I can focus on and pursue my graduate studies, , for painstakingly putting pieces together when they fall apart and for your dedication towards my best well being, you are my nikmah, my miraculous blessing. The pursuit of knowledge at its core is a commitment to apply new discoveries for the transformation and betterment of the society. May this be the beginning of such an endeavour. ii TABLE OF CONTENTS I. Acknowledgements .ii II. Summary .ix III. List of Figures .xi IV. List of Abbreviations .xiv 1.0 Introduction .1 1.1 Cell Death .1 1.1.1 Overview of Cell Death 1.1.2 Apoptotic Cell Death .1 1.1.3 Molecular Mechanisms Mediating Apoptosis .3 1.1.4 Extrinsic, Intrinsic And Granzyme B Cell Death Pathways 1.1.5 Caspase-Independent Cell Death 1.2 Apoptosis In the Immune System 10 1.2.1 Activated Induced Cell Death in T cells (AICD) 11 1.2.2 Activated Cell Autonomous Death in T cells (ACAD) .12 1.2.3 Monocyte-Dependent Cell Death in T cells (MDCD) .13 1.3 CD137/CD137 Ligand, TNFR/TNF Superfamily Members .15 1.3.1 Molecular Characteristics and Expression Patterns of CD137 .17 1.3.2 Effects of CD137 Signalling 18 1.3.2.1 CD137 Signalling in T cells .18 1.3.2.2 CD137 Signalling in B cells .19 iii 1.3.2.3 CD137 Signalling in Dendritic Cells 20 1.3.2.4 CD137 Signalling in Granulocytes and Natural Killer Cells .20 1.3.3 Clinical Significance of CD137 21 1.3.4 Molecular characteristics and expression pattern of CD137 Ligand .23 1.3.5 Effects of CD137 Ligand Signalling .24 1.3.5.1 CD137L signalling in Monocytes/Macrophages 24 1.3.5.2 CD137L signalling in Dendritic Cells .26 1.3.5.3 CD137L signalling in B cells 27 1.3.5.4 CD137/CD137L mediated inhibitory signalling .27 1.4 Reactive Oxygen Species 31 1.4.1 Major Types of ROS and their derivative species 32 1.4.2 Superoxide Anion 32 1.4.3 The family of NADPH Oxidases .33 1.4.4 Hydrogen Peroxide 38 1.4.5 Hydrogen Peroxide-Mediated Cell Death 39 1.4.6 Oxidative Stress and Endogenous Antioxidant Defence Mechanisms 41 1.4.7 Redox Signalling in Peripheral T cells 43 1.4.7.1 ROS-mediated T cell Proliferation .43 1.4.7.2 ROS-mediated T cell Death 44 1.4.7.3 Control of the Extrinsic Apoptotic Pathway by ROS-dependent Expression of FasL .44 1.4.7.4 Control of the Intrinsic Apoptotic Pathway by the ROS-dependent Suppression of Bcl-2 Expression 45 iv 1.4.7.5 ROS-mediated effects on T cell fate by Accessory Immune Cells 49 1.5 Aim Of Study 50 2.0 Materials and Methods 51 2.1 Chemical Reagents .51 2.2 Antibodies for Immunoblotting 52 2.3 Fluorescent dyes used for Flow Cytometry 52 2.4 Induction of CD137L signalling in Target Cells .53 2.5 Cells and Cell Culture 54 2.5.1 Isolation of Peripheral Blood Mononuclear Cells 54 2.5.2 Isolation of Mice splenocytes 55 2.5.3 SGH-MM6 cell line .56 2.6 Assays for proliferation and apoptosis .56 2.6.1 MTT Proliferation Assay 56 2.6.2 Morphological and quantitative analysis of cell size .57 2.6.3 Analysis of Phosphatidyl Serine Externalisation 58 2.6.4 Determination of Mitochondrial Transmembrane Potential 58 2.6.5 Analysis of Caspase Activity 59 2.7 Determination of protein expression levels by Immunoblotting .60 2.8 Mitochondrial and Cytosolic Isolation by differential centrifugation 62 2.9 Nuclear and cytosolic extraction………………………………………… ……………………………62 2.10 p65NF-κB Transcriptional (DNA Binding) Assay .63 2.11 TNF Enzyme –Linked Immunosorbent Assay (ELISA) .64 2.12 Assays for the Determination of Reactive Oxygen Species .64 2.12.1 Analysis of intracellular hydrogen peroxide production .64 v 2.12.2 Analysis of intracellular superoxide anion production 65 2.12.3 Analysis of mitochondrial superoxide production .65 2.12.4 Cell specific depletion of ROS in a mixed T cell and Monocyte reaction 66 2.13 Statistics………………………………………………………………………………… ………………….66 3.0 Results………………………………………………………………………………… ……….………………….67 3.1 Induction of CD137L signalling by immobilised CD137-Fc inhibits proliferation and stimulates apoptosis in three different cell types .67 3.1.1 CD137L signalling inhibits proliferation of activated human PBMCs 67 3.1.2 CD137L signalling also inhibits proliferation in activated mouse splenocytes and in multiple myeloma cell line, SGH-MM6 .68 3.1.3 CD137L mediated decrease in cell proliferation is due to induction of apoptosis .74 3.2 CD137L induced cell death is caspase independent and is mediated by the intrinsic not the extrinsic death pathway .78 3.2.1 CD137L signalling increases expression levels of death receptor TNFR1 and stimulates secretion of TNF, but is not critical to cell death .78 3.2.2 Inhibiting TRAIL-mediated signals with DR4 and DR5 blocking antibodies was unable to abrogate CD137-induced cell death .82 3.2.3 Ligation by CD137-Fc disrupts the mitochondrial transmembrane potential, and induces translocation of Cytochrome C into the cytosol 84 3.2.4 CD137L downregulates Bcl-2 protein levels at early time points, while maintaining Bim levels thus tilting the Bcl-2: Bim ratio towards favouring apoptosis 87 vi 3.2.5 CD137L signalling induces slight caspase-3 and-8 activity at 24h but cell death is caspase independent .89 3.2.6 CD137-induced cell death is independent of casein kinase (CK1) but is dependent on the MAPK pathway .92 3.3 CD137 induces apoptosis specifically within the CD3 + T cell subpopulation of the PBMCs and T cell death is mediated by monocytes 95 3.4 Cell death induced by CD137 is critically regulated by Reactive Oxygen Species (ROS) .100 3.4.1 CD137-induced cell death can be abrogated by scavengers of ROS, Catalase and Tiron 100 3.4.2 CD137 induces significant production of ROS in whole PBMCs 104 3.4.3 CD137L signalling induces ROS in both T cells and monocytes .107 3.5 It is ROS from T cells and not monocytes that is critical for the induction of CD137-mediated cell death 113 3.6 The source of ROS in T cells that activates the cell death program could be the NADPH Oxidase (NOX) or the mitochondria .115 3.6.1 Production of ROS critical to apoptosis is upstream of the disruption in mitochondrial transmembrane potential .115 3.6.2 ROS in T cells originates from NOX 115 3.6.3 Inhibiting superoxide dismutases (SOD) with DDC sensitised PBMCs to cell death 122 3.6.4 ROS in T cells may originate from mitochondria .128 4.0 Discussion .131 vii 4.1 CD137L-induced cell death is mediated via a caspase-independent intrinsic (mitochondrial-mediated) pathway .132 4.2 CD137L mediated cell death signalling in PBMCs is targeted to T cells .137 4.3 CD137/CD137L induced cell death is critically mediated by Reactive Oxygen Species (ROS) 141 4.3.1 ROS is the upstream molecular event crucial for stimulating the CD137-induced death signalling cascade leading to T cell apoptosis 142 4.3.2 ROS potentially mediates CD137-induced T cell apoptosis by downregulating the expression of Bcl-2 via the activity of ERK 143 4.4 Critical ‘killer’ ROS is produced by T cells not monocytes, though cell death is monocyte dependent .146 4.5 ROS species that is ultimately responsible for the induction of cell death signalling is H2O2 which is produced from the activity of NOX 149 4.6 So how is CD137/CD137L induced cell death physiologically relevant? .153 4.6.1 CD137/CD137L: The missing link in monocyte regulated T cell death? 154 4.7 Summary and Conclusion .159 5.0 References 164 6.0 Appendix 185 6.1 Supplementary Figures 185 6.2 Publication and Poster .192 viii SUMMARY CD137-INDUCED CELL DEATH IN PERIPHERAL BLOOD MONONUCLEAR CELLS Nurulhuda Mustafa National University of Singapore, 2010 CD137/CD137L are members of the TNFR/TNF superfamily that have exhibited significant immuno-modulatory effects in healthy and pathogenic states. Anti-CD137 antibodies have shown great promise in murine therapeutic models of cancer and autoimmunity. Generally, CD137/CD137L bi-directional signal transduction greatly amplifies the ongoing immune response as CD137 signalling is co-stimulatory for T-cells while CD137 Ligand (CD137L) signalling is activating for antigen presenting cells (APCs). In spite of the solid evidence for CD137-induced survival signalling in T-cells, it was found when CD137 was knocked out of splenocytes (1-3), splenic T cells responded not by hypoproliferation, but by hyper-proliferating instead (4). We set out to clarify this paradox and identified that when immobilised CD137-Fc cross-links its corresponding ligand (CD137L) on PBMCs, it activates an inhibitory signal which results in a reduced proliferative response to anti-CD3. This potentially explains why deficiency in CD137 would conversely allow cells to hyper-proliferate. We further determined that the lack of proliferative response is actually a function of cell death in PBMCs and that apoptosis is targeted to T cells. T cell death peaks at 24h, and there is a dose-dependent increase in apoptosis with increasing concentrations of CD137-Fc added to the PBMC culture. We found contrary to expectation that although there is a CD137-mediated upregulation of death receptors, CD137-induced apoptosis is not promoted by the extrinsic pathway but by the intrinsic pathway. We found a CD137ix mediated downregulation of Bcl-2 levels, while exerting no effect on Bim levels thus favouring an increase in the Bim: Bcl-2 ratio which subsequently leads to mitochondrial membrane permeabilization, the release of Cytochrome C and cell death. Despite the translocation of Cytochrome C into the cytosol, we observed only low levels of caspase and caspase activity and confirmed with a pan-caspase inhibitor that indeed that cell death is caspase- independent. We discovered instead that induction of cell death is critically dependent on reactive oxygen species (ROS) production. We report that ROS production is not a secondary effect of mitochondrial permeabilization but an early event initiating a death signalling cascade. ROS production was stimulated as early as 2h which returns to baseline at 6h but subsequently rises stably from 12h till 24h. We confirm that the key ROS species involved in cell death is hydrogen peroxide (H2O2) and that H2O2 production is NADPH oxidase (NOX)-dependent. Thus, superoxide produced by NOX is potentially converted to H2O2 by superoxide dismutases in the cell. Directly ligating purified T cells with CD137-Fc does not induce cell death. Concomitant studies demonstrated that CD137-mediated cell death is induced only in the presence of monocytes and increases with increasing monocyte concentrations. 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Virol. 76:829– 840 Xu, L.; Zhang, L.; Yi, Y.; Kang, H. K.; Datta, S. K. (2004) Human lupus T cells resist inactivation and escape death by upregulating COX-2. Nat. Med. 10(4):411-5 Fotin-Mleczek M., Henkler F., Samel D., Reichwein M., Hausser A., Parmryd I., Scheurich P., Schmid J. A., Wajant H. (2002) Apoptotic crosstalk of TNF receptors: TNF-R2-induces depletion of TRAF2 and IAP proteins and accelerates TNF-R1-dependent activation of caspase-8. Journal of Cell Science 115 (13) 2757-2770 184 6.0 Appendix 6.1 Supplementary Figures S1) 87.8% 92.6% 86.3% 66.5% 78.6% 67.7% 54.0% 74.7% Supplementary Figure 1. CD137L induced phosphatidyl serine (PS) externalisation in multiple myeloma cell line SGH-MM6. SGH-MM6 cells were treated with immobilised CD137-Fc, Fc or PBS for 12h and 24h and then were stained with Annexin V-FITC and Propidium Iodide (PI) and analysed by flow cytometry. Dot plots are representative of at least independent experiments 185 S2) Supplementary Figure 2. CD137L signalling significantly elevates cytokine TNF secretion from SGH-MM6 cells. SGH-MM6 cells were treated with immobilised CD137-Fc, Fc or PBS. After 4h, 12h, 24h and 48h, PBMCs were harvested. The supernatants were isolated from the cells and tested for cytokine TNF levels via ELISA. Data shown is mean + SD of two independent experiments done in triplicates. 186 S3) 86.1% 82.7% 61.4% 76.2% 74.2% 64.3% 76.2% 74.6% Untreated 66.8% Supplementary Figure 3. CD137L-induced apoptosis in SGH-MM6 cells are TNFR-independent SGH-MM6 cells were pre-incubated with TNFR blocking antibody (0.5μg/ml and 1μg/ml for 30 minutes before the cells were cross-linked with immobilised CD137-Fc, Fc or PBS. After 24h, the cells were loaded with Annexin V-FITC and Propidium Iodide (PI) and was analysed by flow cytometry. Dot plots are representative of at least independent experiments. 187 S4) Supplementary Figure 4. CD137L induces translocation of p65NF-κB from the cytosol into the nucleus in PBMCs. PBMCs were treated with immobilised CD137-Fc, Fc or PBS. After 24h, cells were harvested for nuclear isolation as described in Materials and Methods. Nuclear and Cytosolic lysates were collected and used for western blot analysis. 60μg of protein lysates were separated on SDS-PAGE to detect for p65NF-κB expression. PARP is the loading control utilised for the nuclear fraction. A representative blot is shown from two independent experiments. 188 189 S5) 0. 0. 0. 0. 1. 1. 1. 1. Fc 4h C D 3-F7c 12 h PB S 24 h 48 h 72 h (+ ) T N- F tr e a t e d H e l a WC L (+ ) c o m p e t i ti v e in h i b i to (+ r p) r noebgea t i v e c o n t r o l p r o b e PBMCs were treated with immobilised CD137-Fc, Fc and PBS. After 4h, 12h, 24h, 48h and 72h cells were harvested for nuclear isolation as described in Materials and Methods. Nuclear lysates were then collected and used for an ELISA based p65 transcription Factor assay as described in Materials and Methods. A representative graph plot is shown from two independent experiments. Supplementary Figure 5. CD137 induces activation of p65 NF-κB transcriptional binding activity in PBMCs. A b so rb an ce valu e p ro p o rtio n al to th e p 65 N F kB tran scrip tio n al b in d in g activity to targe t D N A in n u cle u s S6) 62.4% 69.8% 44.9% 65.8% 66.3% 51.0% Supplementary Figure 6. CD137L induced phosphatidyl serine (PS) externalisation in both OKT3 activated and non activated PBMCs PBMCs activated with 0.5ng/ml of OKT3 or not activated at all were treated with immobilised CD137-Fc, Fc or PBS for 24h and then were stained with Annexin V-FITC and Propidium Iodide (PI) and analysed on the flow cytometry. Dot plots are representative of at least independent experiments. 190 S7) Supplementary Figure 7. There is a dose dependent increase in cell death induced in PBMCs with increasing concentrations of CD137-Fc PBMCs activated with 0.5ng/ml of OKT3 were ligated with 5μg/ml, 10μg/ml, 20 μg/ml, 50 μg/ml and 100 μg/ml immobilised CD137-Fc, Fc or PBS for 24h and then were stained with Annexin V-FITC and Propidium Iodide (PI) and analysed by flow cytometry. 191 6.2 Publication and Poster PUBLICATION Nurulhuda Mustafa, Shaqireen D/O Kwajah M M, Andrea L. Holme, Shazib Pervaiz and Herbert Schwarz (2010). CD137-activated monocytes mediate T cell apoptosis through induction of reactive oxygen species. Manuscript in progress. POSTER AND ABSTRACT PRESENTED Nurulhuda Mustafa, Shaqireen D/O Kwajah M M, Shazib Pervaiz and Herbert Schwarz. CD137-induced cell death in Peripheral Blood Mononuclear Cells Abstract accepted for poster presentation at the 12th International TNF Conference 2009 held at Madrid, Spain. 192 . of CD137 Signalling 18 1.3.2.1 CD137 Signalling in T cells 18 1.3.2.2 CD137 Signalling in B cells 19 iv 1.3.2.3 CD137 Signalling in Dendritic Cells 20 1.3.2.4 CD137 Signalling in Granulocytes. of monocytes and increases with increasing monocyte concentrations. CD137-signalling actually induces increase in ROS levels in both monocytes and T cells and by specifically depleting ROS from T cells. dependent increase in cell death induced in PBMCs with increasing concentrations of recombinant CD137-Fc. xiv LIST OF ABBREVIATIONS AIDS Acquired Immunodeficiency Syndrome AIF Apoptosis Inducing Factor Apaf-1