CHARACTERIZATION OF CD137 LIGAND MEDIATED HUMAN MONOCYTE DIFFERENTIATION AND THEIR EFFECTS ON T CELL ACTIVITIES SHAQIREEN D/O KWAJAH M. M. (B.Sc (Hons), NUS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHYSIOLOGY YONG LOO LIN SCHOOL OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE 2010 ACKNOWLEDGEMENTS I am deeply thankful to my supervisor, Associate Professor Herbert Schwarz, for his constant help, guidance and invaluable advices which has enabled me to develop an understanding for the project. I truly appreciate the encouragement and support that he has given me throughout the course of my study. Special thanks also go to my colleagues who have helped me in one way or another; to Jeanette and Ariel, for their guidance and mentorship when I first joined the lab; to Sun Feng, Doddy, Liang Kai and Weng Tong for their help in the radioactive work; to Shao Zhe, Julia, Dongsheng, Nurulhuda and Zulkarnain for the invaluable and fruitful discussions. I would also like to express my gratitude to all past and present members of the lab for their support and friendship and for the laughter that we shared during my time in the lab. Lastly, I would like to thank my family for their constant and unwavering support. i    TABLE OF CONTENTS ACKNOWLEDGEMENTS i TABLE OF CONTENTS ii ABSTRACT vii LIST OF TABLES ix LIST OF FIGURES x LIST OF ABBREVIATIONS xii 1. INTRODUCTION 1.1 The CD137 biology 1.1.1 The CD137 protein 1.1.2 Expression of CD137 and its role in immunity 1.1.3 CD137 and T cell responses 1.1.4 Immunotherapeutic potential of CD137 1.1.4.1 CD137 and tumour immunotherapy 1.1.4.2 CD137 and autoimmune diseases 1.2 The CD137 ligand biology 11 1.2.1 The CD137 ligand 11 1.2.2 Reverse signalling in the TNF superfamily 12 1.2.3 Expression of CD137L 14 1.2.4 Biological activities of CD137L signalling 14 1.2.4.1 CD137L stimulation and its role in myelopoiesis 14 1.2.4.2 Effects of monocytes in 16 1.2.4.3 Effects of CD137L stimulation in B cells and DCs 18 CD137L stimulation ii    1.2.5 1.2.4.4 Effects of CD137L stimulation in T cells 20 CD137L signal transduction 21 1.3 Immune responses in CD137 / CD137L deficient mice 22 1.4 Myeloid cells, its differentiation and function 24 1.4.1 Macrophage classifications and funtions 25 1.4.2 Monocyte derived DCs 27 1.4.3 DC immunotherapy and its challenges 29 1.4.4 Interactions between T cells and myeloid cells 32 1.4.4.1 T cell co-stimulation 32 1.4.4.2 Suppressive action on T cells 34 35 1.5 Research objectives 2. MATERIALS & METHODS 2.1 Recombinant protein, antibodies and reagents 38 2.2 Protein immobilization on tissue culture plates 39 2.3 Cells and cell culture 40 2.3.1 Cell lines 40 2.3.2 Isolation of PBMCs, monocytes and T cells 40 2.3.3 In vitro generation macrophages 2.4 2.5 of classical DCs and 42 Monocyte – T cell co-culture systems 43 2.4.1 Syngeneic monocyte – T cell co-cultures 43 2.4.2 Allogeneic MLR 43 Quantification of cell proliferation 44 2.5.1 Thymidine incorporation 44 2.5.2 CFSE dilution 45 iii    2.6 Determining suboptimal OKT3 concentration 45 2.7 Flow cytometric analysis 46 2.7.1 CFSE staining 46 2.7.2 Surface marker expression 47 2.7.3 Intracellular cytokine staining 47 2.7.4 Identification of transcription factors 48 2.7.5 Death receptor and ligand expression 48 2.7.6 Apoptosis assay 49 2.8 Identification of cell type undergoing apoptosis 49 2.9 Functional assays 50 2.9.1 Phagocytosis 50 2.9.2 Endocytosis 50 2.9.3 Oxidative burst 51 2.10 Potential cytolytic activity of T cells 51 2.10.1 Perforin expression 51 2.10.2 Flow cytometry based killing assay 52 2.11 Determination of the importance of soluble factors 53 2.11.1 Transwell assays 53 2.11.2 Transfer of soluble factors 53 2.12 Neutralization assays 54 2.12.1 Neutralization of death inducing factors 54 2.12.2 Neutralization of H2O2 by catalase 54 2.12.3 Cytokine neutralization 55 2.13 ELISA 55 2.14 Morphological changes 55 iv    2.15 Statistics 56 3. RESULTS 3.1 CD137L mediated inhibition of PBMC subsets 57 3.1.1 CD137L activation inhibits PBMC proliferation 57 3.1.2 CD137L activation selectively induce T cell apoptosis 59 3.1.3 CD137 mediated T cell apoptosis is dependent on monocytes 61 3.1.4 CD137 induced, monocyte dependent T cell apoptosis requires cell to cell contact 66 3.1.5 CD137 induced T cell apoptosis is independent of CD95 and TRAIL 68 3.1.6 CD137 mediated T cell apoptosis is dependent on ROS 75 3.2 3.3 Characterization of CD137L activated monocytes 77 3.2.1 CD137L signalling activates monocytes 77 3.2.2 CD137L signalling inhibits macrophage activities 78 3.2.3 CD137L activation promotes monocyte to DC differentiation 86 3.2.4 CD137L DCs and classical DCs are functionally different 94 Characterization of CD137L DC – T cell interactions 96 3.3.1 CD137L DC – T cell interactions are biphasic in nature 96 3.3.2 CD137L DCs promote a pro-inflammatory T cell phenotype 101 3.3.3 CD137L DC induce production of IFN- and IL-13 but not IL-17 in naïve T cells 110 3.3.4 CD137L DCs increase the killing activity of CD8 T cells 111 v    118 3.4 Summary 4. DISCUSSION 4.1 CD137L activated monocytes and their inhibitory activities on T cells 121 4.1.1 CD137 induces global inhibition of proliferation but selectively mediates T cell apoptosis 121 4.1.2 CD137 induced T cell apoptosis requires monocyte help 124 4.1.3 CD137 mediates T cell apoptosis independently of the extrinsic pathway of apoptosis 126 4.1.4 ROS in T cells is important in mediating CD137 induced MDCD 129 4.1.5 Physiological MDCD induced 131 CD137L activation induces monocyte differentiation that is stimulatory to T cells 134 4.2.1 CD137L signalling inhibits macrophage differentiation to 134 4.2.2 CD137 promote monocyte to differentiate to inflammatory DCs 138 4.2.3 CD137L DCs are phenotypically and functionally different from classical DCs 141 4.2.4 CD137L DCs promote inflammatory T cells with a high killing activity 144 4.2.5 The potential of using CD137L DCs as a tool in human immunotherapy 147 4.2 significance of CD137 monocyte 4.3 Conclusion 149 4.4 Future work 151 REFERENCES 154 APPENDICES 171 vi    ABSTRACT CD137 is a member of the tumour necrosis factor receptor (TNFR) superfamily that is expressed on a variety of immune cells. Its activation provides co-stimulatory signals to T cells and preferentially induces proliferation and activation of CD8 T cells. Similar to CD137, its ligand (CD137L), is also a membrane bound protein. Interaction between CD137 and CD137L not only induces signalling into the receptor bearing cell, it also initiates signalling into the ligand bearing cell. Hence, CD137 and its ligand participate in bidirectional signalling and stimulation of CD137L which results in signalling into ligand bearing cells has been termed as CD137 reverse signalling. In human monocytes, CD137L signalling delivers a potent activating signal. However, characterization of monocyte activation by CD137L has been restricted to the production of pro-inflammatory cytokines, adherence and morphological changes. This study shows that treatment of monocytes with recombinant CD137 protein, which stimulates CD137L on monocytes, also induces their differentiation to dendritic cells (DCs). This is evidenced by the increased endocytic capacity, up-regulation of co-stimulatory molecules and the ability to induce proliferation of naïve T cells. CD137 not only induces monocyte to DC differentiation but also promotes DC maturation. These DCs in turn inhibit development of regulatory T cells but induce T cell expression of perforin, IFN-, IL-13 and IL-17 and T cells with a high killing activity. Hence, recombinant CD137 protein as a sole factor can induce differentiation of monocytes to mature, inflammatory DCs that have a potent T cell vii    stimulatory capacity. As such, CD137 show potential in generating DCs that can be used in human immunotherapy. In contrast to these T cell stimulatory activities, CD137L stimulated monocytes also induce T cell apoptosis. CD137 mediated, monocyte dependent T cell apoptosis requires direct cell to cell contact and occurs independently of the extrinsic pathway of apoptosis. Instead, ROS are implicated in mediating T cell apoptosis. 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J Immunol. 167, 2671-2676. 170    APPENDICES     Appendix APPENDIX TISSUE CULTURE MEDIA A1.1 RPMI 1640 For a L preparation, A1.2 RPMI powder (Sigma-Aldrich) 16.35 g L-glutamine (200mM) (Gibco, Invitrogen) 10 ml Sodium bicarbonate (Sigma-Aldrich) 2.0 g MilliQ water Top up to 1L R10 For a L preparation, A1.3 RPMI-1640 medium 900 ml FBS (Biowest) 100 ml R10 + P/S For a L preparation, A1.4 R10 990 ml Penicillin / Streptomycin (Gibco, Invitrogen) 10 ml IMDM IMDM powder (Sigma-Aldrich) 17.7 g L-glutamine (200mM) 10 ml Sodium bicarbonate 3.02 g MilliQ water Top up to 1L 171    Appendix A1.5 I10 IMDM medium 900 ml FBS 100 ml The pH of all culture media was adjusted to 7.4. Medium was sterile filtered through a 0.22µm filter membrane before use. 172    Appendix APPENDIX BUFFERS FOR ISOLATION OF PBMCS AND PBMC SUBSETS A2.1 PBS + 2mM EDTA For a L preparation, A2.2 10 X PBS (1st Base) 100 ml EDTA (0.5M) (1st Base) ml MilliQ water Top up to 1L RBC lysis buffer For a L preparation, A2.3 NH4Cl (Sigma-Aldrich) 8.29 g NaHCO3 (Sigma-Aldrich) 0.84 g EDTA (0.5M) 23 µl MilliQ water Top up to 1L MACS buffer For a L preparation, 10 X PBS 100 ml EDTA (0.5M) ml 0.5% BSA (Biowest) 5g The pH of buffers was adjusted to 7.4. Buffers were sterile filtered through a 0.22µm filter membrane before use. 173    Appendix APPENDIX MISCELLANEOUS BUFFERS A3.1 Trypsin-EDTA For a 50 ml preparation, A3.2 0.5% Trypsin-EDTA (Gibco, Invitrogen) ml 10 X PBS ml MilliQ water Top up to 50 ml 10 mM EDTA in PBS For a 50 ml preparation, A3.3 EDTA (0.5M) ml 10 X PBS ml MilliQ water Top up to 50 ml FACS buffer For a L preparation, FBS ml Sodium azide (Sigma-Aldrich) 0.2 g 10 X PBS 100 ml MilliQ water Top up to 1L The pH of buffers was adjusted to 7.4. Trypsin-EDTA and 10 mM EDTA in PBS were sterile filtered through a 0.22µm filter membrane before use. 174    [...]... activation on monocytes, a subtype of myeloid cells Hence, this introduction gives an overview of the protein of interest, CD137, and its interaction with its ligand The concept of reverse signalling will be addressed and current literature on the effects of CD137 reverse signalling is highlighted Further, a review on the current knowledge of monocytes, their differentiation, function and interactions... contact between monocytes and T cells 67 8 CD137 treatment increases expression of death receptors on T cells 70 9 CD137 treatment increases expression of CD95L and TRAIL on T cells 71 10 CD137 mediated T cell apoptosis is independent of the CD95 receptor / ligand and TRAIL / death receptor systems 74 11 Inhibition of ROS completely blocks CD137 induced T cell apoptosis 76 12 CD137L signalling activates... Introduction 1.2.4.2 Effects of CD137L stimulation in monocytes Studies have shown that the activation of CD137L on monocytes induces production of pro-inflammatory cytokines: IL-8, IL-6 and TNF (Langstein et al, 1998; Langstein et al, 2000; Sollner et al, 2007) and a down-regulation of IL-10, a cytokine associated with monocyte deactivation (Langstein et al, 1998) The cytokine profile of CD137L activated... proliferation and cytokine production (Pollok et al, 1993; Alderson et al, 1993; Hurtado et al, 1995) Further, CD137 has been shown to inhibit activation induced cell death (AICD) during restimulation of activated T cells by up-regulation of the antiapoptotic protein, Bcl-xL (Hurtado et al, 1997; Starck et al, 2005) Within the T cell population, most of the studies on CD137 co-stimulation have focused on the... regulatory function of NK cells and that CD137 is important in mediating cross talk between the innate and adaptive immune responses Apart from immune cells, CD137 expression has been reported on other cell types Drenkard et al have reported that CD137 is expressed on blood vessel walls at sites of inflammation where CD137 is thought to facilitate the extravasation of monocytes into the inflammatory tissues... without doubt, that CD137L stimulation activates monocytes However, more studies need to be done to more closely determine how monocyte functions and differentiation are affected by CD137 This may provide an insight as to how CD137L signalling in monocytes can influence the bigger immunological network 1.2.4.3 Effects of CD137L stimulation in B cells and DCs Unlike studies on CD137L activation in monocytes,... B cell activation (Pauly et al, 2002) CD137 stimulation has also been shown to induce proliferation of NK cells and their production of IFN- without increasing the cytolytic potential of these cells Rather, CD137 activated NK cells seem to be important in enhancing cytolytic potential of CD8 T cells (Melero et al, 1998; Wilcox et al, 2002) These studies show that CD137 stimulation affects the immune... all, these data suggest that CD137L stimulation activates peripheral monocytes Apart from being an activating factor, CD137L activation enhanced monocyte survival, proliferation and endomitosis Primary peripheral monocytes that were treated with CD137 showed an ability to survive even after twelve days of culture while untreated monocytes could only survive for up to 6 days post isolation (Langstein and. .. important in eradicating tumours once the tumour has been established Activated, tumour specific T cells would then express CD137 and systemically administered anti -CD137 antibodies can then activate the CD137 expressing T cells, enhancing the CTL 7    Introduction activity and therapeutic potential of the cells This was supported by studies which showed that administration of antibodies late in the tumour... mediate production of pro-inflammatory cytokines In this case, interaction of CD137L seems to be independent of its receptor but depends on TLR agonists (Kang et al, 2007) Independent interaction from the receptor further suggests the possibility of an alternative ligand However, the identity of this alternative ligand has yet to be determined 1.2.2 Reverse signalling in the TNF superfamily Most ligands . ligand Treg Regulatory T cell  INTRODUCTION Introduction 1  This thesis focuses on the activities of CD137 ligand (CD137L) activation on monocytes, a subtype of myeloid cells contrasting activities of CD137L stimulated monocytes that are temporally separated; early induction of T cell apoptosis that is followed by its T cell stimulatory function. The pattern of CD137 induced,. used in human immunotherapy. In contrast to these T cell stimulatory activities, CD137L stimulated monocytes also induce T cell apoptosis. CD137 mediated, monocyte dependent T cell apoptosis