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CD137L SIGNALLING INDUCES DIFFERENTIATION OF PRIMARY ACUTE MYELOID LEUKAEMIA CELLS CHENG CHEONG KIN (B. Biomed. Sc. (Hons.), Monash University) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHYSIOLOGY YONG LOO LIN SCHOOL OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE 2014 DECLARATION I hereby declare that this thesis is my original work and it has been written by me in its entirety. I have duly acknowledged all the sources of information which have been used in this thesis. This thesis has also not been submitted for any degree in any university previously. _________________________________________________ Cheng Cheong Kin 24 January 2014 i ACKNOWLEDGEMENTS I am deeply indebted to my supervisor, Associate Professor Herbert Schwarz, for his constant guidance, support, instruction and unwavering patience throughout the duration of my PhD. This work would have been impossible without his leadership and I am deeply grateful for the difference he has made. I am thankful also for my colleagues and the members of my lab, for their constant assistance, encouragement and for telling me I worry too much. I am grateful to my friends and family for bearing my burdens and without whom I could not have made it this far. And thanks be to God, with whom all things are possible. ii TABLE OF CONTENTS SUMMARY vii LIST OF TABLES viii LIST OF FIGURES ix LIST OF ABBREVIATIONS xi LIST OF PUBLICATIONS xiv INTRODUCTION 1.1 CD137 biology 1.1.1 The CD137 protein 1.1.2 CD137 expression and function 1.1.2 Dual roles of CD137 signalling in anti-tumour immunity and autoimmune disease 1.2 1.3 CD137L biology 1.2.1 The CD137L protein 1.2.2 CD137L expression 1.2.3 Bi-directional signalling of CD137/CD137L 1.2.4 CD137L signalling in monocytes 1.2.5 CD137L signalling in dendritic cells 1.2.6 CD137L signalling in B cells 1.2.7 CD137L signalling in T cells Influence of CD137L signalling on myelopoiesis 10 1.3.1 Myelopoiesis and myeloid cells 10 1.3.2 CD137L signalling and myelopoiesis 12 1.4 Trogocytosis 13 1.5 Activity of soluble and immobilized CD137 15 1.6 Acute myeloid leukaemia 15 1.6.1 Classification and subtypes of AML 16 1.6.2 Patient prognosis and AML classification 17 1.6.3 Runx1 and Cbfb murine models of AML 19 1.6.4 Emerging therapies in AML 20 iii 1.6.5 Immunotherapy of AML 21 1.6.6 Differentiation therapy of AML 23 1.7 CD137-CD137L interactions in AML 24 1.8 Research objectives 25 MATERIALS AND METHODS 27 2.1 Recombinant proteins 28 2.2 Cells and cell culture 28 2.2.1 Patient samples 28 2.2.2 Recovery of cryopreserved AML cells 28 2.2.3 Cell lines 28 2.2.4 Murine AML cells 29 2.3 Immunophenotypic analysis by flow cytometry 29 2.4 Functional assays 30 2.4.1 Assessment of phagocytosis 30 2.4.2 Transwell migration assay 30 2.4.3 Assessment of AML proliferation 31 2.5 Detection of cytokine secretion by ELISA 31 2.6 Allogeneic mixed lymphocyte reaction 32 2.7 Cell morphology 32 2.8 Transfer of CD137 from donor to recipient cells 32 2.9 Allogeneic MLR with monocytes possessing transferred CD137 33 2.10 CD137/CD137L localization by confocal microscopy 33 2.11 Immobilization of CD137-Fc on red blood cell membrane 34 2.12 Statistics 34 RESULTS 3.1 35 Effects of CD137L signalling in cryopreserved acute myeloid leukaemia cells 3.1.1 36 CD137L signalling induces little to no change in the immunophenotype of AML cells iv 36 3.1.2 CD137L signalling induces cytokine secretion from a proportion of AML cells 3.2 37 Effects of CD137L signalling in freshly isolated AML cells 3.2.1 CD137L signalling induces immunophenotypic changes in AML cells consistent with differentiation 43 3.2.2 CD137L signalling induces cytokine secretion by AML cells 46 3.2.3 CD137L signalling induces morphological changes in AML cells that are consistent with DC differentiation 3.2.4 phagocytosis in monocytic AML cells 46 3.2.5 AML cells treated with CD137 exhibit enhanced migratory ability 50 3.2.6 AML cells treated with CD137 exhibit enhanced T cell co-stimulatory activity 50 3.2.7 AML cells treated with CD137 possess reduced proliferative capacity 50 3.2.8 Patterns of observed changes induced by CD137L signalling depend Transfer of CD137 to AML cells 3.3.1 CD137 is transferred from cell lines to monocytes and inhibits subsequent cytokine release from T cells 61 3.3.2 CD137 is transferred from activated T cells to monocytes 61 3.3.3 CD137 from activated T cells and immobilized recombinant CD137 3.3.4 64 The CD137-CD137L complex is internalized into AML cells that are sensitive to CD137-induced differentiation 3.5 53 61 is transferred to AML cells 3.4 46 CD137L signalling increases CD83 expression and reduces on cell type from which AML is derived 3.3 43 64 CD137L signalling induces differentiation of myeloid cells in a murine model of leukaemia 67 Immobilization of recombinant CD137 protein for in vivo applications 72 3.5.1 CD137-Fc immobilized onto red blood cells induces IL-8 secretion and immunophenotypic changes in monocytes v 72 DISCUSSION 4.1 4.2 78 Previously cryopreserved AML cells are resistant to CD137-induced differentiation 79 CD137L signalling induces differentiation of freshly obtained AML cells 80 4.2.1 CD137-treated AML cells undergo immunophenotypic changes consistent with differentiation 4.2.2 80 Monocytic AML cells acquire DC-like characteristics in response to treatment with recombinant CD137 82 4.2.3 AML heterogeneity and sensitivity to recombinant CD137 85 4.2.4 Utility of CD137-treated AML cells in immunotherapy 86 4.3 Transfer of CD137 to AML cells 89 4.4 CD137L signalling induces differentiation of myeloid cells in a murine model of leukaemia 91 4.5 Immobilization of recombinant CD137 protein for in vivo applications 92 4.6 Conclusion 94 4.7 Future work 94 4.7.1 Further characterization of T cell responses to CD137-treated AML cells 4.7.2 4.7.3 94 Immobilization of recombinant CD137 protein or anti-CD137L antibodies 95 Effects of CD137L signalling on AML cells in vivo 96 REFERENCES 97 APPENDICES 109 vi SUMMARY The ligand for CD137 is expressed on hematopoietic progenitor cells and antigen-presenting cells such as monocytes, dendritic cells and B cells. Reverse signalling of CD137 ligand into the cell delivers a potent activating signal that results in the differentiation of hematopoietic progenitor cells into macrophages, and monocytes into dendritic cells. CD137 ligand is also expressed on acute myeloid leukaemia cells, which characteristically possess a maturation block that leads to arrested differentiation and malignancy. We hypothesized that CD137 ligand reverse signalling via stimulation with CD137 may also induce differentiation of the transformed myeloid cells in acute myeloid leukaemia. Primary acute myeloid leukaemia blasts isolated from either the bone marrow or peripheral blood of patients at time of diagnosis were stimulated with a recombinant CD137 protein in vitro. Reverse signalling through CD137 ligand induced differentiation of these leukemic blasts based on morphology, immunophenotype, cellular functions such as phagocytosis and proliferation, and cytokine release. These differentiated cells functionally demonstrated a more potent T cell co-stimulatory capacity as evidenced by up-regulation of co-stimulatory molecules, induction of increased T cell proliferation and cytokine release. These results suggest that CD137, as a single factor, is able to induce differentiation of the immature blasts in acute myeloid leukaemia into more effective antigen-presenting cells with enhanced T cell co-stimulatory potential. The ability to overcome the block in myeloid maturation and drive differentiation of acute myeloid leukaemia cells has implications for the development of differentiation therapies and anti-leukaemia vaccines. vii LIST OF TABLES 1. Frozen AML samples tested 38 2. Immunophenotypic changes and cytokine secretion in AML samples, without maturation 54 3. Immunophenotypic changes and cytokine secretion in AML samples, with maturation 55 4. Immunophenotypic changes and cytokine secretion in AML samples, monocytic 56 5. Freshly obtained AML samples tested 57 viii LIST OF FIGURES 1. Gating strategy used to identify myeloid population 35 2. CD137 induces only minor in immunophenotype of frozen AML samples 36 3. CD137 induces cytokine secretion from a proportion of frozen AML samples 37 4. Majority of frozen AML samples die within days of culture 39 5. CD137 induces immunophenotypic changes in AML cells consistent with differentiation 41 6. CD137 induces secretion of cytokines by AML cells 44 7. CD137 induces adherence and morphological changes in AML cells 45 8. CD137 induces up-regulation of CD83 and decreases phagocytosis in monocytic AML cells 46 9. CD137-treated AML cells demonstrate increased invasiveness 48 10. CD137-treated AML cells enhance allogeneic T cell activation 49 11. CD137-treated AML cells demonstrate reduced proliferation 51 12. Side scatter characteristics and CD45 expression of bone marrow mononuclear cells from representative samples of different FAB subtypes 52 13. CD137 is transferred from CD137-expressing L428 cells to monocytes 59 14. CD137 is transferred from activated T cells to monocytes 60 15. CD137 is transferred to AML cells from activated T cells and from the surface of a culture dish 62 16. The CD137L-CD137 complex is internalized into AML cells 63 17. Haematopoietic stem cells from Cbfb knockout mice spontaneously differentiate in vitro 65 18. CD137 induces immunophenotypic changes consistent with myeloid differentiation in a mouse model of AML 67 19. CD137 induces apoptosis of bone marrow myeloid cells from a Cbfb knock-out mouse 68 ix REFERENCES 97 Alderson, M.R., Smith, C.A., Tough, T.W., Davis-Smith, T., Armitage, R.J., Falk, B., Roux, E., Baker, E., Sutherland, G.R. & Din, W.S. (1994) Molecular and biological characterization of human 41BB and its ligand. Eur J Immunol, 24, 2219-2227. Anderson, C.F., Gerber, J.S. & Mosser, D.M. (2002) Modulating macrophage function with IgG immune complexes. J Endotoxin Res, 8, 477-481. 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MACS buffer (1 L) 10X PBS 100 mL EDTA (0.5 M) mL 0.5% BSA (Biowest) 5g pH of all buffers adjusted to 7.4. Buffers were sterile filtered through a 0.22 µm filter membrane before use. 111 APPENDIX 3: MISCELLANEOUS BUFFERS 1. 10 mM EDTA in PBS (50 mL) EDTA (0.5 M) mL 10X PBS mL MilliQ water Top up to 50 mL 2. FACS buffer (1 L) Fetal bovine serum mL Sodium azide (Sigma-Aldrich) 0.2 g 10X PBS 100 mL MilliQ water Top up to L pH of all buffers adjusted to 7.4. Buffers were sterile filtered through a 0.22 µm filter membrane before use. 112 [...]... Methods for immobilization of CD137-Fc 70 21 Conjugation of CD137-Fc to surface membrane of red blood cells 72 22 RBC-CD137-Fc induces changes in monocytes consistent with differentiation 73 23 Schematic showing CD137-induced myelopoiesis of primary AML cells 79 x LIST OF ABBREVIATIONS AICD Activation-induced cell death AML Acute myeloid leukaemia APC Antigen presenting cell APL Acute promyelocytic leukaemia... levels of CD137L on T cells is so low that detection via flow cytometry or other commonly used methods is not possible Unlike the activating abilities of CD137L signalling in APCs, or the co-stimulating abilities of CD137 on T cells, CD137L signalling in T cell lines appears to be inhibitory Coculture of CD137-expressing transfected CHO cells with anti-CD3-activated human peripheral blood mononuclear cells. .. (Lippert 2008) 8 1.2.6 CD137L signalling in B cells Studies on CD137L signalling in B cells have produced somewhat conflicting results While CD137L is expressed on B cells, whether this expression is induced or constitutive is unclear (Jung, et al 2004, Zhou, et al 1995) In a transgenic mouse model, constitutive expression of CD137L on APCs resulted in gradual depletion of peripheral B cells over time (Zhu,... 2000), it is also possible that activated T cells, which express CD137, may become over-stimulated by the CD137L- expressing B cells, leading to B cell elimination CD137 -CD137L interactions also likely play important roles in regulating maturation and activation of B cells (Pauly, et al 2002) 1.2.7 CD137L signalling in T cells CD137L is not expressed on primary T cells, or constitutively expressed at very... constitutively expressed on mature B cells and activated B cells (Pollok, et al 1994) Like CD137, low levels of CD137L are inducible on T cells, with expression being strictly activation-dependent (Goodwin, et al 1993, Polte, et al 2007) 1.2.3 Bi-directional signalling of CD137 /CD137L CD137L exerts its effects on CD137-expressing cells, like activated T cells, via engagement of CD137, leading to signal transduction... 1999) 1.3 Influence of CD137L signalling on myelopoiesis 1.3.1 Myelopoiesis and myeloid cells All white blood cells are derived from the haematopoietic stem cells (HSCs) found in the bone marrow Being undifferentiated and pluripotent, HSCs have the ability to differentiate into any cell of the lymphoid or myeloid lineage of white blood cells, thereby replenishing or increasing the size of the relevant... Reed-Sternberg cells inhibits T-cell activation by eliminating CD137 ligand expression Cancer Research, 73, 652-661 xiv INTRODUCTION 1 This thesis focusses the effects of CD137L signalling on acute myeloid leukaemia and its potential therapeutic application The following introduction, therefore, presents an overview of CD137 and CD137L biology Acute myeloid leukaemia and its current state of therapy will... cytolytic potential of CD8+ T cells via massive induction of IFN-γ and TNF-α (Shuford, et al 1997, Takahashi, et al 1999), and induces differentiation to CD8+ memory T cells, as suggested by up-regulation of the memory CD8+ T cell marker, CD45RO, the CC chemokine receptor 6 and the contents of granzyme B (Kim, et al 2002) By comparison, studies on the effects of CD137 signalling in CD4+ T cells have been... organization xiii LIST OF PUBLICATIONS Cheng, K., Wong, S.C., Linn, Y.C., Ho, L.P., Chng, W.J & Schwarz, H (2014) CD137 ligand signalling induces differentiation of primary acute myeloid leukaemia cells British Journal of Haematology, 165, 134-144 Ho, W.T., Pang, W.L., Chong, S.M., Castella, A., Al-Salam, S., Tan, T.E., Moh, M.C., Koh, L.K., Gan, S.U., Cheng, C.K & Schwarz, H (2013) Expression of CD137 on Hodgkin... 1998) Compared to CD137 signalling, less is currently known about the effects of CD137L signalling into the cells on which it is expressed 1.2.4 CD137L signalling in monocytes 7 Cross-linking of CD137L on the monocyte cell surface by recombinant CD137 protein or anti -CD137L antibody results in increased adherence within just a few hours (Langstein and Schwarz 1999), secretion of pro-inflammatory cytokines . 1.2.4 CD137L signalling in monocytes 8 1.2.5 CD137L signalling in dendritic cells 8 1.2.6 CD137L signalling in B cells 9 1.2.7 CD137L signalling in T cells 9 1.3 Influence of CD137L signalling. AML cells in immunotherapy 86 4.3 Transfer of CD137 to AML cells 89 4.4 CD137L signalling induces differentiation of myeloid cells in a murine model of leukaemia 91 4.5 Immobilization of recombinant. proportion of AML cells 37 3.2 Effects of CD137L signalling in freshly isolated AML cells 43 3.2.1 CD137L signalling induces immunophenotypic changes in AML cells consistent with differentiation