Characterization of murine soluble CD137 and its biological activities

CHARACTERIZATION OF MURINE SOLUBLE CD137 AND ITS BIOLOGICAL ACTIVITIES SHAO ZHE (Bsc.) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN SCIENCE DEPARTMENT OF PHYSIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2009 ACKNOWLEDGEMENTS I would first like to express my heartfelt gratitude to my supervisor, Associate Professor Herbert Schwarz, for his firm guidance and invaluable advices throughout the course of this project. I truly appreciate the encouragement and support that he gave me. Next, I would like to thank Poh Cheng and Teng Ee for teaching me the essential tissue culture techniques, Doddy and Sun Feng for helping me with the molecular and radioactive work, and Zulkarnain for supporting me with the tumor project. I would also like to thank our collaborator A/P Koh Daw Rhoon for providing mouse models for the testing of soluble CD137. Lastly, I offer my regards and blessings to all the members of A/P Herbert Schwarz’s lab who have supported me in any respect during the completion of the project. i TABLE OF CONTENTS ABSTRACT . v LIST OF TABLES . vii LIST OF FIGURES viii LIST OF ABBREVIATIONS x CHAPTER INTRODUCTION . 1.1 Biology of the CD137 receptor/ligand system . 1.1.1 Expression of CD137 . 1.1.2 Expression of CD137 ligand 1.1.3 Costimulatory activities of CD137 1.1.4 CD137 as a coinhibitory molecule . 1.1.5 Reverse signaling through CD137 ligand 1.2 Involvement of CD137 receptor/ligand in cancer 11 1.2.1 Expression of CD137 receptor/ligand in cancer 11 1.2.2 Possible roles of CD137 as a neoantigen on cancer cells 12 1.3 Soluble CD137 . 13 1.3.1 Expression of soluble CD137 13 1.3.2 Soluble CD137 as an antagonist to membrane-bound CD137 . 14 1.3.3 Soluble CD137 in diseases . 16 1.4 Research objectives 18 CHAPTER MATERIALS AND METHODS 20 2.1 Animals 20 2.2 Cells and cell culture 20 2.3 Antibodies and reagents . 21 2.4 Isolation of murine splenocytes . 22 2.5 Induction of murine soluble CD137 . 22 2.6 Measurement of soluble CD137 by ELISA . 22 2.7 Measurement of membrane-bound CD137 by flow cytometry . 23 2.8 Measurement of stability of murine soluble CD137 23 2.9 Reverse transcription polymerase chain reaction (RT-PCR) . 23 2.9.1 Isolation of total RNA from cells . 23 2.9.2 Reverse transcription . 24 2.9.3 Polymerase chain reaction (PCR) 25 ii 2.10 Isolation of CD4+ and CD8+ cells from mouse spleen . 26 2.11 Size exclusion chromatography (SEC) 26 2.12 Western blot . 27 2.13 Measurement of binding of soluble CD137 to CD137L recombinant protein . 27 2.14 Measurement of binding of soluble CD137 to CD137L-expressing cells . 28 2.15 Depletion of soluble CD137 28 2.16 Isolation of regulatory T cells 28 2.17 Isolation of dendritic cells from mouse spleen 29 2.18 Differentiation of dendritic cells from bone marrow . 29 2.19 Generation of stable, CD137-expressing cell lines 30 2.19.1 Plasmids . 30 2.19.2 Transfection . 31 2.19.3 Measurement of membrane-bound CD137 expression on transfected cells 32 2.19.4 Selection of stably-transfected clones 32 2.20 Measurement of cell viability by manual cell counting . 33 2.21 Measurement of cell proliferation via 3H-thymidine incorporation . 33 2.22 Lymphokine activated killer (LAK) cells assay for A20 cells . 34 2.23 Coating of proteins or antibodies in tissue culture plate 35 2.24 Treatment of A20 cell with agonistic anti-CD137 antibodies . 35 2.25 Measurement of cytokine secretion by ELISA 35 2.26 Nuclear Factor κB (NF-κB) Assay . 36 2.27 Induction of subcutaneous tumor in syngeneic mouse models 36 2.28 Statistics . 37 CHAPTER RESULTS 38 3.1 Generation of murine soluble CD137 38 3.1.1 Soluble CD137 is secreted by activated splenocytes . 39 3.1.2 Soluble CD137 is released by T cells 45 3.1.3 Expression of soluble CD137 by regulatory T cells 48 3.1.4 Expression of soluble CD137 by DC . 52 3.1.5 Summary 58 3.2 Potential agonistic function of murine soluble CD137 59 3.2.1 Examination of the size of soluble CD137 59 3.2.2 Soluble CD137 can bind to CD137L . 66 3.3 Regulatory function of murine soluble CD137 69 3.3.1 Correlation of soluble CD137 with AICD . 69 iii 3.3.2 Soluble CD137 regulates cytokine secretion . 72 3.4 Correlation of soluble CD137 levels with diseases 76 3.5 Effects of soluble CD137 in tumorigenesis . 78 3.5.1 Screening of tumor cell lines for CD137 expression . 79 3.5.2 Generation of stable, CD137-expressing B16 cell lines 82 3.5.3 Generation of stable, CD137-expressing A20 cell lines 87 3.5.4 Morphological changes of A20 cells upon agonistic antibody stimulation . 91 3.5.5 CD137 signaling into A20 cells activates the NF-κB pathway 93 3.5.6 Cytokine secretion of A20 cells upon stimulation of CD137 signaling . 96 3.5.7 CD137 expression and protection against lymphokine activated killer (LAK) cells-mediated cytotoxicity 101 3.5.8 in vivo tumor assays . 104 3.5.9 Summary 109 CHAPTER DISCUSSION 110 4.1 Summary of results 110 4.2 Expression and generation of soluble CD137 112 4.3 Soluble CD137 antagonizes membrane-bound CD137 . 114 4.3.1 Mechanisms of action of soluble CD137 . 114 4.3.2 Soluble CD137 as a general immunomodulator 116 4.4 Applications of soluble CD137 118 4.5 CD137 as a neoantigen 120 4.5.1 Signaling into CD137-expressing tumor cells . 121 4.5.2 Effects of tumor-expressing CD137 on host immune cells . 124 4.6 Future work 127 4.7 Conclusion . 128 REFERENCES 130 APPENDIX I MEDIA AND BUFFERS . 138 APPENDIX II MOCOPLASMA TEST 146 iv ABSTRACT CD137 is a member of the tumor necrosis factor receptor family, and is involved in the regulation of a range of immune activities. Soluble forms of CD137 may antagonize membrane-bound CD137 and regulate host immune responses. Here we report in this study that soluble CD137 can be generated by differential splicing and is mainly released by activated T cells. While CD8+ T cells express significantly more membrane-bound CD137 than CD4+ T cells, both T cell subsets express similar levels of sCD137, resulting a two-fold increased ratio of soluble to membrane-bound CD137 for CD4+ T cells. Other immune cells that express soluble CD137 include Treg and dendritic cells. Expression levels of soluble CD137 correlate with those of membranebound CD137 in most cases except for DC. Soluble CD137 exists as a trimer and a higher order multimer and can bind to CD137 ligand, suggesting it has antagonistic effect on membrane-bound CD137. Levels of soluble CD137 correlate with activation induced cell death and depletion of soluble CD137 results in increase of IL-10 and IL12. Soluble CD137 is present in sera of mice with autoimmune disease but is undetectable in sera of healthy mice. Besides its expression in immune cells, CD137 was also found to be expressed in certain cancer cells. The correlation of CD137 expression and malignancy points to a selection advantages that CD137 expression provides to the tumor. The potential role of CD137 as a cancer neoantigen was characterized in this study. Using cell lines which overexpress CD137, it was found that CD137 signaling in B cell lymphoma A20 induces activation of the NF-κB pathway, which is accompanied by changes of cell morphology and IL-10 production. However, the effect of CD137 was not observed in vivo, as no significant difference could be found between the growth rates v of tumors formed by CD137-expressing and control A20 cells. Further studies will be needed to characterize the role of CD137 in tumorigenesis and possible antagonistic effect of soluble CD137 in this process. vi LIST OF TABLES Table 1. Reverse Transcription Reaction Mix . 24 Table 2. Standard PCR Reaction Mix 25 Table 3. RT-PCR thermal cycling program for examination of CD137 mRNA expression . 25 Table 4. Primers for RT-PCR for examination of CD137 expression . 26 Table 5. Primers for constructing expression vectors for CD137 protein . 31 Table 6. Stimuli used for splenocytes activation . 40 Table 7. Comparison of membrane-bound CD137 and soluble CD137 between CD137-expressing A20 and B16 variants 100 vii LIST OF FIGURES Figure 1. CD137 (4-1BB) signaling pathways in T cells . Figure 2. Bidirectional signal transduction and reverse signaling in the CD137 receptor/ligand system . 11 Figure 3. Schematic depiction of possible mechanisms of soluble CD137 action 16 Figure 4. Induction of soluble CD137 expression . 42 Figure 5. Time course of CD137 expression . 43 Figure 6. In vitro stability of soluble CD137 . 43 Figure 7. Splenocytes express two forms of CD137 mRNA . 44 Figure 8. CD137 is expressed by activated T cells 47 Figure 9. Expression of CD137 mRNA isoforms by T cells . 47 Figure 10. Expression of membrane-bound CD137 by different subsets of CD4+ T cells 50 Figure 11. Expression of soluble CD137 by different subsets of CD4+ T cells 51 Figure 12. Flow cytometry analysis of splenic DC 54 Figure 13. Expression of soluble CD137 by splenic DC . 55 Figure 14. Flow cytometry analysis of BMDC 57 Figure 15. Expression of soluble CD137 by BMDC . 58 Figure 16. Determination of the size of membrane-bound CD137 by Western blot . 61 Figure 17. Calculation of the size of soluble CD137 by SEC . 64 Figure 18. Detection of soluble CD137 and CD137L complexes . 65 Figure 19. Determination of the binding of soluble CD137 to recombinant CD137L 67 Figure 20. Determination of the binding of soluble CD137 to cell surface CD137L 68 Figure 21. Dose dependence of soluble CD137 expression 71 Figure 22. Schematic depictation of the model to deplete soluble CD137 from splenocytes . 73 Figure 23. Density dependence of soluble CD137 expression 75 viii Figure 24. Regulation of cytokine secretion by soluble CD137 75 Figure 25. Levels of soluble CD137 are enhanced in sera of mice with autoimmune disease 77 Figure 26. Screening of CD137 expression of tumor cell lines by RT-PCR . 81 Figure 27. Wild type B16.F0 cells express CD137L but not CD137 83 Figure 28. Expression of CD137 on B16 variants . 84 Figure 29. CD137 expression on B16 variants does not affect cell proliferation 86 Figure 30. Wild type A20 cells express CD137L but not CD137 . 88 Figure 31. Expression of CD137 on A20 variants . 89 Figure 32. CD137 expression on A20 variants does not affect cell proliferation 90 Figure 33. Morphological changes of A20/muCD137 cells 92 Figure 34. NF-κB p65 activation by CD137 signaling in tumor cells . 94 Figure 35. Expression of CD137L by A20 variants 95 Figure 36. Secretion of IL-10 and soluble CD137 by A20/muCD137 cells 98 Figure 37. B16 variants secrete large amounts of soluble CD137 . 99 Figure 38. CD137-expressing A20 cells are less susceptible to LAK cells-induced cytotoxicity 103 Figure 39. In vivo tumor models of A20 cells . 105 Figure 40. In vivo tumor models of B16 cells 106 Figure 41. Expression of soluble CD137 in in vivo tumor models 108 ix Kwon,B.S. and Weissman,S.M. (1989). cDNA sequences of two inducible T-cell genes. Proc. Natl. Acad. Sci. U. S. A 86, 1963-1967. Laderach,D., Movassagh,M., Johnson,A., Mittler,R.S., and Galy,A. (2002). 4-1BB costimulation enhances human CD8(+) T cell priming by augmenting the proliferation and survival of effector CD8(+) T cells. Int. Immunol. 14, 1155-1167. Laderach,D., Wesa,A., and Galy,A. (2003). 4-1BB-ligand is regulated on human dendritic cells and induces the production of IL-12. Cell Immunol. 226, 37-44. Langstein,J., Becke,F.M., Sollner,L., Krause,G., Brockhoff,G., Kreutz,M., Andreesen,R., and Schwarz,H. (2000). Comparative analysis of CD137 and LPS effects on monocyte activation, survival, and proliferation. Biochem. Biophys. Res. Commun. 273, 117-122. Langstein,J., Michel,J., Fritsche,J., Kreutz,M., Andreesen,R., and Schwarz,H. (1998). CD137 (ILA/4-1BB), a member of the TNF receptor family, induces monocyte activation via bidirectional signaling. J. Immunol. 160, 2488-2494. Langstein,J., Michel,J., and Schwarz,H. (1999). CD137 induces proliferation and endomitosis in monocytes. Blood 94, 3161-3168. Langstein,J. and Schwarz,H. (1999). Identification of CD137 as a potent monocyte survival factor. J. Leukoc. Biol. 65, 829-833. Lee,H.W., Park,S.J., Choi,B.K., Kim,H.H., Nam,K.O., and Kwon,B.S. (2002). 4-1BB promotes the survival of CD8+ T lymphocytes by increasing expression of Bcl-xL and Bfl-1. J. Immunol. 169, 4882-4888. Lee,P.K., Chang,C.J., and Lin,C.M. (2003). Lipopolysaccharide preferentially induces 4-1BB ligand expression on human monocyte-derived dendritic cells. Immunol. Lett. 90, 215-221. Lindstedt,M., Johansson-Lindbom,B., and Borrebaeck,C.A. (2003). Expression of CD137 (41BB) on human follicular dendritic cells. Scand. J. Immunol. 57, 305-310. Lippert,U., Zachmann,K., Ferrari,D.M., Schwarz,H., Brunner,E., Mahbub-Ul Latif,A.H., Neumann,C., and Soruri,A. (2008). CD137 ligand reverse signaling has multiple functions in human dendritic cells during an adaptive immune response. Eur. J. Immunol. 38, 1024-1032. Lisignoli,G., Toneguzzi,S., Cattini,L., Pozzi,C., and Facchini,A. (1998). Different expression pattern of cytokine receptors by human osteosarcoma cell lines. Int. J. Oncol. 12, 899-903. Lotz,M., Setareh,M., von Kempis,J., and Schwarz,H. (1996). The nerve growth factor/tumor necrosis factor receptor family. J. Leukoc. Biol. 60, 1-7. Mantovani,A., Allavena,P., and Sica,A. (2004). Tumour-associated macrophages as a prototypic type II polarised phagocyte population: role in tumour progression. Eur. J. Cancer 40, 1660-1667. Masood,R., Zhang,Y., Bond,M.W., Scadden,D.T., Moudgil,T., Law,R.E., Kaplan,M.H., Jung,B., Espina,B.M., and Lunardi-Iskandar,Y. (1995). Interleukin-10 is an autocrine growth factor for acquired immunodeficiency syndrome-related B-cell lymphoma. Blood 85, 34233430. Maus,M.V., Thomas,A.K., Leonard,D.G., Allman,D., Addya,K., Schlienger,K., Riley,J.L., and June,C.H. (2002). Ex vivo expansion of polyclonal and antigen-specific cytotoxic T 133 lymphocytes by artificial APCs expressing ligands for the T-cell receptor, CD28 and 4-1BB. Nat. Biotechnol. 20, 143-148. Melero,I., Johnston,J.V., Shufford,W.W., Mittler,R.S., and Chen,L. (1998). NK1.1 cells express 4-1BB (CDw137) costimulatory molecule and are required for tumor immunity elicited by anti-4-1BB monoclonal antibodies. Cell Immunol. 190, 167-172. Melero,I., Shuford,W.W., Newby,S.A., Aruffo,A., Ledbetter,J.A., Hellstrom,K.E., Mittler,R.S., and Chen,L. (1997). Monoclonal antibodies against the 4-1BB T-cell activation molecule eradicate established tumors. Nat. Med. 3, 682-685. Michel,J., Langstein,J., Hofstadter,F., and Schwarz,H. (1998). A soluble form of CD137 (ILA/4-1BB), a member of the TNF receptor family, is released by activated lymphocytes and is detectable in sera of patients with rheumatoid arthritis. Eur. J. Immunol. 28, 290-295. Michel,J., Pauly,S., Langstein,J., Krammer,P.H., and Schwarz,H. (1999). CD137-induced apoptosis is independent of CD95. Immunology 98, 42-46. Michel,J. and Schwarz,H. (2000). Expression of soluble CD137 correlates with activationinduced cell death of lymphocytes. Cytokine 12, 742-746. Mittler,R.S., Bailey,T.S., Klussman,K., Trailsmith,M.D., and Hoffmann,M.K. (1999). Anti-41BB monoclonal antibodies abrogate T cell-dependent humoral immune responses in vivo through the induction of helper T cell anergy. J. Exp. Med. 190, 1535-1540. Morris,G.P., Chen,L., and Kong,Y.C. (2003). CD137 signaling interferes with activation and function of CD4+CD25+ regulatory T cells in induced tolerance to experimental autoimmune thyroiditis. Cell Immunol. 226, 20-29. Myers,L.M. and Vella,A.T. (2005). Interfacing T-cell effector and regulatory function through CD137 (4-1BB) co-stimulation. Trends Immunol. 26, 440-446. Olofsson,P.S., Soderstrom,L.A., Wagsater,D., Sheikine,Y., Ocaya,P., Lang,F., Rabu,C., Chen,L., Rudling,M., Aukrust,P., Hedin,U., Paulsson-Berne,G., Sirsjo,A., and Hansson,G.K. (2008). CD137 is expressed in human atherosclerosis and promotes development of plaque inflammation in hypercholesterolemic mice. Circulation 117, 1292-1301. Orabona,C., Grohmann,U., Belladonna,M.L., Fallarino,F., Vacca,C., Bianchi,R., Bozza,S., Volpi,C., Salomon,B.L., Fioretti,M.C., Romani,L., and Puccetti,P. (2004). CD28 induces immunostimulatory signals in dendritic cells via CD80 and CD86. Nat. Immunol. 5, 11341142. Palma,C., Binaschi,M., Bigioni,M., Maggi,C.A., and Goso,C. (2004). CD137 and CD137 ligand constitutively coexpressed on human T and B leukemia cells signal proliferation and survival. Int. J. Cancer 108, 390-398. Pauly,S., Broll,K., Wittmann,M., Giegerich,G., and Schwarz,H. (2002). CD137 is expressed by follicular dendritic cells and costimulates B lymphocyte activation in germinal centers. J. Leukoc. Biol. 72, 35-42. Pollok,K.E., Kim,Y.J., Hurtado,J., Zhou,Z., Kim,K.K., and Kwon,B.S. (1994). 4-1BB T-cell antigen binds to mature B cells and macrophages, and costimulates anti-mu-primed splenic B cells. Eur. J. Immunol. 24, 367-374. 134 Pollok,K.E., Kim,Y.J., Zhou,Z., Hurtado,J., Kim,K.K., Pickard,R.T., and Kwon,B.S. (1993). Inducible T cell antigen 4-1BB. Analysis of expression and function. J. Immunol. 150, 771781. Polte,T., Jagemann,A., Foell,J., Mittler,R.S., and Hansen,G. (2007). CD137 ligand prevents the development of T-helper type cell-mediated allergic asthma by interferon-gammaproducing CD8+ T cells. Clin. Exp. Allergy 37, 1374-1385. Reali,C., Curto,M., Sogos,V., Scintu,F., Pauly,S., Schwarz,H., and Gremo,F. (2003). Expression of CD137 and its ligand in human neurons, astrocytes, and microglia: modulation by FGF-2. J. Neurosci. Res. 74, 67-73. Salih,H.R., Kosowski,S.G., Haluska,V.F., Starling,G.C., Loo,D.T., Lee,F., Aruffo,A.A., Trail,P.A., and Kiener,P.A. (2000). Constitutive expression of functional 4-1BB (CD137) ligand on carcinoma cells. J. Immunol. 165, 2903-2910. Schmidt-Wolf,G.D., Negrin,R.S., and Schmidt-Wolf,I.G. (1997). Activated T cells and cytokine-induced CD3+CD56+ killer cells. Ann. Hematol. 74, 51-56. Schwarz,H. (2005). Biological activities of reverse signal transduction through CD137 ligand. J. Leukoc. Biol. 77, 281-286. Schwarz,H., Arden,K., and Lotz,M. (1997). CD137, a member of the tumor necrosis factor receptor family, is located on chromosome 1p36, in a cluster of related genes, and colocalizes with several malignancies. Biochem. Biophys. Res. Commun. 235, 699-703. Schwarz,H., Blanco,F.J., von,K.J., Valbracht,J., and Lotz,M. (1996). ILA, a member of the human nerve growth factor/tumor necrosis factor receptor family, regulates T-lymphocyte proliferation and survival. Blood 87, 2839-2845. Schwarz,H., Tuckwell,J., and Lotz,M. (1993). A receptor induced by lymphocyte activation (ILA): a new member of the human nerve-growth-factor/tumor-necrosis-factor receptor family. Gene 134, 295-298. Schwarz,H., Valbracht,J., Tuckwell,J., von,K.J., and Lotz,M. (1995). ILA, the human 4-1BB homologue, is inducible in lymphoid and other cell lineages. Blood 85, 1043-1052. Seo,S.K., Choi,J.H., Kim,Y.H., Kang,W.J., Park,H.Y., Suh,J.H., Choi,B.K., Vinay,D.S., and Kwon,B.S. (2004). 4-1BB-mediated immunotherapy of rheumatoid arthritis. Nat. Med. 10, 1088-1094. Setareh,M., Schwarz,H., and Lotz,M. (1995). A mRNA variant encoding a soluble form of 41BB, a member of the murine NGF/TNF receptor family. Gene 164, 311-315. Sharief,M.K. (2002). Heightened intrathecal release of soluble CD137 in patients with multiple sclerosis. Eur. J. Neurol. 9, 49-54. Shuford,W.W., Klussman,K., Tritchler,D.D., Loo,D.T., Chalupny,J., Siadak,A.W., Brown,T.J., Emswiler,J., Raecho,H., Larsen,C.P., Pearson,T.C., Ledbetter,J.A., Aruffo,A., and Mittler,R.S. (1997). 4-1BB costimulatory signals preferentially induce CD8+ T cell proliferation and lead to the amplification in vivo of cytotoxic T cell responses. J. Exp. Med. 186, 47-55. Siebenlist,U., Franzoso,G., and Brown,K. (1994). Structure, regulation and function of NFkappa B. Annu. Rev. Cell Biol. 10, 405-455. 135 So,T., Lee,S.W., and Croft,M. (2008). Immune regulation and control of regulatory T cells by OX40 and 4-1BB. Cytokine Growth Factor Rev. 19, 253-262. Sollner,L., Shaqireen,D.O.K., Wu,J.T., and Schwarz,H. (2007). Signal transduction mechanisms of CD137 ligand in human monocytes. Cell Signal. 19, 1899-1908. Son,J.H., Lee,U.H., Lee,J.J., Kwon,B., Kwon,B.S., and Park,J.W. (2004). Humanization of agonistic anti-human 4-1BB monoclonal antibody using a phage-displayed combinatorial library. J. Immunol. Methods 286, 187-201. Starck,L., Scholz,C., Dorken,B., and Daniel,P.T. (2005). Costimulation by CD137/4-1BB inhibits T cell apoptosis and induces Bcl-xL and c-FLIP(short) via phosphatidylinositol 3kinase and AKT/protein kinase B. Eur. J. Immunol. 35, 1257-1266. Sznol,M., Hodi,F.S., Margolin,K., McDermott,D.F., Ernstoff,M.S., Kirkwood,J.M., Wojtaszek,C., Feltquate,D., and Logan,T. (2008). Phase I study of BMS-663513, a fully human anti-CD137 agonist monoclonal antibody, in patients (pts) with advanced cancer (CA). J. Clin. Oncol. 26 (May 20 suppl. abstr 3007). Tamada,K. and Chen,L. (2006). Renewed interest in cancer immunotherapy with the tumor necrosis factor superfamily molecules. Cancer Immunol. Immunother. 55, 355-362. Thum,E., Shao,Z., and Schwarz,H. (2009). CD137, implications in immunity and potential for therapy. Front Biosci. 14, 4173-4188. van Kooten,C., Rensink,I., Aarden,L., and van Oers,R. (1993). TNF-alpha induces spreading of B-CLL via the CD11c/CD18 molecule. Am. J. Hematol. 44, 221-228. Vinay,D.S. and Kwon,B.S. (2006). Genes, transcripts and proteins of CD137 receptor and ligand. In CD137 Pathway: Immunology and Diseases. L.Chen, ed. (NY: Springer). von Kempis,J., Schwarz,H., and Lotz,M. (1997). Differentiation-dependent and stimulusspecific expression of ILA, the human 4-1BB-homologue, in cells of mesenchymal origin. Osteoarthritis. Cartilage. 5, 394-406. Wang,C., Lin,G.H., McPherson,A.J., and Watts,T.H. (2009). Immune regulation by 4-1BB and 4-1BBL: complexities and challenges. Immunol. Rev. 229, 192-215. Watts,T.H. (2005). TNF/TNFR family members in costimulation of T cell responses. Annu. Rev. Immunol. 23, 23-68. Wen,T., Bukczynski,J., and Watts,T.H. (2002). 4-1BB ligand-mediated costimulation of human T cells induces CD4 and CD8 T cell expansion, cytokine production, and the development of cytolytic effector function. J. Immunol. 168, 4897-4906. Whiteside,T.L. (2007). The role of death receptor ligands in shaping tumor microenvironment. Immunol. Invest 36, 25-46. Wilcox,R.A., Chapoval,A.I., Gorski,K.S., Otsuji,M., Shin,T., Flies,D.B., Tamada,K., Mittler,R.S., Tsuchiya,H., Pardoll,D.M., and Chen,L. (2002). Cutting edge: Expression of functional CD137 receptor by dendritic cells. J. Immunol. 168, 4262-4267. Wilkinson,D.G. (2000). Eph receptors and ephrins: regulators of guidance and assembly. Int. Rev. Cytol. 196, 177-244. 136 Zhang,B., Maris,C.H., Foell,J., Whitmire,J., Niu,L., Song,J., Kwon,B.S., Vella,A.T., Ahmed,R., Jacob,J., and Mittler,R.S. (2007a). Immune suppression or enhancement by CD137 T cell costimulation during acute viral infection is time dependent. J. Clin. Invest 117, 3029-3041. Zhang,G.B., Dong,Q.M., Hou,J.Q., Ge,Y., Ju,S.G., Lu,B.F., and Zhang,X.G. (2007b). Characterization and application of three novel monoclonal antibodies against human 4-1BB: distinct epitopes of human 4-1BB on lung tumor cells and immune cells. Tissue Antigens 70, 470-479. Zhang,J., Xu,X., and Liu,Y. (2004). Activation-induced cell death in T cells and autoimmunity. Cell Mol. Immunol. 1, 186-192. Zheng,G., Wang,B., and Chen,A. (2004). The 4-1BB costimulation augments the proliferation of CD4+CD25+ regulatory T cells. J. Immunol. 173, 2428-2434. Zhou,Z., Kim,S., Hurtado,J., Lee,Z.H., Kim,K.K., Pollok,K.E., and Kwon,B.S. (1995). Characterization of human homologue of 4-1BB and its ligand. Immunol. Lett. 45, 67-73. 137 APPENDIX I MEDIA AND BUFFERS RPMI-1640 To prepare L of medium: Item Quantity Source RPMI powder 16.35 g Sigma-Aldrich (St Louis, MO) L-glutamine (200 mM) 10 ml Gibco, Invitrogen (Carlsbad, CA) Sodium bicarbonate 2.0 g Sigma-Aldrich (St Louis, MO) MilliQ water Top up to L - The medium was then sterile-filtered through a 0.22 µm filter membrane (Millipore, Billerica, MA) RPMI-S2-10 To prepare L of medium: Item Quantity Source RPMI-1640 medium 900 ml - Fetal Bovine Serum (FBS) 100 ml Biowest (Nuaville, France) Sodium pyruvate (100 mM) 10 ml Gibco, Invitrogen (Carlsbad, CA) 2-mercaptoethanol 3.5 µl Sigma-Aldrich (St Louis, MO) Item Quantity Source RPMI-S2-10 990 ml - Penicillin(10,000 U/ml)/Streptomycin(10 mg/ml) 10 ml Gibco, Invitrogen (Carlsbad, CA) RPMI-S2-10-P/S To prepare L of medium: 138 Dulbecco's Modified Eagle Medium (DMEM) To prepare L of medium: Item Quantity Source DMED powder 17.6 g Sigma-Aldrich (St Louis, MO) L-glutamine (200 mM) 10 ml Gibco, Invitrogen (Carlsbad, CA) Sodium bicarbonate 3.7 g Sigma-Aldrich (St Louis, MO) MilliQ water Top up to L - The medium was then sterile-filtered through a 0.22 µm filter membrane (Millipore, Billerica, MA) DMEM-10 To prepare L of medium: Item Quantity Source DMED 900 ml - Foetal Bovine Serum (FBS) 100 ml Biowest (Nuaville, France) Item Quantity Source Murine GM-CSF (100 µg/ml) 200 µl PeproTech Inc. (Rocky Hill, NJ) Murine IL-4 (100 µg/ml) 125 µl PeproTech Inc. (Rocky Hill, NJ) RPMI-S2-10-P/S Top up to 500 ml - DC Medium To prepare 500 ml of medium: The solution was then sterile-filtered through a 0.22 µm filter membrane (Millipore, Billerica, MA) 139 Selection Medium for Transfected A20 Cells To prepare 100 ml of medium: Item Quantity Source RPMI-S2-10 100 ml - ZeocinTM (100 mg/ml) 100 µl Invitrogen (Carlsbad, CA) Selection Medium for Transfected B16 Cells To prepare 100 ml of medium: Item Quantity Source DMEM-10 99 ml - Geneticin® (100 mg/ml) ml Gibco, Invitrogen (Carlsbad, CA) Item Quantity Source NaCl 8g Sigma-Aldrich (St Louis, MO) KCl 0.2 g Sigma-Aldrich (St Louis, MO) Na2HPO4 1.44 g Sigma-Aldrich (St Louis, MO) KH2PO4 0.24 g Sigma-Aldrich (St Louis, MO) MilliQ water Top up to 1L - PBS To prepare L of solution: The solution was sterilized by autoclaving after its pH was adjusted to 7.4. 140 Trypsin-EDTA To prepare 50 ml of solution: Item Quantity Source 0.5% Trypsin-EDTA (10 ×) ml Gibco, Invitrogen (Carlsbad, CA) PBS 45 ml - The solution was then sterile-filtered through a 0.22 µm filter membrane (Millipore, Billerica, MA) 10mM EDTA in PBS To prepare 50 ml of solution: Item Quantity Source 0.5 M EDTA solution ml NUMI store, NUS PBS 49 ml - The solution was then sterile-filtered through a 0.22 µm filter membrane (Millipore, Billerica, MA) RBC Lysis Buffer To prepare 500 ml of solution: Item Quantity Source NH4Cl 4.145 g Sigma-Aldrich (St Louis, MO) NaHCO3 0.42 g Sigma-Aldrich (St Louis, MO) 0.5 M EDTA solution 11.5 ml NUMI store, NUS MilliQ water Top up to 500 ml - 141 Digestion buffer for DC To prepare 100 ml of solution: Item Quantity Source Liberase CI 50 mg Roche (Mannheim, Germany) FBS ml Biowest (Nuaville, France) RPMI-1640 medium Top up to 100 ml - The buffer was then aliquoted and frozen at -20 ºC OptiPrep® diluent To prepare 100 ml of solution: Item Quantity Source NaCl 0.85 g Sigma-Aldrich (St Louis, MO) 0.5 M EDTA solution 200 µl NUMI store, NUS HEPES 0.48 g Sigma-Aldrich (St Louis, MO) NaOH Adjust pH to 7.4 Sigma-Aldrich (St Louis, MO) Quantity Source OptiPrep density gradient medium 1.852 ml Sigma-Aldrich (St Louis, MO) OptiPrep® diluent 8.148 ml - 10.88% OptiPrep® solution To prepare 10 ml of solution: Item ® 142 MACS Buffer To prepare 500 ml of solution: Item Quantity Source FBS 10 ml Biowest (Nuaville, France) 0.5 M EDTA ml NUMI store, NUS PBS Top up to 500 ml - The solution was then sterile-filtered through a 0.22 µm filter membrane (Millipore, Billerica, MA) FACS Buffer To prepare 500 ml of solution: Item Quantity Source FBS 2.5 ml Biowest (Nuaville, France) NaH3 0.1 g Sigma-Aldrich (St Louis, MO) PBS Top up to 500 ml - Item Quantity Source FBS 25 ml Biowest (Nuaville, France) PBS Top up to 500 ml - PBSF To prepare 500 ml of solution: The solution was then sterile-filtered through a 0.22 µm filter membrane (Millipore, Billerica, MA) 143 Lysis Buffer for LAK Assay To prepare 50 ml of solution: Item Quantity Source Triton X-100 50 µl Bio-Rad (Hercules, CA) MilliQ water Top up to 50 ml - The solution was then sterile-filtered through a 0.22 µm filter membrane (Millipore, Billerica, MA) 0.05 M Phosphate Citrate Buffer (for TMB Substrate) To prepare 100 ml of solution: Item Quantity Source 0.2 M Na2HPO4 solution 25.7 ml Sigma-Aldrich (St Louis, MO) 0.1 M Citric acid 24.3 ml Sigma-Aldrich (St Louis, MO) MilliQ water Top up to 100 ml - The pH of the solution was adjusted to 5.0. TMB Substrate Solution To prepare 10 ml of solution: Item Quantity Source TMB tablet tablet Sigma-Aldrich (St Louis, MO) 0.05 M Phosphate citrate 10 ml buffer - 30% H2O2 Kanto Chemicals (Japan) µl 144 PBST To prepare L of solution: Item Quantity Source Tween-20 500 µl Bio-Rad, Hercules, CA PBS Top up to L - 145 APPENDIX II MOCOPLASMA TEST To prevent Mycoplasma contamination, all cell lines were routinely checked by PCRbased tests using the MycoSensorTM PCR Assay kit (Stratagene, La Jolla, CA). The detailed protocol is listed as following. 1. Sample preperation 50 µl of supernatant from cell culture was transferred to a microcentrifuge tube and boiled at 95 ºC for min. The supernatant was then mixed with 10 µl of StrataClean resin, followed by a short centrifugation. The treated supernatant was harvested as template of the PCR tests. 2. PCR mixture preperation The reaction mixture was prepared by combining the corresponding components in order listed in the table below. Components Stock concentration Sterile water Final concentration Volume/Reaction - - 13.75 µl 20 mM mM 2.5 µl 10 × 1× 2.5 µl dNTP/dUTP mix - - 0.5 µl Mycoplasma primer mix - - µl Internal control template - - µl U/µl 0.05 U/µl 0.25 µl Positive control template/test sample - - 2.5 µl Total reaction volumn - - 25 µl MgCl2 Taq buffer Taq DNA polymerase 3. PCR thermal cycling program 146 Step Process Temperature (ºC) Time Initial melting 94 10 Melting 94 30 sec Primer annealing 55 Extension 72 Repeat step 2-4 for 34 cycles - - The PCR products were loaded into 2% agarose gel for electrophoresis and the bands were visualized with GelRedTM (Biotrium, Hayward, CA). 4. Typical results The Mycoplasma primer mix was designed to amplify a 315-bp PCR product from the most common species of Mycoplasma. A 500-bp internal control product was also included to ensure the reaction was successful. Typical results from the PCR tests were summarized in the table below. PCR template Cell culture extract + internal control template Positive control template + internal control template Negative control template + internal control template PCR product(s) Result None Inhibited PCR 315 bp and 500 bp Mycoplasma infection 315 bp only Heavy Mycoplasma infection 500 by only No Mycoplasma infection None Failed PCR 315 bp only Failed PCR 315 bp and 500 bp Expected control result None Failed PCR 500 bp only Expected control result 315 bp and 500 bp Contaminated reagents 147 5. Treatment of Mycoplasma contamination Cell lines tested positive for Mycoplasma contamination were treated with 0.5 µg/ml Mycoplasma removal reagent (AbD Serotec, MorphoSys, Germany) for three passages and tested again for the clearance of Mycoplasma contamination. 148 [...]... into CD137expressing cells 15 Figure 3 Schematic depiction of possible mechanisms of soluble CD137 action In the absence of soluble CD137, signals can go through membrane-bound CD137 (mCD137) upon crosslinking of CD137L (A) To antagonize mCD137-induced signaling, soluble CD137 (sCD137) may (B) compete with mCD137 for binding to CD137L or (C) insert into mCD137 trimers 1.3.3 Soluble CD137 in diseases Soluble. .. Levels of soluble CD137 will be compared with membrane-bound CD137 in each cell type ii Antagonistic mechanism(s) of soluble CD137 The multimeric status of soluble CD137 will be examined by testing its size by Western blot and size exclusion chromatography (SEC) Naturally occurring soluble CD137 will be used to test the binding ability of soluble CD137 to CD137L iii Regulatory functions of soluble CD137. .. cancer cells 1.3 1.3.1 Soluble CD137 Expression of soluble CD137 Soluble isoforms are a common occurence of the members of the TNFR family These soluble isoforms are generated either by alternative splicing of the mRNA or by cleavage of membrane-bound receptors by matrix metalloproteinase Soluble forms of CD95, CD40 and CD137 are generated by differential splicing while the rest of the family are by... possible role of soluble CD137 in antagonizing membrane-bound CD137 and in regulating immune responses, it is important to examine the expression and regulation of soluble CD137 In this chapter, the biology of the CD137 receptor/ligand system will be introduced first This will be followed by a description of its involvement in cancer Finally the biology and possible functions of soluble CD137 will be... concept of reverse signaling relies on very conventional molecules (Sollner et al., 2007) 10 Figure 2 Bidirectional signal transduction and reverse signaling in the CD137 receptor/ligand system Crosslinking of CD137 and CD137L leads to activation of APC and costimulation of T cells simultaneously 1.2 1.2.1 Involvement of CD137 receptor/ligand in cancer Expression of CD137 receptor/ligand in cancer CD137. .. soluble CD137 Levels of soluble CD137 will be compared with the activation status of the cells by testing proliferation and cell death The potential regulatory function of soluble CD137 will be examined by testing the cytokine profile in a cell culture system after depletion of soluble CD137 iv Correlation of soluble CD137 and disease activity in mouse models Sera from murine disease models and healthy controls... differential splicing of CD137 mRNA (Michel et al., 1998) Studies using recombinant CD137 proteins indicate that soluble CD137 can antagonize the costimulatory activities of the membrane-bound CD137 and reduce T cell proliferation Interestingly, enhanced levels of soluble CD137 can be detected in sera of autoimmune, leukemia and lymphoma patients Compared to its membranebound counterpart, soluble CD137 has been... The objective of this study was to characterize the expression and functions of murine soluble CD137 More specifically, the following points will be examined in this thesis: i Expression and regulation of membrane-bound and soluble forms of CD137 The presence of membrane-bound and soluble CD137 in different immune cell populations will be tested at the protein level by flow cytometry and ELISA, respectively... antagonistic effects of soluble CD137 will then be determined by applying soluble CD137 in these assays This thesis presents the first systematic study on murine soluble CD137 Results of the present study should contribute to the understanding of the expression and regulation of soluble CD137 in the murine system The studies on the antagonistic mechanisms may also shed some light on how CD137 regulates... for further studies to explain the presence of soluble CD137 in the respective diseases and its possible implications Because of the bidirectional signaling of CD137 receptor/ligand interaction, the inhibitory effect of soluble CD137 on its membrane-bound counterpart could block the signal into both T lymphocytes and APC simultaneously In that case, soluble CD137 may be helpful to treat autoimmune diseases . agonistic function of murine soluble CD137 59 3.2.1 Examination of the size of soluble CD137 59 3.2.2 Soluble CD137 can bind to CD137L 66 3.3 Regulatory function of murine soluble CD137 69 3.3.1. Summary of results 110 4.2 Expression and generation of soluble CD137 112 4.3 Soluble CD137 antagonizes membrane-bound CD137 114 4.3.1 Mechanisms of action of soluble CD137 114 4.3.2 Soluble CD137. Determination of the binding of soluble CD137 to recombinant CD137L 67 Figure 20. Determination of the binding of soluble CD137 to cell surface CD137L 68 Figure 21. Dose dependence of soluble CD137