臺灣博碩士論文加值系統

The emerging of cancer immune therapies with immune checkpoint inhibitors (ICIs) have resulted in significant improvement of cancer patients’ clinical outcome with traditionally poor prognosis. Programmed cell death protein-1 (PD-1) is an immune checkpoint where its ligation with the ligand, programmed cell death ligand-1 (PD-L1) dampen the immune response. Nivolumab is the anti-PD-1 antibody to inhibit the interaction between PD-1 and PD-L1 to prevent cancer cells escape immune surveillance. However, PD-1/PD-L1 axis plays a critical role in the regulation of peripheral immune tolerance to prevent autoimmune diseases. Therefore, inhibiting this pathway by anti-PD-1 ICIs can lead to immune-related-myocarditis in cancer patients because of off-target effects of immune response on the heart. There were increasing fatal myocarditis cases in recent year due to broaden Nivolumab indication. In our project, we tend to investigate cardiomyopathy resulted from PD-1/PD-L1 axis blockade by anti-PD-1 antibody using Rockefeller University embryonic stem cells derived cardiomyocytes (RUES2-CMs) and melanoma tumor-bearing murine model. We found that nivolumab alone did not induce inflammatory related protein, STAT1 and NFκB, did not increase PD-L1 expression and did not induce apoptosis marker, cleaved caspase-3 which contrary with doxorubicin, a cardiotoxic chemotherapy drugs. However, nivolumab was able to exacerbate immune response by increasing interferon-gamma cytokine and by inducing PD-L1, STAT1 and NFκB expression in Rockefeller University embryonic stem cell derived cardiomyocytes (RUES2-CMs) when RUES2-CMs co-cultured with CD4+ T lymphocytes. Cleaved caspase-3 was also induced when RUES2-CMs co-cultured with CD4+ T lymphocytes but the above results were not significant when co-cultured with CD8+ T lymphocytes. In in vivo model, Heart function of tumor-bearing mice was decreased after treated with anti-PD-1 antibody. Cardiac histology of anti-PD-1-treated tumor-bearing mice demonstrated dilated left ventricle, which was resulted by infiltration of CD4+ and CD8+ T lymphocytes into the myocardium. PD-L1, STAT1, NFκB, interferon-gamma and tumor necrosis factor-alpha protein expression was increased significantly in anti-PD-1-treated tumor bearing mice. Cleaved caspase-3 expression and mouse plasma cardiac troponin I were also raised significantly in anti-PD-1-treated tumor group. This study provides in vitro and in vivo platform to study the role of PD-1/PD-L1 axis and how anti-PD-1 inhibition increases T lymphocytes-mediated cardiomyopathy. Our data provide preclinical evidence of the immune-related cardiomyopathy when administered anti-PD-1 ICIs, and thus, the cardiac condition should be concerned and monitored in cancer patients treated with anti-PD-1 ICIs therapy.

Abstract I
Table of Contents III
List of Figures V
Abbreviations VI
Introduction 1
1.1 Cancer therapy-related cardiotoxicity 1
1.2 Cancer immunotherapy 3
1.3 Immune checkpoint inhibitors 4
1.4 Role of the immune checkpoint in homeostasis 6
1.5 Immune-related adverse events 7
1.6 Immune-related cardiotoxicity 9
1.7 Cardiotoxicity in PD-1/PD-L1 deficient mice 10
1.8 Research motivation 11
1.9 Hypothesis 12
Materials and methods 13
2.1 Cell culture 13
2.2 Cardiomyocytes differentiation 13
2.3 MTT assay 14
2.4 Isolation of peripheral blood mononuclear cells (PBMCs) 15
2.5 Isolation, activation, and expansion of CD4+ and CD8+ T lymphocytes 16
2.6 Co-culture RUES2-CMs with isolated CD4+ or CD8+ T lymphocytes 16
2.7 Flow Cytometry 17
2.8 Immunocytochemistry 18
2.9 Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end-labeling (TUNEL) assay 18
2.10 Protein extraction and Bicinchoninic acid (BCA) protein assay 19
2.11 Western blot 20
2.12 RNA isolation and reverse transcription 21
2.13 Polymerase chain reaction (PCR) and real-time quantitative PCR (RT-qPCR) 22
2.14 Enzyme-linked immunosorbent assay (ELISA) 23
2.15 Animals 24
2.15.1 B16-F10 mouse melanoma tumor xenograft model 24
2.16 Echocardiography 25
2.17 Mouse facial vein blood collection 25
2.18 Tissue processing and immunohistochemistry staining 26
2.19 Statistical Analysis 27
Results 28
3.1 Generation and characterization of hESC-derived cardiomyocytes 28
3.2 Nivolumab treatment did not induce apoptosis and inflammation in cardiomyocytes 28
3.3 Nivolumab treatment induces cardiomyocyte inflammation and apoptosis in co-culture model 29
3.4 Anti-PD-1 immunotherapy reduces tumor growth in tumor-bearing mice 31
3.5 Anti-PD-1 immunotherapy impairs heart function in tumor-bearing mice 32
3.6 Anti-PD-1 immunotherapy increases myocardium T lymphocytes infiltration in tumor-bearing mice 32
3.7 Anti-PD-1 immunotherapy induces cardiac inflammation and apoptosis in tumor-bearing mice 33
Discussion 34
References 41
Figures 50

1.Sheng CC, Amiri-Kordestani L, Palmby T, Force T, Hong CC, Wu JC, Croce K, Kim G and Moslehi J. 21st Century Cardio-Oncology: Identifying Cardiac Safety Signals in the Era of Personalized Medicine. JACC Basic Transl Sci. 2016;1:386-398.
2.Lenneman CG and Sawyer DB. Cardio-Oncology: An Update on Cardiotoxicity of Cancer-Related Treatment. Circ Res. 2016;118:1008-20.
3.Ewer MS and Ewer SM. Cardiotoxicity of anticancer treatments. Nat Rev Cardiol. 2015;12:547-58.
4.Hahn VS, Lenihan DJ and Ky B. Cancer therapy-induced cardiotoxicity: basic mechanisms and potential cardioprotective therapies. J Am Heart Assoc. 2014;3:e000665.
5.Bellinger AM, Arteaga CL, Force T, Humphreys BD, Demetri GD, Druker BJ and Moslehi JJ. Cardio-Oncology: How New Targeted Cancer Therapies and Precision Medicine Can Inform Cardiovascular Discovery. Circulation. 2015;132:2248-58.
6.Varricchi G, Galdiero MR, Marone G, Criscuolo G, Triassi M, Bonaduce D, Marone G and Tocchetti CG. Cardiotoxicity of immune checkpoint inhibitors. ESMO Open. 2017;2:e000247.
7.Larsen CM and Mulvagh SL. Cardio-oncology: what you need to know now for clinical practice and echocardiography. Echo Res Pract. 2017;4:R33-R41.
8.Campia U, Moslehi JJ, Amiri-Kordestani L, Barac A, Beckman JA, Chism DD, Cohen P, Groarke JD, Herrmann J, Reilly CM and Weintraub NL. Cardio-Oncology: Vascular and Metabolic Perspectives: A Scientific Statement From the American Heart Association. Circulation. 2019;139:e579-e602.
9.Zhang H and Chen J. Current status and future directions of cancer immunotherapy. J Cancer. 2018;9:1773-1781.
10.Darvin P, Toor SM, Sasidharan Nair V and Elkord E. Immune checkpoint inhibitors: recent progress and potential biomarkers. Exp Mol Med. 2018;50:165.
11.Rosenberg SA, Mule JJ, Spiess PJ, Reichert CM and Schwarz SL. Regression of established pulmonary metastases and subcutaneous tumor mediated by the systemic administration of high-dose recombinant interleukin 2. J Exp Med. 1985;161:1169-88.
12.Atkins MB, Lotze MT, Dutcher JP, Fisher RI, Weiss G, Margolin K, Abrams J, Sznol M, Parkinson D, Hawkins M, Paradise C, Kunkel L and Rosenberg SA. High-dose recombinant interleukin 2 therapy for patients with metastatic melanoma: analysis of 270 patients treated between 1985 and 1993. J Clin Oncol. 1999;17:2105-16.
13.White RL, Jr., Schwartzentruber DJ, Guleria A, MacFarlane MP, White DE, Tucker E and Rosenberg SA. Cardiopulmonary toxicity of treatment with high dose interleukin-2 in 199 consecutive patients with metastatic melanoma or renal cell carcinoma. Cancer. 1994;74:3212-22.
14.Lee RE, Lotze MT, Skibber JM, Tucker E, Bonow RO, Ognibene FP, Carrasquillo JA, Shelhamer JH, Parrillo JE and Rosenberg SA. Cardiorespiratory effects of immunotherapy with interleukin-2. J Clin Oncol. 1989;7:7-20.
15.Yu Y and Cui J. Present and future of cancer immunotherapy: A tumor microenvironmental perspective. Oncol Lett. 2018;16:4105-4113.
16.Miliotou AN and Papadopoulou LC. CAR T-cell Therapy: A New Era in Cancer Immunotherapy. Curr Pharm Biotechnol. 2018;19:5-18.
17.Buchbinder EI and Desai A. CTLA-4 and PD-1 Pathways: Similarities, Differences, and Implications of Their Inhibition. Am J Clin Oncol. 2016;39:98-106.
18.Leach DR, Krummel MF and Allison JP. Enhancement of antitumor immunity by CTLA-4 blockade. Science. 1996;271:1734-6.
19.Yang Y. Cancer immunotherapy: harnessing the immune system to battle cancer. J Clin Invest. 2015;125:3335-7.
20.Ishida Y, Agata Y, Shibahara K and Honjo T. Induced expression of PD-1, a novel member of the immunoglobulin gene superfamily, upon programmed cell death. EMBO J. 1992;11:3887-95.
21.Dong H, Strome SE, Salomao DR, Tamura H, Hirano F, Flies DB, Roche PC, Lu J, Zhu G, Tamada K, Lennon VA, Celis E and Chen L. Tumor-associated B7-H1 promotes T-cell apoptosis: a potential mechanism of immune evasion. Nat Med. 2002;8:793-800.
22.Iwai Y, Ishida M, Tanaka Y, Okazaki T, Honjo T and Minato N. Involvement of PD-L1 on tumor cells in the escape from host immune system and tumor immunotherapy by PD-L1 blockade. Proc Natl Acad Sci U S A. 2002;99:12293-7.
23.Lee HT, Lee SH and Heo YS. Molecular Interactions of Antibody Drugs Targeting PD-1, PD-L1, and CTLA-4 in Immuno-Oncology. Molecules. 2019;24.
24.Mall C, Sckisel GD, Proia DA, Mirsoian A, Grossenbacher SK, Pai CS, Chen M, Monjazeb AM, Kelly K, Blazar BR and Murphy WJ. Repeated PD-1/PD-L1 monoclonal antibody administration induces fatal xenogeneic hypersensitivity reactions in a murine model of breast cancer. Oncoimmunology. 2016;5:e1075114.
25.Collins AV, Brodie DW, Gilbert RJ, Iaboni A, Manso-Sancho R, Walse B, Stuart DI, van der Merwe PA and Davis SJ. The interaction properties of costimulatory molecules revisited. Immunity. 2002;17:201-10.
26.Tivol EA, Borriello F, Schweitzer AN, Lynch WP, Bluestone JA and Sharpe AH. Loss of CTLA-4 leads to massive lymphoproliferation and fatal multiorgan tissue destruction, revealing a critical negative regulatory role of CTLA-4. Immunity. 1995;3:541-7.
27.Waterhouse P, Penninger JM, Timms E, Wakeham A, Shahinian A, Lee KP, Thompson CB, Griesser H and Mak TW. Lymphoproliferative disorders with early lethality in mice deficient in Ctla-4. Science. 1995;270:985-8.
28.Wei SC, Duffy CR and Allison JP. Fundamental Mechanisms of Immune Checkpoint Blockade Therapy. Cancer Discov. 2018;8:1069-1086.
29.Nishimura H, Nose M, Hiai H, Minato N and Honjo T. Development of lupus-like autoimmune diseases by disruption of the PD-1 gene encoding an ITIM motif-carrying immunoreceptor. Immunity. 1999;11:141-51.
30.Nishimura H, Okazaki T, Tanaka Y, Nakatani K, Hara M, Matsumori A, Sasayama S, Mizoguchi A, Hiai H, Minato N and Honjo T. Autoimmune dilated cardiomyopathy in PD-1 receptor-deficient mice. Science. 2001;291:319-22.
31.Wang J, Yoshida T, Nakaki F, Hiai H, Okazaki T and Honjo T. Establishment of NOD-Pdcd1-/- mice as an efficient animal model of type I diabetes. Proc Natl Acad Sci U S A. 2005;102:11823-8.
32.Guleria I, Khosroshahi A, Ansari MJ, Habicht A, Azuma M, Yagita H, Noelle RJ, Coyle A, Mellor AL, Khoury SJ and Sayegh MH. A critical role for the programmed death ligand 1 in fetomaternal tolerance. J Exp Med. 2005;202:231-7.
33.Myers G. Immune-related adverse events of immune checkpoint inhibitors: a brief review. Curr Oncol. 2018;25:342-347.
34.Davar D and Kirkwood JM. PD-1 Immune Checkpoint Inhibitors and Immune-Related Adverse Events: Understanding the Upside of the Downside of Checkpoint Blockade. JAMA Oncol. 2019.
35.Kumar V, Chaudhary N, Garg M, Floudas CS, Soni P and Chandra AB. Current Diagnosis and Management of Immune Related Adverse Events (irAEs) Induced by Immune Checkpoint Inhibitor Therapy. Front Pharmacol. 2017;8:49.
36.Winer A, Bodor JN and Borghaei H. Identifying and managing the adverse effects of immune checkpoint blockade. J Thorac Dis. 2018;10:S480-S489.
37.Moslehi JJ, Salem JE, Sosman JA, Lebrun-Vignes B and Johnson DB. Increased reporting of fatal immune checkpoint inhibitor-associated myocarditis. Lancet. 2018;391:933.
38.Horvat TZ, Adel NG, Dang TO, Momtaz P, Postow MA, Callahan MK, Carvajal RD, Dickson MA, D'Angelo SP, Woo KM, Panageas KS, Wolchok JD and Chapman PB. Immune-Related Adverse Events, Need for Systemic Immunosuppression, and Effects on Survival and Time to Treatment Failure in Patients With Melanoma Treated With Ipilimumab at Memorial Sloan Kettering Cancer Center. J Clin Oncol. 2015;33:3193-8.
39.Heinzerling L, Ott PA, Hodi FS, Husain AN, Tajmir-Riahi A, Tawbi H, Pauschinger M, Gajewski TF, Lipson EJ and Luke JJ. Cardiotoxicity associated with CTLA4 and PD1 blocking immunotherapy. J Immunother Cancer. 2016;4:50.
40.Varricchi G, Galdiero MR and Tocchetti CG. Cardiac Toxicity of Immune Checkpoint Inhibitors: Cardio-Oncology Meets Immunology. Circulation. 2017;136:1989-1992.
41.Raschi E, Diemberger I, Poluzzi E and De Ponti F. Reporting of immune checkpoint inhibitor-associated myocarditis. Lancet. 2018;392:383.
42.Johnson DB, Balko JM, Compton ML, Chalkias S, Gorham J, Xu Y, Hicks M, Puzanov I, Alexander MR, Bloomer TL, Becker JR, Slosky DA, Phillips EJ, Pilkinton MA, Craig-Owens L, Kola N, Plautz G, Reshef DS, Deutsch JS, Deering RP, Olenchock BA, Lichtman AH, Roden DM, Seidman CE, Koralnik IJ, Seidman JG, Hoffman RD, Taube JM, Diaz LA, Jr., Anders RA, Sosman JA and Moslehi JJ. Fulminant Myocarditis with Combination Immune Checkpoint Blockade. N Engl J Med. 2016;375:1749-1755.
43.Matson DR, Accola MA, Rehrauer WM and Corliss RF. Fatal Myocarditis Following Treatment with the PD-1 Inhibitor Nivolumab. J Forensic Sci. 2018;63:954-957.
44.Neilan TG, Rothenberg ML, Amiri-Kordestani L, Sullivan RJ, Steingart RM, Gregory W, Hariharan S, Hammad TA, Lindenfeld J, Murphy MJ, Moslehi JJ and Checkpoint Inhibitor Safety Working G. Myocarditis Associated with Immune Checkpoint Inhibitors: An Expert Consensus on Data Gaps and a Call to Action. Oncologist. 2018;23:874-878.
45.Freeman GJ, Long AJ, Iwai Y, Bourque K, Chernova T, Nishimura H, Fitz LJ, Malenkovich N, Okazaki T, Byrne MC, Horton HF, Fouser L, Carter L, Ling V, Bowman MR, Carreno BM, Collins M, Wood CR and Honjo T. Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation. J Exp Med. 2000;192:1027-34.
46.Okazaki T, Tanaka Y, Nishio R, Mitsuiye T, Mizoguchi A, Wang J, Ishida M, Hiai H, Matsumori A, Minato N and Honjo T. Autoantibodies against cardiac troponin I are responsible for dilated cardiomyopathy in PD-1-deficient mice. Nat Med. 2003;9:1477-83.
47.Wang J, Okazaki IM, Yoshida T, Chikuma S, Kato Y, Nakaki F, Hiai H, Honjo T and Okazaki T. PD-1 deficiency results in the development of fatal myocarditis in MRL mice. Int Immunol. 2010;22:443-52.
48.Lucas JA, Menke J, Rabacal WA, Schoen FJ, Sharpe AH and Kelley VR. Programmed death ligand 1 regulates a critical checkpoint for autoimmune myocarditis and pneumonitis in MRL mice. J Immunol. 2008;181:2513-21.
49.Tarrio ML, Grabie N, Bu DX, Sharpe AH and Lichtman AH. PD-1 protects against inflammation and myocyte damage in T cell-mediated myocarditis. J Immunol. 2012;188:4876-84.
50.Grabie N, Gotsman I, DaCosta R, Pang H, Stavrakis G, Butte MJ, Keir ME, Freeman GJ, Sharpe AH and Lichtman AH. Endothelial programmed death-1 ligand 1 (PD-L1) regulates CD8+ T-cell mediated injury in the heart. Circulation. 2007;116:2062-71.
51.Golde WT, Gollobin P and Rodriguez LL. A rapid, simple, and humane method for submandibular bleeding of mice using a lancet. Lab Anim (NY). 2005;34:39-43.
52.Unverferth BJ, Magorien RD, Balcerzak SP, Leier CV and Unverferth DV. Early changes in human myocardial nuclei after doxorubicin. Cancer. 1983;52:215-21.
53.Eppihimer MJ, Gunn J, Freeman GJ, Greenfield EA, Chernova T, Erickson J and Leonard JP. Expression and regulation of the PD-L1 immunoinhibitory molecule on microvascular endothelial cells. Microcirculation. 2002;9:133-45.
54.Yang W, Chen PW, Li H, Alizadeh H and Niederkorn JY. PD-L1: PD-1 interaction contributes to the functional suppression of T-cell responses to human uveal melanoma cells in vitro. Invest Ophthalmol Vis Sci. 2008;49:2518-25.
55.Hu JR, Florido R, Lipson EJ, Naidoo J, Ardehali R, Tocchetti CG, Lyon AR, Padera RF, Johnson DB and Moslehi J. Cardiovascular toxicities associated with immune checkpoint inhibitors. Cardiovasc Res. 2019;115:854-868.
56.Sun C, Mezzadra R and Schumacher TN. Regulation and Function of the PD-L1 Checkpoint. Immunity. 2018;48:434-452.
57.Lundy DJ, Lee DS and Hsieh PCH. Solving the puzzle of pluripotent stem cell-derived cardiomyocyte maturation: piece by piece. Ann Transl Med. 2017;5:143.
58.Baban B, Liu JY, Qin X, Weintraub NL and Mozaffari MS. Upregulation of Programmed Death-1 and Its Ligand in Cardiac Injury Models: Interaction with GADD153. PLoS One. 2015;10:e0124059.
59.Honda T, Egen JG, Lammermann T, Kastenmuller W, Torabi-Parizi P and Germain RN. Tuning of antigen sensitivity by T cell receptor-dependent negative feedback controls T cell effector function in inflamed tissues. Immunity. 2014;40:235-247.
60.Sury K, Perazella MA and Shirali AC. Cardiorenal complications of immune checkpoint inhibitors. Nat Rev Nephrol. 2018;14:571-588.
61.Dobrzanski MJ. Expanding roles for CD4 T cells and their subpopulations in tumor immunity and therapy. Frontiers in oncology. 2013;3:63.
62.Krown KA, Page MT, Nguyen C, Zechner D, Gutierrez V, Comstock KL, Glembotski CC, Quintana PJ and Sabbadini RA. Tumor necrosis factor alpha-induced apoptosis in cardiac myocytes. Involvement of the sphingolipid signaling cascade in cardiac cell death. J Clin Invest. 1996;98:2854-65.
63.Chen Y, Pat B, Zheng J, Cain L, Powell P, Shi K, Sabri A, Husain A and Dell'italia LJ. Tumor necrosis factor-alpha produced in cardiomyocytes mediates a predominant myocardial inflammatory response to stretch in early volume overload. J Mol Cell Cardiol. 2010;49:70-8.
64.Maisch B. Cardio-Immunology of Myocarditis: Focus on Immune Mechanisms and Treatment Options. Front Cardiovasc Med. 2019;6:48.
65.Gui J, Yue Y, Chen R, Xu W and Xiong S. A20 (TNFAIP3) alleviates CVB3-induced myocarditis via inhibiting NF-kappaB signaling. PLoS One. 2012;7:e46515.
66.Knowlton KU. Myocarditis: An Intersection Between Genetic and Acquired Causes of Human Cardiomyopathy. J Am Coll Cardiol. 2017;69:1666-1668.
67.Henke A, Huber S, Stelzner A and Whitton JL. The role of CD8+ T lymphocytes in coxsackievirus B3-induced myocarditis. J Virol. 1995;69:6720-8.
68.Grabie N, Delfs MW, Westrich JR, Love VA, Stavrakis G, Ahmad F, Seidman CE, Seidman JG and Lichtman AH. IL-12 is required for differentiation of pathogenic CD8+ T cell effectors that cause myocarditis. J Clin Invest. 2003;111:671-80.
69.Du S, Zhou L, Alexander GS, Park K, Yang L, Wang N, Zaorsky NG, Ma X, Wang Y, Dicker AP and Lu B. PD-1 Modulates Radiation-Induced Cardiac Toxicity through Cytotoxic T Lymphocytes. J Thorac Oncol. 2018;13:510-520.
70.Eriksson U and Penninger JM. Autoimmune heart failure: new understandings of pathogenesis. Int J Biochem Cell Biol. 2005;37:27-32.
71.Huber SA, Sartini D and Exley M. Vgamma4(+) T cells promote autoimmune CD8(+) cytolytic T-lymphocyte activation in coxsackievirus B3-induced myocarditis in mice: role for CD4(+) Th1 cells. J Virol. 2002;76:10785-90.
72.Wiseman AC, Pietra BA, Kelly BP, Rayat GR, Rizeq M and Gill RG. Donor IFN-gamma receptors are critical for acute CD4(+) T cell-mediated cardiac allograft rejection. J Immunol. 2001;167:5457-63.