1. 100 60 10 0 year Cancer/ Neuronal disorder Infection/Development 1 Tumor immunology BC Yang Expected life-span Immune capability 2010

2. 衛生署資料，台灣人十大死因 2 排名 年度 8889909192939495 96 1 惡性腫瘤 135.32142.23147.68152.88156.01160.54163.8166.5 175.9 2 腦血管疾病 57.3960.158.8253.4654.9854.4857.855.2 56.2 3 心臟疾病 51.3347.5649.2550.9352.2356.7957.153.8 56.7 4 糖尿病 40.9942.640.7939.2644.3840.5846.242.5 44.6 5 事故傷害 58.8847.442.5837.7936.337.3336.835.1 31.1 6 慢性病跟肝硬化 23.5323.3223.4521.3522.9823.6324.722.1 22.5 7 肺炎 18.214.8816.7720.1722.624.4425.023.6 25.7 8 腎病徵候群及腎病性病 15.7817.4518.1518.5519.0820.6721.220.6 22.2 9 自殺 10.3611.1412.4513.5914.1615.3118.819.3 17.2 10 高血壓性疾病 8.43--7.98.678.177.978.38.0 8.6 98 年 癌症、心臟疾病（不含高血壓）、腦血管疾病、肺炎、糖尿病、事故傷害、慢性下呼吸道疾病、 慢性肝病及肝硬化，自殺、腎臟疾病。

3. 1960 Nobel prize in physiology or Medicine for discovery of acquired immunological tolerance 3

4. THE JOURNAL OF EXPERIMENTAL BIOLOGY 207 (23): 4013-4014 4 There are very few scientific papers that continue to have an impact on research more than 50 years after their initial publication. A paper published in J. Exp. Biol. in 1951 by Rupert Billingham and Peter Medawar is one such paper. THE BIRTH OF TRANSPLANTATION IMMUNOLOGY: THE BILLINGHAM–MEDAWAR EXPERIMENTS AT BIRMINGHAM UNIVERSITY AND UNIVERSITY COLLEGE LONDON (The technique of free skin grafting in mammals)

5. Evidence for active host defense against cancer 5 80 years of immunotherapy. (Currie GA, 1972, Br. J. Cancer 26: 141) A critique of the evidence for active host defense against cancer, based on personal studies of 27 murine tumors of spontaneous origin. (Hewitt HB, et al. 1976, Br. J. Cancer 33:241)

6. Circumvent Evidence The high frequency of cancers in immunosuppressed patients  Extremes of age  Immunosuppressive drugs Tumors that are infiltrated by T cells have an improved prognosis Spontaneous regression occurs  Melanoma, breast, lung cancers, etc Circulating tumor antibodies MHC-I down-regulation 6

7. The ways to prove effective tumor immunity ： 7

8. Immune surveillance, animal model 8

9. Full immunity needed to fight cancer 9 CD4 + T cells contribute to the remodeling of the microenvironment required for sustained tumor regression upon oncogene inactivation (Cancer Cell, 2010, 18:485-498)

10. 10 Full immunity needed to fight cancer

11. 11 A fight between immune cells and cancer But, sometimes we lose

12. THE GREAT ESCAPE: IMMUNE EVASION VERSUS TUMOR PROGRESSION 12

13. 13 有些事只有在變動的狀況才看得見事物的本質

14. 14 Difficulties due to study model

15. Tumor formation of Ras-over-expression cells in BALB/c 15 It is a dynamic process death survival

16. 16 Immune selection in the development of cancer: no two tumors are alike Initiation, proliferation diversification Microevolution, selection of immune resistance Fey MF & Tobler A 1996 Immune escape and unchecked proliferation

17. Use of tumor cell lines 17 Commonly derived from advanced tumors Retain the genetic instability and lose the ability of adaptations introduced in large numbers (more than 10 6 cells)

18. 18 Successful immunization for cancer

19. Immune therapy (T cell-based) 19 Recombinant and synthetic vaccination Cytokine treatments (IL-2; GM-CSF; IFN) Cellular therapy with tumor-specific CTL Engineered macrophages (Th1…?) Antigen-pulsed macrophages or dendritic cells

20. Tumor associated antigens 20 Tumor-specific shared antigens: restricted in expression to tumors and immune privilege sites. Tissue-specific differentiation antigens: tyrosinase. (melanoma) Tumor-specific antigens: mutated, tranlocated genes. Ubiquitous antigens with over-expression in tumors. Rosenberg SA. 1999, Immunity 10:281

21. 21 Peptide epitopes for melanoma Nicholaas P, et al. 1999, Curr Opin Oncol 11:50

22. 22 Tumor and activated T cells Two major pathways for TCL: Fas-mediated and perforrin-mediated

23. HLA class I molecules 23 Antigen presentation for T cells, cytotoxic Inhibitory signals for NK cells

24. Immune escape/erosion 24 Making invisible Resistance to death Repelling Active suppression ….

25. An overview 25

26. Examples Down-regulation of the class I presentation pathway Resistance to killing Antigen-specific mechanisms (Treg?) Global mechanisms 26

27. Antigen-specific mechanisms 27 Tumor antigen-loss variants Loss of the melanoma tumor-associated antigen in patients with recurrent metastatic melanoma (J Clin Invest 1996, 98:1633) B cell tumors expressing class II induced a rapid tolerance of cognate CD4 T cell carrying a transgenic TCR. (PNAS, 1998, 95:1178) Tolerance

28. Down regulation of the MHC class presentation pathway 28 Downregulation of MHC class I expression is frequently seen in human tumors. Loss of MHC-I as a mechanism for tumor escape from CTL-mediated elimination (longitudinal study of melanoma patients) Five major HLA altered phenotypes found in tumor tissues (Human Immunol. 2000, 61:65)

29. The five altered phenotypes 29 Normal A1A2B8B35Cw7Cw4 1. Total loss - 2. Haplotype loss A1B8Cw7 3. Locus loss A1A2B8B35 4. Allelic loss A2B8B35Cw7Cw4 5. Compound phenotype A1 (Human Immunol. 2000, 61:65)

30. Resistance to killing 30 Defective Fas pathway Resistance to Granzyme B  Fas-L and Neutrophil Cytotoxic T cells Innate immunity

31. 31 Caspase 3 Apaf-1 Caspase 9 Death substrates Apoptosis Nature 407, 789 5(2000) Fas signal

32. Key person in Fas signal study 32 Shigekazu Nagata For a man who spends most of his time thinking about death, Shigekazu Nagata is remarkably upbeat. For over a decade, he has been making a name for himself with research on apoptosis, the mechanism of programmed cell death, and during that time he has watched the field come alive. Nature Medicine 7, 759 (2001) Peter H. Krammer BC Yang 2008/2/20

33. Loss sensitivity to Fas-mediated apoptosis 33 FLICE-like inhibitory proteins Bcl-2, Bcl-XL defected sphingomyelinase activation (ceramide) decoy receptor 3 (DcR3), soluble Fas

34. Fas-L + -melanoma cells are relatively resistant to killing of neutrophils 34 B16F10 Chen YL, et al J. Immunol. 171:1183-1192.

35. Global Mechanisms 35 TGF-beta IL-10 Growth in immune privilege sites Mucin production: interfering intercellular adhesion Fas-L? (Fas counterattack) Tumor extracellular matrix

36. 36 TGF-  signaling in tumor signaling and cancer progression

37. 37 IL-10 (Th3 or Treg) Tumors or other cells in environments

38. Shaping T cell functions by extracellular matrix. 38 NPC Done by 張逸如 (1998) Hematoxylin/eosin stainingCD3 + cells Immune cells are there! But, in peripheral stroma area (cell cuffing)

39. 39 How can tumor cells highjack immune cells? Extracellular matrix shapes Fas-mediated apoptosis. Protective: U-118, Huh-7, HeLa, A549, NIH3T3 Non-protective: MCF-7, HepG2 Su CC, et al. 2007 Phosphatidylinositol 3-Kinase/Akt activation by integrin-tumor matrix interaction suppresses Fas-mediated apoptosis in T cells. J Immunol 179:4589-4597.

40. 40 Coculture with tumor cells inhibits the Fas-mediated apoptosis.

41. 41 Direct cell contact is required.

42. 42 Caspase 3 Apaf-1 Caspase 9 Death substrates Apoptosis Apoptosis program is inhibited.

43. 43 Fas-mediated death is not suppressed by components of MAPK, NFkB, PKA, HSP70.

44. 44 Fas Fas-L Immune cells Tumor Tissue environment ? Cytokines ? What will happen when Fas-stimulated immune cells resist to die?

45. 45 J Immunol, 2003, 171: 3947 – 3954. Jurkat and Molt-4 cells were cultured alone (lane 1) or in the presence of U118(V), U118(R), U373(V), or U373(R) (lanes 2, 3, 4, and 5, respectively) for 24 h. T cells in tumor microenvironment 林亨楷

46. Regulatory T cells: the third man 46 Shimon Sakaguchi (2000) Regulatory T Cells Key Controllers of Immunologic Self-Tolerance Cell 101: 455-458

47. A very dynamic interactions 47 Tumor microenviroment Individual properties

48. 48 A FasL mystery 1.Tissue dependant 2.Reverse signaling? 3.Other than death-triggering

49. 49 Joe O’connell, 2002, Fas ligand and the fate of antitumorur cytotoxic T lymphocytes. Immunology 105:263-266.

50. In vivo consequences of Fas-L expression by tumors (using over-expression system) 50 Renca, MH134, L5178Y, B16- BL6, CT26* Note: *:syngenic, nude, SCID # : allogenic, lpr Ref: Lejeune FJ et al., 1998, Curr. Op. Immunol.  Enhanced rejection  Delayed rejection CT26# B16-F10

51. Distinct structure of tumor mass Glioma in Nude mice May be Fas-L associated? Why TIL in particular sites? Penetration of tumor by immune cells 51 Fas-L R Control

52. 52 B16-F10 (melanoma) in B6 mice Control Fas-L Ribozyme

53. 53 Granulocytes mediates the Fas-L-associated apoptosis during lung metastasis of melanoma that determines the metastatic behavior (B16F10 in C57BL/6) Br J Cancer, 87: 359 (2002) i.v. 5 x 10 5 cells /mouse; Observed at day 14; (bar = 1 cm) Kang, S. M. et al. 1997. Fas ligand expression in islets of Langerhans does not confer immune privilege and instead targets them for rapid destruction. Nature Med. 3:738.

54. The structure of FasL 54

55. FasL reverse signaling 55 Suzuki I. and Fink PJ.. Maximal Proliferation of Cytotoxic T lymphocytes Requires Reverse Signaling Through Fas Ligand. J. Exp. Med., 187:123, 1998. Boursalian TE, Fink PJ, Mutation in Fas Ligand Impairs Maturation of Thymocytes Bearing Moderate Affinity T Cell Receptors, J. Exp. Med., 198, 349, 2003. Sun M, Ames KT, Suzuki I,. Fink PJ, The Cytoplasmic Domain of Fas Ligand Costimulates TCR Signals, J. Immunol., 177, 1481, 2006. Pamela J. Fink, h.D. Department of Immunology University of Washington

56. 56 Fas ligand (FasL/CD95L) is best known for its role in delivering apoptotic signals through its receptor, Fas (APO-1/CD95). In this study, we present evidence that FasL has a second role as a signaling receptor. …….(abstract) Suzuki I. and Fink PJ. (1998) J. Exp. Med., 187:123. Depressed proliferation and cell recovery of FasL-deficient CTLs relative to FasL+ CTLs over the course of an MLC. (a and b) [3H]TdR uptake and viable cell counts over days 1-5 from long- term CTL lines from B6, B6.lpr, and B6.gld mice cultured with allogeneic H-2k splenocytes. (c) CTL responders were purified CD8+ T cells derived from B6 and B6.gld mice cultured with H- 2bm1 splenocytes. (d) [3H]TdR uptake by CTL responders used in a, cultured with C3H.gld splenocytes.

57. 57 Fas ligand (FasL/CD95L) is best known for its role in delivering apoptotic signals through its receptor, Fas (APO-1/CD95). In this study, we present evidence that FasL has a second role as a signaling receptor. …….(abstract) Suzuki I. and Fink PJ. (1998) J. Exp. Med., 187:123.

58. Ectopic expression of FasL in NIH3T3 58 (A). Full-length and truncated FasL. Adapted from Orlinick JR, et al. J Biol Chem 1997, 272:32221-29. Jodo S, et al. J Immunol 2005, 174:4470-4474. FasL- △ 70: deleting the N-terminal 2-70 aa; FasL- △ 33: deleting the N-terminal 2-33 aa.

59. 59 (A). Nude mice were injected with 5x105 cells of NIH3T3 cells carrying FasL as indicated via tail vein. After 4 weeks, spontaneous transformed NIH3T3 tumor nodules formed in the lung. Ling sections were stained with H/E. Tumor nodules show dark purple stain. (B). FasL constructs were tranfected into B16F10 cells and injected into B6 mice. After 2 weeks, tumor nodules on the lung surface were counted. N1: B16F10-transfected with plasmid vector, Robozyme: B16F10-transfected with FasL-ribozyme plasmid. Full-length FasL suppresses lung metastasis, while truncated FasL promotes tumor formation. A B

60. 60 FasL forms complex with Met and activate authentic Met signaling pathway 林煥晴

61. 61 A new paradigm for FasL signaling 林煥晴

62. Alternative paradigm 62 A tumor is a local growth of abnormal tissue consisting of genetic-altered transformed cells and a number of other cell types and connective tissue components characteristic of each tumor type. No host, as a tissue, no fighting. Seljelid R et al 1999, Anticancer Res 19:4809

63. Tumor Stroma 63 Fibroblasts Macrophages mast cells Endothelium lymphoid cells (T, B, granulocytes, NK cells) intercellular substance; extracellular matrix

64. Ctrl. shCol Fibroblast attraction 林若曦 Effects of ECM on the tumor microenviroment: Collagen and tumor fibroblast 64

65. 65 A. tumor as abnormal growth of transformed cells. B. Tumor as malignant tissue. C. Tumor hijacks macrophage to direct growth. A B C Black squares: tumor cells; Round: lymphocytes; Oval: macrophages; small circles: mast cells Seljelid R. 1997, Scan J Immunol 46:437