100 60 10 0 year

Cancer/ Neuronal disorder Infection/Development

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Tumor immunologyBC Yang

Expected life-span

2010

衛生署資料，台灣人十大死因2

排名 年度 88 89 90 91 92 93 94 95 96

1 惡性腫瘤 135.32 142.23 147.68 152.88 156.01 160.54 163.8 166.5 175.9

2 腦血管疾病 57.39 60.1 58.82 53.46 54.98 54.48 57.8 55.2 56.2

3 心臟疾病 51.33 47.56 49.25 50.93 52.23 56.79 57.1 53.8 56.7

4 糖尿病 40.99 42.6 40.79 39.26 44.38 40.58 46.2 42.5 44.6

5 事故傷害 58.88 47.4 42.58 37.79 36.3 37.33 36.8 35.1 31.1

6 慢性病跟肝硬化 23.53 23.32 23.45 21.35 22.98 23.63 24.7 22.1 22.5

7 肺炎 18.2 14.88 16.77 20.17 22.6 24.44 25.0 23.6 25.7

8 腎病徵候群及腎病性病 15.78 17.45 18.15 18.55 19.08 20.67 21.2 20.6 22.2

9 自殺 10.36 11.14 12.45 13.59 14.16 15.31 18.8 19.3 17.2

10 高血壓性疾病 8.43 -- 7.9 8.67 8.17 7.97 8.3 8.0 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。

1960 Nobel prize in physiology or Medicinefor discovery of acquired immunological tolerance

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THE JOURNAL OF EXPERIMENTAL BIOLOGY 207 (23): 4013-4014

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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: THEBILLINGHAM–MEDAWAR EXPERIMENTS AT BIRMINGHAM UNIVERSITY AND UNIVERSITY COLLEGE LONDON

(The technique of free skin grafting in mammals)

Evidence for active host defense against cancer

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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)

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 antibodiesMHC-I down-regulation

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The ways to prove effective

tumor immunity：

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Immune surveillance, animal model8

Full immunity needed to fight cancer9

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

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Full immunity needed to fight cancer

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A fight between immune cells and cancer

But, sometimes we lose

THE GREAT

ESCAPE:

IMMUNE

EVASION

VERSUS TUMOR

PROGRESSION

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有些事只有在變動的狀況才看得見事物的本質

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Difficulties due to study model

Tumor formation of Ras-over-expression cells in BALB/c

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It is a dynamic process

death

survival

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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

Use of tumor cell lines17

Commonly derived from advanced tumors

Retain the genetic instability and lose the ability of adaptations

introduced in large numbers (more than 106 cells)

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Successful immunization for cancer

Immune therapy (T cell-based)19

Recombinant and synthetic vaccinationCytokine treatments (IL-2; GM-CSF; IFN)Cellular therapy with tumor-specific CTLEngineered macrophages (Th1…?)Antigen-pulsed macrophages or dendritic

cells

Tumor associated antigens20

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

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Peptide epitopes for melanoma

Nicholaas P, et al. 1999, Curr Opin Oncol 11:50

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Tumor and activated T cells

Two major pathways for TCL: Fas-mediated and perforrin-mediated

HLA class I molecules23

Antigen presentation for T cells, cytotoxic

Inhibitory signals for NK cells

Immune escape/erosion24

Making invisible Resistance to death Repelling Active suppression ….

An overview25

Examples

Down-regulation of the class I presentation pathway

Resistance to killingAntigen-specific

mechanisms (Treg?)Global mechanisms

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Antigen-specific mechanisms27

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

Down regulation of the MHC class presentation pathway

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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)

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)

Resistance to killing30

Defective Fas pathwayResistance to Granzyme B

Fas-L and Neutrophil

Cytotoxic T cells

Innate immunity

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Caspase 3 Apaf-1Caspase 9Death substrates

Apoptosis

Nature 407, 789 5(2000) Fas signal

Key person in Fas signal study32

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

Loss sensitivity to Fas-mediated apoptosis

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FLICE-like inhibitory proteinsBcl-2, Bcl-XLdefected sphingomyelinase

activation (ceramide)decoy receptor 3 (DcR3), soluble

Fas

Fas-L+-melanoma cells are relatively resistant to killing of neutrophils

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B16F10

Chen YL, et al J. Immunol. 171:1183-1192.

Global Mechanisms35

TGF-betaIL-10Growth in immune privilege

sitesMucin production: interfering

intercellular adhesionFas-L? (Fas counterattack)Tumor extracellular matrix

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TGF- signaling in tumor signaling and cancer progression

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IL-10 (Th3 or Treg)

Tumors or other cells in environments

Shaping T cell functions by extracellular matrix.

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NPC Done by 張逸如 (1998)

Hematoxylin/eosin staining CD3+ cells

Immune cells are there! But, in peripheral stroma area (cell cuffing)

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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.

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Coculture with tumor cells inhibits the Fas-mediated apoptosis.

0.0

10.0

20.0

30.0

40.0

50.0

60.0

J CH-11 U118V U118V+CH-11

U118R U118R+CH-11

HeLa HeLa+CH-11

Huh-7 Huh-7 +CH-11

A549 A549 +CH-11

Apo

ptos

is (%

)

24 h 48 h

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Direct cell contact is required.

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Caspase 3 Apaf-1Caspase 9

Death substrates Apoptosis

Apoptosis program is inhibited.

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0

10

20

30

40

50

60

None Z-vade (10μm)

SP600125 (10μm)

PD98059 (10μm)

SB202190 (5μm)

SN-50 (10μm)

KT5720 (2.5μm)

HSP70 (10μm)

Ap

opto

sis

(%)

Jurkat Jurkat + CH-11 Jurkat + U-118MG Jurkat + U-118MG + CH-11

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

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Fas

Fas-LImmune cells

Tumor

Tissue environment ?

Cytokines ?

What will happen when Fas-stimulated immune cells resist to die?

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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

Regulatory T cells: the third man46

Shimon Sakaguchi (2000) Regulatory T Cells Key Controllers of Immunologic Self-Tolerance Cell 101: 455-458

A very dynamic interactions47

Tumor microenviromentIndividual properties

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A FasL mystery

1. Tissue dependant2. Reverse signaling?3. Other than death-triggering

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

In vivo consequences of Fas-L expression by tumors(using over-expression system)

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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

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

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Fas-LR

Control

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B16-F10 (melanoma) in B6 mice

Control Fas-LRibozyme

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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 105 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.

The structure of FasL54

FasL reverse signaling55

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

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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.

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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.

Ectopic expression of FasL in NIH3T3

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(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. △ △

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(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

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FasL forms complex with Met and activate authentic Met signaling pathway

林煥晴

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A new paradigm for FasL signaling

林煥晴

Alternative paradigm62

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

Tumor Stroma63

Fibroblasts

Macrophages

mast cells

Endothelium

lymphoid cells (T, B, granulocytes, NK cells)

intercellular substance; extracellular matrix

Ctrl.

shCol

Fibroblast attraction

林若曦

Effects of ECM on the tumor microenviroment:Collagen and tumor fibroblast

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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