Immune Regulation

林俊彥醫師Chun-Yen Lin MD PhD

Department of HepatogastroenterologyLinkou Medical Center, Chang Gung

Memorial Hospital

林俊彥醫師Chun-Yen Lin MD PhD

Department of HepatogastroenterologyLinkou Medical Center, Chang Gung

Memorial Hospital

生物醫學的研究

蛋白質核酸

人體

組織器官系統

細胞

物理化學原則（化約原則）

生物原則（渾沌複雜原則）

Immune System

Overview of Immune System

Innate Immune System

Principles of innate immunity

• 不具專一性• 參與之細胞：Macrophage, neutrophils and

NKcell• 產生之反應： inflammatory responses

– 症狀：heat 、 swelling 、 pain

The inter-talk between innate immunity and acquired

immunity

Antigen-Presenting Cells

• Activated innate immunity could activated antigen-specific cells – Three main kinds of antigen-presenting

cells (APC)• Dendritic cells• Macrophages• B- cells

– APCs can in turn activate antigen-specific T cell activity

Antigen-Presenting Cells

Principles of adaptive immunity

• 具專一性及記憶性• 參與細胞： T cells (CD4+T cells and CD8+T

cells)and B Cells• 反應慢，活化時間長

Acquired immunity• Specificity

– Every T cell and B cell has its own unique T cell receptor or B cell receptor

– Every TCR/BCR can recognize one specific antigen

• Clonal expansion– Proliferation of antigen-specific T or B cells after

ligation with APCs

• Memory– After activation, T/B cell can differentiate into

memory T/B cells

Kinetics of host responses during a primary acute viral

infection

• Innate Immunity

• Acquired Immnuity

Natural History of HBV Infection

Chu et al Hepatology 1985

HBe Ag Anti-HBe Ab

Immune Tolerance

Immune Clearance

Residual Intergrated

Cirrhosis

HCC

HBV-replicative phase

Non--replicative phase

ALT/AST

HBV-DNA

SEROLOGY

Stage

Mechanism of action of anti-nucleotide analog for HBV replication

Partially double-stranded DNA

Adefovir

A(n)

Infectious HBV virion

(-)-DNA

Infectious HBV virion

mRNAcccDNA

DNA polRT

Encapsidated pregenomic mRNA

HBsAg envelopes

0

50

100

150

200

250

300

0 4 12 20 28 36 44 52 60 68 76Time (Weeks)

0

0.5

1

1.5

2

2.5

3

3.5

HBV DNA

ALT

HBV DNA (pg/mL) ALT (xULN)

Lamivudine Follow-up

Tassopoulos Tassopoulos et al.et al. 1999b 1999b

Post-treatment Responses in HBeAg (-) CHB Patients Treated with Lamivudine

Therapeutic endpoints over time

Development of Treatment Strategy

Loss of Loss of HBeAgHBeAg

SuppressSuppressHBV replicationHBV replication

Anti-HBe+Anti-HBe+

Loss ofLoss ofHBsAgHBsAg

Anti-HBs+Anti-HBs+ ImprovedImprovedsurvivalsurvival

Inhibit virus replication

Enhance anti-viral immunity

Exhaustion of Virus specific CD8+T cells

Ahmed; 2004; J. Viol.

Malignancy as the leading cause of

mortality93 年台灣十大死因排行

順位 死亡原因 死亡人數 每十萬人口死亡率

死亡百分比

1 惡性腫瘤 3 萬 6357 160.54 27.20 2 心臟疾病 1 萬 2861 56.79 9.62 3 腦血管疾病 1 萬 2329 54.48 9.07 4 糖尿病 9191 40.58 6.88 5 事故傷害 8453 37.33 6.32 6 肺炎 5536 24.44 4.14 7 慢性肝病及肝硬

化 5351 23.63 4.06

8 腎炎、腎徵候群及腎性病變

4680 20.67 3.50

9 自殺 3468 15.31 2.59 10 高血壓性疾病 1806 7.97 1.35

資料來源：衛生署 2004

Cancer Studies in Biological Research

各類藥品之市場佔有率 已進入 phase III 的生技候選藥物

資料來源： IMS Health, Deutsche Bank estimates

T cells can kill the tumor cells

Swann, J. B. et al. J. Clin. Invest. 2007;117:1137-1146

Extrinsic Tumor Suppression by Immune System

Intrinsic tumor

suppression Extrinsic tumor

suppression

Current Paradigm for Tumor Immunotherapy

The not enough of a good thing paradigm

Curiel TJ,. J Clin Invest 2007

Tumor vaccine as immunotherapy

Dendritic-cell based Immunotherapy

Nestle FO; Nature Medicine  7, 761 - 765 (2001)

Tumor-infiltrating lymphocytes as immunotherapy

Intra-tumor Injection to enhance anti-tumor

responses

Dendritic cellCytokine DNA vaccine

Tumor vaccine

Immunotherapies for hepatoma

Greten TF. J Hepatol 2006

Immunotherapies for hepatoma

Greten TF. J Hepatol 2006

Is there any other way to ignite effective anti-

tumor/ anti-viral immune responses?

Homeostasis of Immune System

Regulatory Immune

Responses

Aggressice Immune

Responses

Homeostasis of Immune System

Aggressive Immune

Responses

Regulatory Immune

Responses

(Regulatory T cells)

Papers published in PubMed

Milestone for Treg cell discovery

Evidence supporting existence of Treg cells

Waldmann, J Exp Med, 1989

Sakaguchi, JI, 1995

Evidence supporting existence of Treg cells

Definition of regulatory T cells

• A group of T cells that could prevent the development of immunopathology in vivo

Prolfieration of T cells

0 1/0 1/625 1/125 1/25 1/5 1/1 0

5000

10000

15000

20000

CD4+D25+ T cell dosec.

p.m

.

Proliferation assay

0 1/12500 1/2500 1/500 1/1000

10000

20000

30000CD25(-)

CD25(+)

Concentration of anti-CD3

c.p

.m.

Anergized Inhibition of naïve

T cell proliferation

In vitro Behavior of Treg cells

Characteristics of Treg cells- Dominant Tolerance

• Infectious Tolerance• Linked suppression

Infectious Tolerance

Graft B

Graft (AxB)F1 Graft BGraft A

Linked Suppression

Searching for the markers of Treg cells

The transcription factor Foxp3

• Foxp3 gene : the disease-causative gene in Scurfy mice, which spontaneously develop severe autoimmunity/inflammation as a result of a single gene mutation on the X chromosome

• Mutations of the human FOXP3 gene: the cause of a similar human disease called IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome)

• CD25+CD4+ peripheral T cells and CD25+CD4+CD8- thymocytes specifically expressed Foxp3 mRNA

The transcription factor Foxp3

• Retroviral transduction of Foxp3 to normal CD25-CD4+ T cells converted them into phenotypically and functionally TR-like cells.

• In BM chimera with a mixture of BM cells from wild-type and Foxp3-deficient mice, Foxp3-deficient BM cells failed to give rise to CD25+CD4+ T cells, while Foxp3-intact BM cells generated them and suppressed disease development.

• These findings collectively indicated that the transcription factor Foxp3 could be a master controller of the development and function of natural CD25+CD4+ TR cells.

The transcription factor Foxp3

• Not all Treg cells express Foxp 3 molecule

• Not all cell express Foxp 3 could behave like Treg cell– Human CD4+CD25-T cells could

transiently express low level of Foxp 3 without suppression function

Heterogeneous groups of Treg cells

• Natural Treg cells• Induced Treg cells

– TH3 cells (TGF-)– Tr1 (IL-10)

• Regulatory CD8+T cell• Regulatory NK T cell• CD4-CD8- (DN) T cells• T cells

Multiple favor of Treg cells

Ice-cream flavor

Ethan Shevach 2006 immunity

Treg cell mediated phenomenon

• Bystander suppression

• Infectious tolerance/linked suppression

Treg cell mediated phenomenon

• Bystander suppression– suppressive activity of Treg cells requires

their prior activation through their T cell receptor

– once activated, Treg cells suppress in an antigen-nonspecific way

– Treg cells with one antigen specificity can suppress effector T cells (Teff cells) with many other distinct antigen specificities

Treg cell mediated phenomenon

• Infectious tolerance/linked suppression – population of suppressor T cells creates a

regulatory milieu – promotes the outgrowth of a new population

of Treg cells with antigen specificities distinct from those of the original Treg population

Suppression mechanisms of Treg

cells

• Treg cell influence other T cells• Treg cell alter APCs• T cell derived suppression

molecules

Treg cell influence other T cells

• Suppress the proliferation/cytokine release of both CD4 and CD8 Teff cells in vitro

• Prevent CD8+T cells from differentiation into effector T cells but not proliferation in vivo

• Alter the differentiation of naïve CD4+T cell into IL-10/TGF- secreting T cells

Suppress the proliferation/cytokine release of both CD4 and CD8 Teff cells in

vitro• Direct contact mechanisms

– APC-free in vitro system and transwell system

– Membranous TGF-– cAMP—through gap junction– Granzyme B/perforin

• No direct contact in LN by in vivo two-photon microscopy

Suppress the proliferation/cytokine release of both CD4 and CD8 Teff cells in

vitro

• IL-2 “sink”– CD25: high affinity of IL-2 receptor– Teff cells death depends on pro-

apoptotic factor Bim• Bim difficient Teff or forced expression of

Bcl-2 could render the Teff cell resistent to Treg cell suppression bot in vivo and in vitro

T cells

DisarmedLoss of CTL effect

Loss of IFN- secretion

Loss of graft rejection ability

Lin C-Y; 2002; Nature Immunol

Prevent CD8+T cells from differentiation into effector T

cells but not proliferation in vivo

Prevent CD8+T cells from differentiation into effector T cells but not proliferation in

vivo

Mempel; 2006; Immunity

Alter the differentiation of naïve CD4+T cell into

IL-10/TGF- secreting T cells• Naive CD4+T cell could differentiate

into IL-10 or TGF- secreting induced Treg cells in the presence of Treg cells in in vivo and in vitro study

• Consistent with the phenomenon of infectious tolerance/linked suppression

Treg cell alter APCs• Treg cell could conjugate with dendritic

cells documented by in vivo two-photon microscopy

• Treg cells could alter the dendritic cells functions in the following ways– DC silencing: increase IDO expression in DC– Local TGF- activation (latent TGF- bind to DC

surface for later activation)– Treg cell expansion: with the help of to TGF-

the differentiation of Teff to Treg cells)

Multiple molecular mechanisms that involved in

Treg cell suppression• Suppressive cytokines

– IL10; TGF-; IL35

• Killing molecule– Perforin/granzyme B/HO-1(produce CO)

• Suppression molecules– cAMP/CD39-CD73--adenosine/Galectins

Multiple molecular mechanisms that involved in

Treg cell suppression

Multiple Arena of Treg cells (Gelati Misti

model)

Dario Vignali 2008 EJI

Three tiered model of functions of Treg cells

• Homeostatic control– Prevent potential outgrowth of auto-reactive

T cells

• Damage control– Limit the tissue injury during inflammation

caused by infection/autoimmune/transplantation

• Infectious Tolerance– Stabilized tolerance during the final stage of

inflammation

Homeostatic control• Prevent the potential outgrowth of

auto-reactive T cells• Treg cell “sop up” IL2 produced by local

incidental auto-reactive Teff to inhibit the Teff and enahnce Treg cell functions/number

• Treg cells also target the self-antigen presenting DC to silence DC

Damage control• Infection/autoimmune reaction/transplantation

cause tissue damage• Activated Treg cells loss the CD62L and

upregulate chemokine receptors to migrate into inflammed tissue

• Highly expressed IL-10/CTLA-4/IL35/TGF-/HO-1/CD39-CD73 and granzyme B/perforin to inhibit Teff function

Infectious Tolerance• Stabilized tolerance during the final

stage of inflammation• Tissue damage lead to the expose

of newly exposed antigen on Treg cells

• Expanding Treg cell receptor repertoire through DC and TGF-and retinoic acid

Three tiered model of functions of Treg cells

Bluestone J; 2008 Nat Immunol

Importance of Treg cells

• Benefits– T cell homeostasis– Prevent autoimmune

disease– Tolerance after

transplantation– Prevent GVHD– Prevent hypersensitivity

• Detrimental Effects– Down-regulation of tumor

immunity– Down-regulation of

immunity to infection

C57BL/6 mice with tumor vaccination and then challenge with melanoma tumor cell line

Associated autoimmune skin depigmentation in mice with successful tumor eradication

Survival

Days

Depleting CD25+Cells

+Anti-CTLA4

Anti-CTLA4No Tx

Sutmuller, J. Exp.Med.; 2002

Treg cells in Tumor Immunology

Bhardwaj N,Bhardwaj N,. J Clin Invest 2007

Anti-Tumor Immune Tolerance for Tumor

Development

Treg cell could protecting tumor from

immune attack

Sheva E,Sheva E,. Nature Medicine; 2004

Naïve Treg Cells

Tissue-protecting Treg cells

A

B

Tumor-protecting Treg cells

Inflammation

?

YC Lin submitted for publication

Changing Paradigm Thinking for Tumor

immunology

The not enough of a good thing paradigm

The too much of a bad thing paradigm.

Curiel TJ,. J Clin Invest 2007

Changing Strategies about Tumor

ImmunotherapyEnhance tumor-specific CD8+T

cells

Block the Regulator

y T cell function

Natural History of HBV Infection

Chu et al Hepatology 1985

HBe Ag Anti-HBe Ab

Immune Tolerance

Immune Clearance

Residual Intergrated

CirrhosisHCC

HBV-replicative phase

Non--replicative phase

ALT/AST

HBV-DNA

SEROLOGY

Stage

Aggressive T cell responses

Treg

慢性 B 型肝炎的免疫機轉

ALT

Liver injury could enhance the frequency of

Treg cells

Normal 1-2X 2-5X >5X0.0

2.5

5.0

7.5

10.0

12.5

2.17¡Ó0.18 2.85¡Ó0.50 3.62¡Ó0.29 5.05¡Ó0.55

Freq

uen

cy o

f T

reg

cell

(%)

P<0.01

P<0.05

p<0.001

P<0.001

n=33 n=21 n=37 n=21

Lin C-Y; 2007; J Viral Hepat

Aggressive T cells

Lin C-Y; 2007; J Viral Hepat

Treg cells could inhibit the HBV specific CD8+T cell responses

Regulatory T cells(Treg cells)

Cure

Chronic Infection

Treg cells

Belkaid 2003 Nature

Treg cells in Leishmania infection

Manipulation of natural Treg cells as a therapeutic

approach during infection

Treatment strategy for autoimmune disease focusing on Treg cells

Larche, M. Chest 2007;132:1007-1014

Treg inhibit the effector phase of the allergic

response

動態不平衡

Aggressive Immune

Responses

Regulatory Immune

Responses

自體免疫疾病移植失敗

動態不平衡 Aggressive Immune

Responses

Regulatory Immune

Responses

癌症慢性感染