control of immune response by regulatory t cells

© 2015 Osaka University. All rights reserved.

Control of immune responsesby regulatory T cells

Shimon SakaguchiWPI Immunology Frontier Research Center

Osaka University

Immunological Self-Tolerance

Autoimmune Disease

Tumor ImmunityChronic microbial infection

Allergy

Organ transplantationFeto-maternal tolerance

Immunesystem

Self Non-self


Possible Mechanisms of Immunological Self-Tolerance

Deletion (Apoptosis)

Self1

Inactivation (Anergy)

Self2

Suppression Self3Treg

Autoimmune diseases in humans

Autoimmune diseases

Organ-specific Non-organ-specific


5% of the population is afflicted with autoimmune disease

Organ-specific

Two types of autoimmune disease

Non-organ-specific

brainMultiple sclerosis(?)

thyroidHashimoto’s thyroiditisprimary myxoedemathyrotoxicosis

stomachpernicious anaemia

adrenalAddison’s disease

pancreasInsulin-dependentDiabetes mellitus (TYPE I)

muscledermatomyositis

kidneySLE

skinsclerodermaSLE

joints

rheumatoid arthritis

Grave’s disease

Systemic Lupus Erythematosus

Overlapping of affected organs as a characteristic of organ-specific autoimmune diseases

Reference: Irvine (1979) Medical Immunology

Clinical Level

Subclinical Level

Patients with TYPE I diabetes


Overlapping of affected organs as a characteristic of organ-specific autoimmune diseases

Type 1 diabetes, Thyroiditis

NOD mice BB rats

Type 1 diabetes, Thyroiditis, Gastritis

Thymectomy

normal mouse

Removal of the thymus


Induction of autoimmune diseases by manipulatingthe T cell immune system

MiceTx

Day 3 after birth

Autoimmune gastritis,oophoritis, thyroiditis, etc.

Rats6 week

TxX-irradiation

Autoimmune thyroiditis, Type 1 diabetes

Induction of autoimmune diseases by manipulatingthe T cell immune system

MiceTx

Day 3 after birth

Autoimmune gastritis,oophoritis, thyroiditis, etc.

Rats6 week

TxX-irradiation

Autoimmune thyroiditis, Type 1 diabetes


Thyroiditis Oophoritis Orchitis

Post-thymectomy organ-specific autoimmune diseases

BALB/c

Day 0-Tx 0/35 1/35(2.9%) 0/35 0/35

Day 3-Tx 0/45 15/45(33.3%) 12/45(26.7%) 0/45

Day 7-Tx 0/35 0/35 0/350/35

A Day 3-Tx 3/50(6.0) 5/50(10.0%) 44/50(88.0%) 8/50(16.0%)

C57BL/6 Day 3-Tx 0/20 0/200/200/20Reference: Sakaguchi et al. J. Exp. Med. 1982

Asano et al., J. Exp. Med. 1996

Gastritis

MICE

Autoimmune diseases

Tx：Thymectomy

Findings

BALB/c A C57BLACK

Date ofthymectomy

1 2

Genetic backgroundof mice


DAY 0 3

Tx17/20

Tx

Tx

Tx

Tx

Tx

0

10

17

24

31

5x106 T cells

0/5

0/10

0/10

2/10

8/10

Incidence ofAutoimmune

disease

Tx：Thymectomy

Prevention of NTx-induced autoimmune disease by T cells from normal adult mice

Sakaguchi et al. J. Exp. Med. 1982

60

0-1 3 4 5 6 7-2 1 2Day Regulatory T cells

NTx

How does NTx cause autoimmune disease?

Autoimmune T cells

Autoimmune Disease

Thymus


Induction of autoimmune disease in normal animals by depleting a T-cell subpopulation (1)

BALB/c

CD4+ T-cell suspensions eliminated of

CD5high, CD45RClow, or CD25+ cells

BALB/c nude

Induction of autoimmune disease in normal animals by depleting a T-cell subpopulation (2)

Sakaguchi S, et al. J. Immunol. 1995CD25 (IL-2Rα)

CD25 (IL-2Rα)

CD

4


Induction of autoimmune disease in normal animals by depleting a T-cell (3)

Sakaguchi S, et al. J. Exp. Med. 1985

Sakaguchi S, et al. J. Immunol. 1995

Powrie F & Mason D. J. Exp. Med. 1990

Thyroid

Sislet

Stomach

Salivary gland

Langerhans islets

Overies

Testes

Inflammatory

bowl disease

develop very similar autoimmune diseases in various organs

BALB/c nude

Remove CD25+ cells Then transfer remaining cells

into nude mice

Induction of autoimmune disease in normal animals by depleting a T-cell subpopulation

Gastritis

Thyroiditis

Insulitis/(type 1 diabetes)

BALB/c Nude(Removed CD25+ cells)


A. 18 0 0 0 0 0 0 0 0Whole(5x107)

Induction of autoimmune disease in nude mice by transferring CD25-CD4+ T cells

Exp.group

Inoculated cells

Total numberof mice

Number of mice with autoimmune disease

Gas Oop Thyr Sial Adr Ins GN Arth

C. CD4+CD25-

(5x107)16 14

(87.5)13

(81.3)7

(43.8)5

(31.3)2

(12.3)0 3

(18.8)0

B. 22 22 (100)

22(100)

16(72.7)

10 (45.1)

7 (31.8)

2 (9.1)

7 (31.8)

2 (9.1)

CD25-

(5x107)

6 1 (16.7)

0 0 0 0 0 0 0E.(2x106)

CD25- CD25++(5x107)

10 0 0 0 0 0 0 0D. 0CD8+CD25-

(5x107)

Ontogeny of CD25+CD4+ T cells(1)

Figure: Asano et al. J. Exp. Med. 1996


Figure: Asano et al. J. Exp. Med. 1996

Ontogeny of CD25+CD4+ T cells(2)

Treg

Altered Treg-mediated immunoregulationas a cause of immunological diseases

Biological (e.g., MTLV)Chemical (e.g., Cyclosporin A)Physical (e.g., ionizing radiation)

Non-genetical insults

Genetic anomaliesFoxp3CTLA-4IL-2, CD25, CD122Runx1/AML1CD40

Sakaguchi et al., 1988; Sakaguchi et al., 1989; Sakaguchi et al., 1994; Morse et al., 1999; Kumanogo et al., 2000; Takahashi et al., 2000; Setoguchi et al., 2005; Hori et al., 2003; Ono et al., 2007; Wing et al., 2008; Kitoh et al., 2009

Self Antigen

Bone MarrowThymus

Periphery

CD25+

CD4+

Genetic anomaliesEnvironmental insults

MF

B

CD25-

CD4+

CD4+ Teff

Examples

CTL


• Autoimmune disease : T1D (>80%),Thyroiditis, etc.

• Inflammatory bowel disease

• Allergy (hyper-IgE)

Foxp3 mutations abrogate Treg cell development, causing autoimmune/inflammatory diseases (1)

IPEX

Immune dysregulation, Polyendocrinopathy,

Enteropathy, X-linked syndrome

Scurfy mice

Foxp3 mutations abrogate Treg cell development, causing autoimmune/inflammatory diseases

Wild type Scurfy

• Lymphoadenopathy

• Hyperactivation of CD4+ T cells

• X-linked disease


Induction of autoimmune disease in normal animals by depleting Foxp3+CD25+ Treg cells (1)

Thymocytesor

Splenic T cells

Depletion of CD25+ Tregs

Autoimmune diseasesInflammatory bowel disease

etc.

Cell transfer

Normal mouse

develop

T cell-deficientmouse

Induction of autoimmune disease in normal animals by depleting Foxp3+CD25+ Treg cells (2)

Thymus Spleen

CD25

Fo

xp3

Sakaguchi S, et al. J. Exp. Med. 1985Sakaguchi S, et al. J. Immunol. 1995Itoh M, et al., J. Immunol. 1999


Inhibition of autoimmune disease and IBD by Foxp3-transduced naïve T cells

Foxp3/MIGR1 MIGR1

CD

4

GFP

LTR Foxp3 IRES GFP LTR

Foxp3/MIGR1

LTR IRES GFP LTR

MIGR1

Hori, et al., Science. 2003

Inhibition of autoimmune disease and IBD by Foxp3-transduced naïve T cells

Colon

Stomach

+Foxp3/MIGR1 None+MIGR1CD25-CD45RBhialone

ga

str

itis

sc

ore

0

1

2

3

co

litis

sc

ore

0

1

2

3

4

5


Dominant self-tolerance in rodents and humans

Teff

Teff

Treg

Nude or SCID mice

No disease

Teff

Treg

Teff

Treg

Mothers of IPEX patients

IPEX patients

WholeT cells

CD25T cells

Induction of autoimmune diseaseand IBD by depleting Treg cells

Normal

Autoimmune disease IBD

Hyperreactivity

Development of autoimmune disease,IBD, and allergy in IPEX

Humans

Autoimmune disease IBD

Hyperreactivity

Sakaguchi, Annu. Rev. Immunol. 2004

Complete depletion of Foxp3+ cells produces fatal autoimmune/ inflammatory diseases


Complete depletion of Foxp3+ cells produces fatal autoimmune/ inflammatory diseases

Non-self

Self

Allergy

Immunopathology

Autoimmunity

Deficiency or dysfunction of Foxp3+ Treg cells produces a variety of autoimmune, immunopathological,

and allergic diseases

CD25+

cellsFoxp3+

cells

Normal Treg Treg deficiency/dysfunction


Induction of tumor immunity by depleting CD25+CD4+ T cells

(n=10 each)

Athymic nude mouse

Shimizu, et al., J. Immunol. 1999

Tumor cells (RLm1)

Cell transfer

Induction of allograft tolerance by graft-specific expansion of CD25+CD4+ Treg cells

Nishimura, et al., Int. Immunol. 2004

B6 skin graft

1 week

CD25+CD4+ T cells Naïve T cells

Graftsurvival

BALB/c

BALB/c nude


Induction of allograft tolerance by graft-specific expansion of CD25+CD4+ Treg cells

100806040200

20

40

60

80

100

Days after cell transfer

Gra

ft s

urv

ival

(%

)

T cells alone

(1:1)

T cells & CD25+ T cells(1:3)

12 0

n=15

n=13n=29

Nishimura, et al., Int. Immunol. 2004

T cells & CD25+ T cells

Self antigen

Tumor antigen Allo antigen

Bone Marrow

Thymus

Foxp3

Foxp3Treg

Foxp3Treg

Th1Th2Th17etc.,

NaïveT cell

CTLA-4+

CD25+TGF-

CTLA-4+

CD25+

Summary: Section 1


Control of cytokines and Treg-associated molecules by Foxp3

CD25 GITR CTLA-4

IL-2R

?

Foxp3IL-2

CD25 (IL-2R -chain) CD122 (IL-2R -chain)CTLA-4 GITR -

Deficiency Autoimmune/inflammatory disease

IL-2 IFN

Repression

Sakaguchi, Nat. Immunol. 2005

++

+

++

Activation

Foxp3

Is CD25 (IL-2R -chain) a mere marker for natural Tregs or an essential molecule for their function?

Figure: R. Setoguchi et al. JEM 2005

IL-2

IL-2R

Treg


CD25+CD4+ CD25-CD4+CD25+CD4+ CD25-CD4+

IL-2Rβ

IL-2R

Tregs constitutively express the high affinity IL-2 receptoralready in the thymus

Figure: Setoguchi et al. J. Exp. Med. 2005

Thymus Spleen

IL-2 neutralization by specific mAb reduces Tregsin the thymus and the periphery

CD25

CD

4

1.7％ 0.3％

2.6％ 0.6％

Saline Anti-IL-2

Setoguchi et al. J. Exp. Med. 2005

Thymus

Spleen


CD25+CD4+ Tregs are physiologically proliferating and IL-2 neutralization selectively inhibits their proliferation

6.47 % 2.22 %

1.79%1.71 %

Saline Anti-IL-2

CD25+CD4+ T cells

CD25-CD4+ T cells

BrdU

Induction of autoimmune disease in normal mice

by IL-2 neutralization

Birth

0 10 20 3 month

Anti-IL-21mg i.p.

Histological Serological

analysis


BALB/cDay


Induction of autoimmune disease in normal mice by IL-2

neutralization

Anti-parietal cell autoantibody

(OD 405 nm)

0

0.2

0.4

0.6

0.8

1.0

1.2

Saline Anti-IL-2

N=6 N=6

: intact gastric mucosa: histologically evident gastritis


APC

CD25lowCD4+ T cells are the principal IL-2 producers in normal naïve mice

25hi

25lo

25-

CD4

CD

25

IL-2IL-2R

0

4

8

12

16

25hi 25lo 25-

IL-2

(p

g/m

l)


NKTCD8+

NK


Crucial roles of IL-2 for self-tolerance

Foxp3+ Tregs

Th17 differentiation

AICD of T cells

NK cells,CD8+ T cells, esp. memory

IL-2

CTLA-4

Two models of CTLA-4-mediated immune regulation

APC

(B)

CD28

B7

Treg TeffB7

APCAPC

(A)

CTLA-4 CD28

APC

B7

Teff

B7

(A) (B)


Treg-specific CTLA-4 conditional KO miceTargeted Foxp3 allele

11.2 0.4

44.42.4

CTLA-4 Cond KOWT littermate

Foxp3

CT

LA

-4

13.35.8

Gated on CD4+ T cells

K. Wing et al., Science 2008

Exon Exon

PGK Neo PA

FRTFRT

IRES Cre PA

10 11 12 13

Targeted CTLA-4 allele

IoxP IoxPFRT

2 3 PGK Neo PA

FRT

1 2 3 4

Reduced survival of BALB/c CTLA-4 CKO mice

CTLA-4

CTLA-4

CTLA-4

Foxp3

Foxp3

Foxp3

FIC/Y

CTLA-4 CKO

CTLA-4 KO


Autoimmune gastritis in CTLA-4 CKO mice

An

ti-p

arie

tal

cell

Ab

(u

nit

)

WT FIC CKO

FIC/Y

CTLA-4 CKO

Autoimmune myocarditis in CTLA-4 CKO mice


Hyperproduction of IgE in CTLA-4 CKO miceIg

E

IgG

Induction of effective tumor immunity by Treg-specific CTLA-4 deficiency

Tumor cells: RLm1 leukemiaK. Wing et al., Science 2008


Cancer regression and autoimmunity induced bycytotoxic T lymphocyte-associsted antigen 4 blockade

in patients with etastatic melanoma

Phan G. Q., Yang J. C., Sherry R. M., et al. Cancer regression and autoimmunity induced by cytotoxic T lymphocyte-associated antigen 4 blockade in patients with metastatic melanoma. Proceedings of the National Academy of Sciences of the United States of America. 2003;100(14):8372–8377. doi: 10.1073/pnas.1533209100


Patient characteristics, clinical response, and toxicity

1 52/M Lung I, S 2 PR(15+) Enteroco;it is; dermatiyis

2 40/F Supraclavicular lymph node C, I, S 1 NR Dermatitis, vitiligo

3 39/M Lung, mediastinum, subcutaneous S 6 NR(Mixed)

4 55/F Skin, subcutaneous I, S 1 NR Pulmonary infiltrates

5 67/M Liver, retroperitoneum, subcutaneous C, I, R, S 4 NR ANA+

6 59/M Lung, subcutaneous I, S 4 NR Vitiligo

7 48/M Lung, brain, adrenal, subcutaneous I, S 2 NR

8 48/M Lung, liver, adrenal, mesentery, subcutaneous C, I, S 2 NR

9 53/M Mediastinum, mesentery, skin I, R, S 2 NR Colitis

10 62/M Lung, hilum C, I, S 2 NR(mixed)

11 54/M Lung, brain, subcutaneous C, S 5 CR(12+) Hypophysitis

12 43/M Subdiaphragm, muscle, subcutaneous I, S 3 NR Hepatitis; ANA+

13 49/F Lung, subcutaneous C, I, S 4 CR(11+) Dematitis

14 63/M Lung, pelvic, lymph node S 4 NR

Patient Age/sex Disease sites Prior therapy

NO. of cyclesreceived

Response(mos.)

Toxicity (grade III / IV)

Tregs down-regulate CD86 on CD86-transfected L cells, whereas CTLA-4 CKO Tregs do not

CD80/CD86CTLA-4

K. Wing et al., Science 2008

APC

Treg

APC?

Trogocytosis Transendocytosis Down-regulation of CD80/CD86

expression by APC Soluble CTLA-4 IDO induction


APC

Foxp3, CTLA-4, and IL-2 in Treg-mediated suppression

IL-2IL-2R

Treg

Suppression

Foxp3

CTLA-4

IL-2IL-2

TCRCD80/86

MHCIL-10etc.

Responder T

Ono et al., Nature 2007Onishi et al., PNAS 2008Wing et al., Science 2008Kitoh et al., Immunity 2009Miyara et al., Immunity 2009Ohkura et al., Immunity 2012Yamaguchi et al., PNAS 2013

How are Foxp3+ Treg function and lineage stability maintained in the immune system?

Bone Marrow

ThymusFoxp3

Foxp3Treg

Foxp3Treg

Th1Th2Th17etc.,

NaïveT cell

CTLA-4+

CD25+TGF-

CTLA-4+

CD25+

Self antigen Tumor antigen Allo antigen


TGF -induced Foxp3+ iTregs are functionally and phenotypically unstable

iTreg or nTreg(Thy1.1+)

CD45RBhighCD4+

(Thy1.2+)

Rag-/-

Ohkura et al., Immunity 2012

nTreg iTreg

TGF -induced Foxp3+ iTregs are functionally and phenotypically unstable

Sur

viva

l rat

e (%

)

100

500 10 20 30

Days

iTreg+naïve T

nTreg+naïve T

iTreg nTreg

Histology (Colon)

Naïve T alone

Unstable expression of Treg-associated molecules in iTregs in vivo



Multiple factors and modifications construct a specific epigenome

DNA

histone

chromatin

chromosome

DNA modification

Histone modification

Chromatin remodeling

Epigenome

Dnmt1TETTFsPolycombHDACHATHMTSWI/SNFBRG1BAFEZH2JHDM1LSD1etc.

AGTTGACGTACGGCAATA

AGTTGACGTACGGCAATA

Me Me

: DNA methylation

DNA methylation

heterochromatin

euchromatin

chromatin structures close→ repressive

→ permissive

chromatin structures open

Highly heritable

RNA pol II

Relatively stable Linked to gene expression


Treg-specific DNA demethylated sites are presentonly in limited regions of the genome

Methylated DNA immunoprecipitation (MeDIP) sequencing

DNA sequencing andAnnotation on the genome

CH3 CH3

CH3 CH3

CH3

CH3

CH3

CH3

CH3

CH3 CH3

CH3

CH3

CH3

CH3

Total methylated regions (156,743)

Treg-specific DNA demethylated sites are presentonly in limited regions of the genome

~300 regions (0.19%)

Treg-specific demethylated regions

Ohkura et al., Immunity, 2012, Morikawa et al., PNAS, 2014

Treg

Naïve T

Foxp3


Detailed CpG methylation examined by bisulfite sequencing

Genomic DNA CGATCCGAAACGCCCCGTTACG

Bisulfite treatment

CGATUCGAAACGUUUCGTTACG

UGATUUGAAAUGUUUUGTTAUG

Methylated DNA

Unmethylated DNA

Target gene TregNaïve T

Foxp3Treg

Naïve T

Foxp3

Detailed CpG methylation examined by bisulfite sequencing

Heat map exhibition of methylation status (%)

● ● ● ● ● ● ● ● ● ● ● ●

● ● ● ● ● ● ● ● ● ● ● ●

● ● ● ● ● ● ● ● ● ● ● ●

● ● ● ○ ● ● ● ● ● ● ● ●

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● ● ● ● ○ ● ● ● ● ● ● ●

1 2 3 4 5 6 7 8 9 10 11 12

○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○

○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○

○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○

○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○

○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○

○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○

○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○

○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○

1 2 3 4 5 6 7 8 9 10 11 12

n=8

CGatcCGaaaCGcccCGttaCG

CpG demethylation

100% 0%

n=8


Foxp3 Cd25

Eos Ctla4

Tregs possess specific epigenetic patterns

Tconv

nTreg

Homology

Bis

ul

Tconv

nTregMe

DIP

Tconv

nTreg

Homology

Bis

ul

Tconv

nTregMe

DIP

Tconv

nTreg

Homology

Bis

ul

Tconv

nTregMe

DIP

Tconv

nTreg

Homology

Bis

ul

Tconv

nTregMe

DIP

Stim

Specificity and stability of Treg-type epigenetic changes

The Treg-type epigenetic pattern is highly specific for natural Tregs

StimCont

StimCont

Cont

StimCont

24h

72h

CpG demethylation

100% 0%

Cont

iTreg

Cont

iTregTconv

nTreg

TGF-

Retinoicacid

Th1Th2

Th17

Central memoryEffector memory

TCR stimulation induced Treg Other T cell subsets

IL-2 expandedTconv

Control

TconvnTreg

transducedFoxp3

Vector

Tconvtransduced

24h

72h


Treg-type epigenetic change begins in the thymus and is progressively established towards the periphery

Thymus

CD25F

oxp

3

Spleen

CD25

Fo

xp3


0

50

100

ThymicDP

ThymicFoxp3+

SplenicFoxp3+

Foxp3

CD25

GITR

CTLA-4

EosC

pG

dem

eth

yla

tio

n(%

)

ThymicCD4SP

Foxp3-

Foxp3+

SplenicCD4+ T

Foxp3-

Foxp3+

Thymic DP

CpG demethylation

100% 0%

CD25-

CD25+

Wild-type CD25+

Scurfy

Foxp3 mRNA 1.00.50

Foxp3 mRNA

Foxp3 protein

Foxp3 demethylation

No

No

YesCD25+

CD25-Scurfy

Treg-type epigenetic changes occur in Scurfy mice

Treg-type epigenetic change is not a consequence of Foxp3 protein expression

Foxp3-null (scurfy) A frame-shift mutation of Foxp3


Foxp3CD25GITRCTLA-4Eos

Thymus

Spleen

ThymusGFP-GFP+

SpleenGFP-GFP+

GFP-GFP+GFP-GFP+

(GFP-marked Treg)

(GFP-marked Foxp3-null Treg)

DP

Thymus DP

Thymus DP

Cp

Gd

em

eth

yla

tio

n (

%)

0

50

100

0

50

100

ThymusGFP+

SpleenGFP+

DEREG♂

DEREG/Scurfy♂

Treg-type epigenetic change is established without Foxp3

CpG demethylation

100% 0%

Foxp3-null TregsnTregs


DPThymus

GFP+SpleenGFP+

nTregs

Foxp3-null Tregs

Treg-specific DNA hypomethylated regions (TSDR) and Foxp3-binding regions are mostly different in nTreg cells

Morikawa et al., PNAS 2014

Foxp3-biding regions

TSDR

Foxp3 CNS2


Foxp3 expression and Treg-type epigenetic changes together establish Treg function and phenotype

nTregs

Foxp3 Epigenome

nTregs

T cell subpopulations delineated by CD25, Foxp3, and the Treg-cell-type epigenome

CD4+

Foxp3Treg epigenome+

CD25+

Natural TregIn vivo iTreg

PotentialTreg

Stable Treg

Foxp3+ TconvIn vitro iTregUnstable Treg-like

Tconv

ActivatedTconv


FoxP3+ Treg subsets in humans

Correlation between CD25 and FOXP3 expressionin CD25+FoxP3+CD4+ T cells in human PBMCs

FoxP3

CD

45R

A

II

I

III

CD25

Fo

xP3 C

D25

-

IIII II

CD

25+

CD

25+

+

CD

25+

++


Fr. I (FoxP3loCD45RAhi) and Fr. II (FoxP3hiCD45Rlo) cells are suppressivewhile Fr. III (FoxP3loCD45RAlo) cells are not

CFSE

CD

45R

A

CD

45R

A

CD25

CD4+ T cellsI

II

Foxp3

IIII

IIIII

Responder alone Fr. l + Responder Fr. ll + Responder Fr. lll + Responder

I IIIII

FOXP3

CD

45R

A

Miyara et al. Immunity 2009

Fr. I and Fr. II Tregs are cytokine hypo-producing, while Fr. III cells are not

I

II

III


FoxP3

CD

45R

AC

TL

A-4

Fr. II (Foxp3hiCD45RA-) Tregs are highly proliferativeand express CTLA-4

Effector Tregs


I

IIIII

FoxP3

CD

45R

AK

i-67

Naïve Tregs

Differentiation and interactions among FoxP3+ subsets

Proliferative

Fr.I

Fr.IIFr.IIICD

45R

A

FoxP3

Epigenome+

Die by apoptosis

Naïve Tregs

Effector Tregs

Thymus

CTLA-4+

CCR4+


Healthy donor

CD

45R

A

FOXP3

FoxP3+ subsets in normal and disease states

nTreg

eTregNon

TregCD

45R

AFoxP3


CD

45R

A

FOXP3

Healthy donor

Sarcoidosis donor

Active SLE patient

PBMC

0.9%

3.6%

CD4 gated

3.25%43.6%

CD

45R

A

Foxp3

CD

4

Foxp3

Predominant infiltration of effector Tregs into tumor tissues

Treg fractions

% o

f CD

4+T

cel

ls

Naïve Effector Naïve Effector

TILPBMC

Tregs:

TIL

14.0%16.8%Foxp3

0.8%

37.6%

CD4 gatedMelanoma

Sugiyama et al., PNAS 2013

CD

4

Foxp3

CD

45R

A

Foxp3


Treg-targeting cancer immunotherapywithout evoking autoimmunity

Effector Treg depletion by anti-CCR4 plus Tumor antigen (e.g., cancer/testis antigen) vaccination

Blood and lymph nodes Tumor tissue

CCR4+CTLA-4+PD-1+

nTreg

eTregNon

TregCD

45R

A

FoxP3

eTregCD

45R

A

FoxP3

Depletion of tumor-infiltrating FoxP3+ Tregsas an immunotherapy of cancer

Nishikawa and Sakaguchi, Int. J. Cancer 2010

CCR4

CCL22

Treg migrationSelf-Ag / Tumor Ag

TGF-β

TolerogenicDendritic cell

Treg expansion

Suppression

Tumor infiltratingmacrophage

Tumor cell

NK

NKT

CTL

Th


CC

R4

(MF

I)

PBMC

CD4+ T-cell subsets

85

IIIIIIIV

Effector Tregs express CCR4, whereas naïve Tregs do not

I

IIIII

IV

I II III IV

Sugiyama et al., PNAS 2013

CD

45R

A

Foxp3

Cel

l num

ber

CCR4

One stone for two birds: Anti-CCR4 mAb depletes both ATLL cells and effector Tregs

Pre-treatment 2nd round injection

CD4

CD

8C

D45

RA

CD

8

Foxp3

CD

45R

A

ATLL patient

6.2%

CD4

80.7%

1.1%

1.4%

98.5%

80.0%Foxp3

Anti-CCR4 mAbtreatment


Anti-CCR4 mAb treatment is able to evoke in vivo anti-tumor responses in ATLL patients

Sugiyama et al., PNAS, 2013.

0.24%

0.46% 0.16%

0.83%

0.51% 0.96%

33.3%

2.9% 10.7%

2.97%0.14%

Pre-treatment Post-treatment

IFN-γ

TN

F-α

B*3

501

/ NY

-ES

O-1

94

-102

Tet

ram

er

CD8

Control

0.01%

NY-ESO-1 staining

Small intestine (positive)

Negative control

Differential control of FoxP3+ subsetsfor immune enhancement

(e.g., to evoke tumor and microbial immunity)

• Reduction of Fr.II by specific mAbs or chemicals• Blockade of Treg differentiation from Fr.I to Fr.II• Blockade of cell differentiation from Fr.III to Fr.II (?)

Naïve TregsFr.I

Fr.IIFr.IIICD

45R

A

FoxP3

Effector Tregs

Thymus

BiologicalsSmall molecules

?


Differential control of FoxP3+ subsetsfor immune suppression

(e.g., to control autoimmunity, allergy, etc.)

• Antigen-specific expansion of Fr.I• Facilitation of Treg differentiation from Fr.I to Fr.II• Induction of cell differentiation from Fr.III to Fr.II (?)

?

Naïve TregsFr.I

Fr.IIFr.IIICD

45R

A

FoxP3

Effector Tregs

Thymus

BiologicalsSmall molecules

?

CTLA-4

CCR4

Control of immune responses by Foxp3+CD25+CD4+ Tregs

CD25

Autoimmune Disease

Tumor Immunity

Microbial infection

Allergy

Organ transplantation

Feto-maternal tolerance

Bone MarrowThymus

Foxp3

APC

TeffTreg

APC

Teff Teff

Production of Tregs Deletion

control of immune response by regulatory t cells

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