control of immune response by regulatory t cells
TRANSCRIPT
© 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
© 2015 Osaka University. All rights reserved.
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
© 2015 Osaka University. All rights reserved.
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
© 2015 Osaka University. All rights reserved.
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
© 2015 Osaka University. All rights reserved.
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
© 2015 Osaka University. All rights reserved.
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
© 2015 Osaka University. All rights reserved.
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
© 2015 Osaka University. All rights reserved.
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
© 2015 Osaka University. All rights reserved.
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)
© 2015 Osaka University. All rights reserved.
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
© 2015 Osaka University. All rights reserved.
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
© 2015 Osaka University. All rights reserved.
• 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
© 2015 Osaka University. All rights reserved.
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
© 2015 Osaka University. All rights reserved.
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
© 2015 Osaka University. All rights reserved.
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
© 2015 Osaka University. All rights reserved.
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
© 2015 Osaka University. All rights reserved.
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
© 2015 Osaka University. All rights reserved.
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
© 2015 Osaka University. All rights reserved.
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
© 2015 Osaka University. All rights reserved.
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
© 2015 Osaka University. All rights reserved.
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
Setoguchi et al. J. Exp. Med. 2005
BALB/cDay
© 2015 Osaka University. All rights reserved.
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
Setoguchi et al. J. Exp. Med. 2005
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)
Setoguchi et al. J. Exp. Med. 2005
NKTCD8+
NK
© 2015 Osaka University. All rights reserved.
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)
© 2015 Osaka University. All rights reserved.
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
© 2015 Osaka University. All rights reserved.
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
© 2015 Osaka University. All rights reserved.
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
© 2015 Osaka University. All rights reserved.
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
© 2015 Osaka University. All rights reserved.
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
© 2015 Osaka University. All rights reserved.
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
© 2015 Osaka University. All rights reserved.
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
Ohkura et al., Immunity 2012
© 2015 Osaka University. All rights reserved.
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
© 2015 Osaka University. All rights reserved.
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
© 2015 Osaka University. All rights reserved.
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 (%)
● ● ● ● ● ● ● ● ● ● ● ●
● ● ● ● ● ● ● ● ● ● ● ●
● ● ● ● ● ● ● ● ● ● ● ●
● ● ● ○ ● ● ● ● ● ● ● ●
● ● ● ● ● ● ● ● ○ ● ● ●
● ○ ● ● ● ● ● ● ● ● ● ●
● ● ● ● ● ● ● ● ● ● ○ ●
● ● ● ● ○ ● ● ● ● ● ● ●
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
© 2015 Osaka University. All rights reserved.
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
© 2015 Osaka University. All rights reserved.
Treg-type epigenetic change begins in the thymus and is progressively established towards the periphery
Thymus
CD25F
oxp
3
Spleen
CD25
Fo
xp3
Ohkura et al., Immunity 2012
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
© 2015 Osaka University. All rights reserved.
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
Ohkura et al., Immunity 2012
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
© 2015 Osaka University. All rights reserved.
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
© 2015 Osaka University. All rights reserved.
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+
++
© 2015 Osaka University. All rights reserved.
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
© 2015 Osaka University. All rights reserved.
FoxP3
CD
45R
AC
TL
A-4
Fr. II (Foxp3hiCD45RA-) Tregs are highly proliferativeand express CTLA-4
Effector Tregs
Miyara et al. Immunity 2009
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+
© 2015 Osaka University. All rights reserved.
Healthy donor
CD
45R
A
FOXP3
FoxP3+ subsets in normal and disease states
nTreg
eTregNon
TregCD
45R
AFoxP3
Miyara et al. Immunity 2009
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
© 2015 Osaka University. All rights reserved.
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
© 2015 Osaka University. All rights reserved.
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
© 2015 Osaka University. All rights reserved.
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
?
© 2015 Osaka University. All rights reserved.
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