Control of immune response by regulatory T cells

1. © 2015 Osaka University. All rights reserved.
Control of immune responses
by regulatory T cells
Shimon Sakaguchi
WPI Immunology Frontier Research Center
Osaka University
Immunological Self-Tolerance
Autoimmune Disease
Tumor Immunity
Chronic microbial infection
Allergy
Organ transplantation
Feto-maternal tolerance
Immune
system
Self Non-self

2. © 2015 Osaka University. All rights reserved.
Possible Mechanisms of
Immunological Self-Tolerance
Deletion
(Apoptosis)
Self
1
Inactivation
(Anergy)
Self
2
Suppression Self
3
Treg
Autoimmune diseases in humans
Autoimmune diseases
Organ-specific Non-organ-specific

3. © 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
brain
Multiple sclerosis(?)
thyroid
Hashimoto’s thyroiditis
primary myxoedema
thyrotoxicosis
stomach
pernicious anaemia
adrenal
Addison’s disease
pancreas
Insulin-dependent
Diabetes mellitus (TYPE I)
muscle
dermatomyositis
kidney
SLE
skin
scleroderma
SLE
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

4. © 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

5. © 2015 Osaka University. All rights reserved.
Induction of autoimmune diseases by manipulating
the T cell immune system
Mice
Tx
Day 3 after birth
Autoimmune gastritis,
oophoritis, thyroiditis, etc.
Rats
6 week
Tx
X-irradiation
Autoimmune thyroiditis,
Type 1 diabetes
Induction of autoimmune diseases by manipulating
the T cell immune system
Mice
Tx
Day 3 after birth
Autoimmune gastritis,
oophoritis, thyroiditis, etc.
Rats
6 week
Tx
X-irradiation
Autoimmune thyroiditis,
Type 1 diabetes

6. © 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 0/200/200/20
Reference: Sakaguchi et al. J. Exp. Med. 1982
Asano et al., J. Exp. Med. 1996
Gastritis
MICE
Autoimmune diseases
Tx：Thymectomy
Findings
BALB/c A
C57
BLACK
Date of
thymectomy
1 2
Genetic background
of mice

7. © 2015 Osaka University. All rights reserved.
DAY 0 3
Tx
17/20
Tx
Tx
Tx
Tx
Tx
0
10
17
24
31
5x106 T cells
0/5
0/10
0/10
2/10
8/10
Incidence of
Autoimmune
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

8. © 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)
CD4

9. © 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)

10. © 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 number
of 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. 0
CD8+CD25-
(5x107)
Ontogeny of CD25+CD4+ T cells(1)
Figure: Asano et al. J. Exp. Med. 1996

11. © 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 immunoregulation
as a cause of immunological diseases
Biological (e.g., MTLV)
Chemical (e.g., Cyclosporin A)
Physical (e.g., ionizing radiation)
Non-genetical insults
Genetic anomalies
Foxp3
CTLA-4
IL-2, CD25, CD122
Runx1/AML1
CD40
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 Marrow
Thymus
Periphery
CD25+
CD4+
Genetic anomalies
Environmental insults
MF
B
CD25-
CD4+
CD4+ Teff
Examples
CTL

12. © 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

13. © 2015 Osaka University. All rights reserved.
Induction of autoimmune disease in normal animals
by depleting Foxp3+CD25+ Treg cells (1)
Thymocytes
or
Splenic T cells
Depletion of
CD25+ Tregs
Autoimmune diseases
Inflammatory bowel disease
etc.
Cell transfer
Normal
mouse
develop
T cell-deficient
mouse
Induction of autoimmune disease in normal animals
by depleting Foxp3+CD25+ Treg cells (2)
Thymus Spleen
CD25
Foxp3
Sakaguchi S, et al. J. Exp. Med. 1985
Sakaguchi S, et al. J. Immunol. 1995
Itoh M, et al., J. Immunol. 1999

14. © 2015 Osaka University. All rights reserved.
Inhibition of autoimmune disease and IBD
by Foxp3-transduced naïve T cells
Foxp3/MIGR1 MIGR1
CD4
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-
CD45RBhi
alone
gastritisscore
0
1
2
3
colitisscore
0
1
2
3
4
5

15. © 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
Whole
T cells
CD25
T cells
Induction of autoimmune disease
and 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

16. © 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+
cells
Foxp3+
cells
Normal Treg Treg deficiency/dysfunction

17. © 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
Graft
survival
BALB/c
BALB/c nude

18. © 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
Graftsurvival(%)
T cells alone
(1:1)
T cells & CD25+
T cells
(1:3)
120
n=15
n=13
n=29
Nishimura, et al., Int. Immunol. 2004
T cells & CD25+
T cells
Self antigen
Tumor antigen
Allo antigen
Bone Marrow
Thymus
Foxp3
Foxp3
Treg
Foxp3
Treg
Th1
Th2
Th17
etc.,
Naïve
T cell
CTLA-4+
CD25+TGF-
CTLA-4+
CD25+
Summary: Section 1

19. © 2015 Osaka University. All rights reserved.
Control of cytokines and Treg-associated
molecules by Foxp3
CD25 GITR CTLA-4
IL-2R
?
Foxp3
IL-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


20. © 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 receptor
already in the thymus
Figure: Setoguchi et al. J. Exp. Med. 2005
Thymus Spleen
IL-2 neutralization by specific mAb reduces Tregs
in the thymus and the periphery
CD25
CD4
1.7％ 0.3％
2.6％ 0.6％
Saline Anti-IL-2
Setoguchi et al. J. Exp. Med. 2005
Thymus
Spleen

21. © 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-2
1mg i.p.
Histological
Serological
analysis
Setoguchi et al. J. Exp. Med. 2005
BALB/c
Day

22. © 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
CD25
IL-2IL-2R
0
4
8
12
16
25hi 25lo 25-
IL-2(pg/ml)
Setoguchi et al. J. Exp. Med. 2005
NKT
CD8+
NK

23. © 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 Teff
B7
APCAPC
(A)
CTLA-4 CD28
APC
B7
Teff
B7
(A) (B)

24. © 2015 Osaka University. All rights reserved.
Treg-specific CTLA-4 conditional KO mice
Targeted Foxp3 allele
11.2 0.4
44.42.4
CTLA-4 Cond KOWT littermate
Foxp3
CTLA-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 IoxP
FRT
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

25. © 2015 Osaka University. All rights reserved.
Autoimmune gastritis in CTLA-4 CKO mice
Anti-parietalcellAb(unit)
WT FIC CKO
FIC/Y
CTLA-4 CKO
Autoimmune myocarditis in CTLA-4 CKO mice

26. © 2015 Osaka University. All rights reserved.
Hyperproduction of IgE in CTLA-4 CKO miceIgE
IgG
Induction of effective tumor immunity
by Treg-specific CTLA-4 deficiency
Tumor cells: RLm1 leukemia
K. Wing et al., Science 2008

27. © 2015 Osaka University. All rights reserved.
Cancer regression and autoimmunity induced by
cytotoxic 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

28. © 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 cycles
received
Response
(mos.)
Toxicity (grade III / IV)
Tregs down-regulate CD86 on CD86-transfected
L cells, whereas CTLA-4 CKO Tregs do not
CD80/CD86
CTLA-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

29. © 2015 Osaka University. All rights reserved.
APC
Foxp3, CTLA-4, and IL-2 in Treg-mediated
suppression
IL-2
IL-2R
Treg
Suppression
Foxp3
CTLA-4
IL-2IL-2
TCR
CD80/86
MHC
IL-10
etc.
Responder T
Ono et al., Nature 2007
Onishi et al., PNAS 2008
Wing et al., Science 2008
Kitoh et al., Immunity 2009
Miyara et al., Immunity 2009
Ohkura et al., Immunity 2012
Yamaguchi et al., PNAS 2013
How are Foxp3+ Treg function and lineage stability
maintained in the immune system?
Bone Marrow
Thymus
Foxp3
Foxp3
Treg
Foxp3
Treg
Th1
Th2
Th17
etc.,
Naïve
T cell
CTLA-4+
CD25+
TGF-
CTLA-4+
CD25+
Self antigen
Tumor antigen
Allo antigen

30. © 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
Survivalrate(%)
100
50
0 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

31. © 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
Dnmt1
TET
TFs
Polycomb
HDAC
HAT
HMT
SWI/SNF
BRG1
BAF
EZH2
JHDM1
LSD1
etc.
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

32. © 2015 Osaka University. All rights reserved.
Treg-specific DNA demethylated sites are present
only in limited regions of the genome
Methylated DNA immunoprecipitation (MeDIP) sequencing
DNA sequencing and
Annotation 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 present
only 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

33. © 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 Treg
Naïve T
Foxp3
Treg
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

34. © 2015 Osaka University. All rights reserved.
Foxp3 Cd25
Eos Ctla4
Tregs possess specific epigenetic patterns
Tconv
nTreg
Homology
Bisul
Tconv
nTreg
MeDIP
Tconv
nTreg
Homology
Bisul
Tconv
nTreg
MeDIP
Tconv
nTreg
Homology
Bisul
Tconv
nTreg
MeDIP
Tconv
nTreg
Homology
Bisul
Tconv
nTreg
MeDIP
Stim
Specificity and stability of Treg-type epigenetic changes
The Treg-type epigenetic pattern is highly specific for natural Tregs
Stim
Cont
Stim
Cont
Cont
Stim
Cont
24h
72h
CpG demethylation
100%0%
Cont
iTreg
Cont
iTregTconv
nTreg
TGF-
Retinoic
acid
Th1
Th2
Th17
Central memory
Effector memory
TCR stimulation induced Treg Other T cell subsets
IL-2 expanded
Tconv
Control
Tconv
nTreg
transduced
Foxp3
Vector
Tconv
transduced
24h
72h

35. © 2015 Osaka University. All rights reserved.
Treg-type epigenetic change begins in the thymus and
is progressively established towards the periphery
Thymus
CD25
Foxp3
Spleen
CD25
Foxp3
Ohkura et al., Immunity 2012
0
50
100
Thymic
DP
Thymic
Foxp3+
Splenic
Foxp3+
Foxp3
CD25
GITR
CTLA-4
Eos
CpGdemethylation(%)
Thymic
CD4SP
Foxp3-
Foxp3+
Splenic
CD4+ 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
Yes
CD25+
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

36. © 2015 Osaka University. All rights reserved.
Foxp3
CD25
GITR
CTLA-4
Eos
Thymus
Spleen
Thymus
GFP-
GFP+
Spleen
GFP-
GFP+
GFP-
GFP+
GFP-
GFP+
(GFP-marked Treg)
(GFP-marked
Foxp3-null Treg)
DP
Thymus DP
Thymus DP
CpGdemethylation(%)
0
50
100
0
50
100
Thymus
GFP+
Spleen
GFP+
DEREG♂
DEREG/Scurfy♂
Treg-type epigenetic change is established without Foxp3
CpG demethylation
100%0%
Foxp3-null TregsnTregs
Ohkura et al., Immunity 2012
DP
Thymus
GFP+
Spleen
GFP+
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

37. © 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+
Foxp3
Treg epigenome+
CD25+
Natural Treg
In vivo iTreg
Potential
Treg
Stable Treg
Foxp3+
TconvIn vitro iTreg
Unstable Treg-like
Tconv
Activated
Tconv

38. © 2015 Osaka University. All rights reserved.
FoxP3+ Treg subsets in humans
Correlation between CD25 and FOXP3 expression
in CD25+FoxP3+CD4+ T cells in human PBMCs
FoxP3
CD45RA
II
I
III
CD25
FoxP3
CD25-
I
III II
CD25+
CD25++
CD25+++

39. © 2015 Osaka University. All rights reserved.
Fr. I (FoxP3loCD45RAhi) and Fr. II (FoxP3hiCD45Rlo) cells are suppressive
while Fr. III (FoxP3loCD45RAlo) cells are not
CFSE
CD45RA
CD45RA
CD25
CD4+ T cells
I
II
Foxp3
III
I
II
III
Responder alone Fr. l + Responder Fr. ll + Responder Fr. lll + Responder
I IIIII
FOXP3
CD45RA
Miyara et al. Immunity 2009
Fr. I and Fr. II Tregs are cytokine hypo-producing,
while Fr. III cells are not
I
II
III

40. © 2015 Osaka University. All rights reserved.
FoxP3
CD45RACTLA-4
Fr. II (Foxp3hiCD45RA-) Tregs are highly proliferative
and express CTLA-4
Effector Tregs
Miyara et al. Immunity 2009
I
II
III
FoxP3
CD45RAKi-67
Naïve Tregs
Differentiation and interactions
among FoxP3+ subsets
Proliferative
Fr.I
Fr.IIFr.III
CD45RA
FoxP3
Epigenome+
Die by apoptosis
Naïve Tregs
Effector Tregs
Thymus
CTLA-4+
CCR4+

41. © 2015 Osaka University. All rights reserved.
Healthy
donor
CD45RA
FOXP3
FoxP3+ subsets in normal and disease states
nTreg
eTreg
Non
Treg
CD45RA
FoxP3
Miyara et al. Immunity 2009
CD45RA
FOXP3
Healthy
donor
Sarcoidosis
donor
Active SLE
patient
PBMC
0.9%
3.6%
CD4 gated
3.25%43.6%
CD45RA
Foxp3
CD4
Foxp3
Predominant infiltration of effector Tregs into tumor tissues
Treg fractions
%ofCD4+Tcells
Naïve Effector Naïve Effector
TILPBMC
Tregs:
TIL
14.0%16.8%
Foxp3
0.8%
37.6%
CD4 gatedMelanoma
Sugiyama et al., PNAS 2013
CD4
Foxp3
CD45RA
Foxp3

42. © 2015 Osaka University. All rights reserved.
Treg-targeting cancer immunotherapy
without 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
eTreg
Non
Treg
CD45RA
FoxP3
eTreg
CD45RA
FoxP3
Depletion of tumor-infiltrating FoxP3+ Tregs
as an immunotherapy of cancer
Nishikawa and Sakaguchi, Int. J. Cancer 2010
CCR4
CCL22
Treg migration
Self-Ag / Tumor Ag
TGF-β
Tolerogenic
Dendritic cell
Treg expansion
Suppression
Tumor infiltrating
macrophage
Tumor cell
NK
NKT
CTL
Th

43. © 2015 Osaka University. All rights reserved.
CCR4(MFI)
PBMC
CD4+ T-cell subsets
85
I
II
III
IV
Effector Tregs express CCR4,
whereas naïve Tregs do not
I
II
III
IV
I II III IV
Sugiyama et al., PNAS 2013
CD45RA
Foxp3
Cellnumber
CCR4
One stone for two birds:
Anti-CCR4 mAb depletes both ATLL cells and effector Tregs
Pre-treatment 2nd round injection
CD4
CD8CD45RA
CD8
Foxp3
CD45RA
ATLL patient
6.2%
CD4
80.7%
1.1%
1.4%
98.5%
80.0%
Foxp3
Anti-CCR4 mAb
treatment

44. © 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
TNF-α
B*3501/NY-ESO-1
94-102Tetramer
CD8
Control
0.01%
NY-ESO-1 staining
Small intestine (positive)
Negative control
Differential control of FoxP3+ subsets
for 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.III
CD45RA
FoxP3
Effector Tregs
Thymus
Biologicals
Small molecules
?

45. © 2015 Osaka University. All rights reserved.
Differential control of FoxP3+ subsets
for 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.III
CD45RA
FoxP3
Effector Tregs
Thymus
Biologicals
Small 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 Marrow
Thymus
Foxp3
APC
Teff
Treg
APC
Teff Teff
Production
of Tregs
Deletion