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IL-1
Th17
(The mechanism of Th17 cell development induced
by excess IL-1 signaling)
23
12
(Il1rn-/-)
IL-1
IL-17
Th17
TGF-β
IL-6
Il1rn-/-
IL-6
Il1rn-/-Il6-/IL-6
naïve CD4+T
Il6-/-
naïve T
Th17
TCR
TGF-β IL-1
TGF-β+IL-1+IL-21
Th17
IL-21
TGF+IL-21
TGF-β
IL-1
Th17
Th17
Th17
Th17
IL-1
IL-1
TGF-β
TGF-β
Foxp3
Th17
IL-6
IL-1
IL-1
Nfkbiz
IL-6
Batf
Th17
Th17
2
IL-21
Abstract
IL-1 receptor antagonist-deficient (Il1rn-/-) mice spontaneously develop autoimmune
arthritis and that IL-17 is crucial for the onset of the disease. Although many studies have
shown that the Th17 cell differentiation is dependent on TGF-β and IL-6, we found that IL-6
deficiency does not affect the development of arthritis in Il1rn-/- mice at all. In vivo, Th17
cells developed normally in Il1rn-/-Il6-/- mice. To elucidate the mechanisms of IL-6
independent Th17 cell differentiation, we purified naïve CD4+ T cells from Il6-/- mice and
cultured with TCR stimulation, TGF-β, IL-1 and/or IL-21. We found that naïve CD4+ cells
efficiently differentiated into Th17 cells when cultured with TGF-β, IL-21 and IL-1 than the
combination of TGF-β and IL-21, however, these cells did not differentiate with TGF-β and
IL-1. In addition, IL-1 maintained Th17 lineage synergistically with TGF-β. Interestingly, we
found that IL-1 inhibited TGF-β-induced Foxp3 expression to promote Th17 cell
differentiation in a proliferation, IL-6 and IL-21 independent manner. Furthermore, IL-1
augments Th17 transcription factors expression such as Nfkbiz and Batf. These results
indicate that IL-1 plays an important role in the Th17 cell differentiation in an IL-6
independent manner.
3
1-1
2-3
1-2
5-8
1-3
9-17
1-4
18-27
1-5
28-37
1-6
38
39-41
42-51
1-7
52-76
4
(McInnes and Schett, 2007)
(McInnes and Schett, 2007) IL-6
IL-6
CIA
HTLV-1 transgenic (Tg)
SKG
(Figure 0-1a)(Ishihara et al., 2004; Iwakura et al., 2008)
IL-6R
(Figure 0-1b) (Jones et al.,
2010; Nishimoto et al., 2009)
IL-1
(Il1rn-/- mouse)
IL-1
IL-1Ra
(Horai et al., 2000)( Figure 0-2a b)
IL-1
CIA HTLV-I Tg
Figure 0-1a
SKG
K/BxN
(Iwakura et al., 2008)
IL-1
IL-1Ra
2002; Furst, 2004)
Th17
IL-1
(Cohen et al.,
IL-1
IL-17
et al., 2009)(Figure 0-3) Th17
IL-6
CD4+
CIA
5
T
(Korn
EAE (experimental autoimmune
encephalomyelitis)
(Iwakura et al., 2011)
IL-17
Th17
(Genovese et al., 2010; Hueber et al., 2010)
naïve T
TGF β IL-6
2007) TGF β
Foxp3
IL-21
(Figure 0-3)(Zhou et al.,
Th17
retinoic
acid receptor-related orphan receptor (ROR)-γt
Zhou et al., 2008) IL-6 IL-21 IL-23
IL-17
STAT3
RORγt
RORγt
(Ichiyama et al., 2008;
(Figure 0-4) STAT3
IL-17
IRF-4
IL-17
(Hirahara et al., 2010) STAT3
Nfkbiz Hif1a Ahr
Th17
(Figure 0-4) (Dang et al., 2011; Kimura et al., 2008;
Okamoto et al., 2010) STAT3
CD4+T
Th17
IL-21
STAT3
IL-23R
Th17
(Durant et al., 2010; Zhou and Littman, 2009)
IL-23
Th17
IL-17A IL-17F IL-22
IL-1β
IL-17
IL-1
PKCθ
T
IL-17
al., 2009; Sutton et al., 2006; Yang et al., 2008a)
(Chung et al., 2009)
IL-1
(Kryczek et al., 2007) IL-6
IL-1R1
mTOR
PI3K NF-κB
IL-2
IL-6
IRF-4
Th17
SIGIRR
Th17
6
(Korn et
IL-1R
(Gulen et al., 2010)
Foxp3
IL-1
Th17
T(Treg)
IRF-4
Blimp-1
IL-10
CTLA-4
ICOS GITR
Treg
(Cretney et al., 2011; de Lafaille and Lafaille,
2009)
Foxp3+T Induced Foxp3+ Treg:iTreg
(de Lafaille and
Lafaille, 2009) TCR
IL-2 TGF-β
Th17
Foxp3
TGF β
TGF-β
Foxp3
IL-6
Foxp3
RORγt
IL-21
Treg
RORγt
RORγt
IL-23
Th17
Foxp3
Th17
RORγt
(Ahern et al., 2010; Ichiyama et
al., 2008; Zhou et al., 2008)(Figure 0-5)
Th17
Treg
(Bettelli et al., 2006)
(Figure 0-2 and Figure 0-3)
Foxp3
RORγt
IL-17
Il1rn-/-
IL-6
Il1rn-/-Il6-/-
IL-6
/
Il1rn-/-Il6-/-
Il1rn-/-
IL-1
IL-6
IL-1
IL-1
IL-6
IL-6
Th17
Th17
Th17
Th17
IL-1
TGF-β
7
Foxp3
Th17
Th17
IL-6
IL-1
8
Il1rn-/- (Il1rntm1Yiw)
Il1ab-/- (Il1atm1Yiw/Il1btm1Yiw)
(Horai et al., 1998; Horai et al., 2000) Il1rn-/-Il6-/(Il6tm1Kopf M. Kopf
Il1rn-/-
Il6-/-
)(Kopf et al., 1994)
Il1r1-/-
Immunex Corporation
Rag2-/-
(Glaccum et al., 1997)
(Shinkai et al., 1992)
8
BALB/cA (
Il1rn-/-
)
Il1rn-/-Il6-/MyD88-/- (MyD88tm1Aki)
C57BL/6J
(Adachi et al., 1998)
C57BL/6J (Nihon SLC; Shizuoka, Japan)
BALB/cA (CLEA Japan, Inc.)
SPF (specific pathogen-free
conditions)
10% FBS RPMI1640:
50 U/ml penicillin
invitrogen
)
50 mM 2-mercaptoethanol (GIBCO;
50 µg/ml streptomycin
10% FBS (w/v)
RPMI1640 (nakarai tesque; Kyoto, Japan)
9
10% FBS RPMI1640
FBS
Hemolysis Buffer: 1.4 M NH4Cl
T
Th17
0.45 mm
Tris-HCl (pH7.2)
170 mM
17mM Tris-HCl (pH7.2)
mM NH4Cl
140
Hemolysis Buffer
MACS Buffer:
(
)
g
1L
5
0.45 µm
MACS Buffer
FACS Buffer: HBSS (Hanks' Balanced Salt Solutions) (Nissui, Tokyo)
FBS 0.1% NaN3(Nakarai tesque, Tokyo)
2%
FACS Buffer
anti-CD3
anti-CD3(clone 145-2C11;
)
48 well/ Flat Bottom (Asahi glass)
PBS
anti-IL-4
37
PBS
4
2
anti-IFN-γ
anti-IL-4
(clone 11B11)
anti-IFN-γ
11B11
(clone R4-6A2)
CeLLine
BD Biosciences
Freund’s Incomplete Adjuvant
0.5 ml
BALB/cA-nu/nu
10
PIERCE Biotechnology
R4-6A2
1
106 cells/
mouse
1~2
1
37
2000rpm
Cleanascite BIOTECH SUPPORT GROUP
0.45 µm
11B11 R4-6A2
HiTrap Protein G
GE Healthcare
Slide-A-Lyzer 10,000 MWCO Dialysis Cassette PIERCE; Thermo Fisher
Scientific
PBS
BCA
Protein Assay Reagent
PIERCE; Thermo Fisher Scientific
CD4+
Il6-/-
10% FBS RPMI1640
cm
3.5
(FALCON; BECTON DICKINSON)
(on ice)
108 µm
1300 rpm
MACS
Buffer
MACS Buffer
Micro beads
CD4
(Miltenyi Biotec GmbH; Bergisch, German)
15
Auto MACS possel program (Miltenyi Biotec GmbH; Bergisch, German)
CD4+
anti-CD3
5
105 cells
(clone 145-2C11)
GLASS; Tokyo, Japan)
San Jose, CA, USA)
500 µl/48well
4 µg/ml
48well/ Flat Bottom (ASAHI
anti-CD28
(1 µg/ml) (clone 37.51; Biolegend;
10 µg/ml anti-IFN-γ (clone R4-6A2) 10 µg/ml anti-IL-4 (clone
X-VIVOTM 20 (Lonza; Basel, Switzerland)
11B11)
CD4+
5
37
/5% CO2
10 ng/ml rmIL-1α (PeproTech; Rocky Hill,
NJ, USA) 10 ng/ml rmIL-1β (PeproTech)
11
naïve T
T
Naive CD4+ T
Il1rn-/-
BALB/cA BALB/cA
Il6-/-
Il1rn-/-Il6-/-
Il1ab-/- C57BL/6J
/
MyD88+/-
MyD88-/-
1300 rpm
Hemolysis Buffer (1~2 ml)
5
10% FBS RPMI1640
MACS Buffer
anti-mouse B220
anti-mouse CD8α anti-mouse CD11b
Biosciences; San Jose, CA, USA)
BlueTM-
anti-mouse DX5 anti-mouse Ter119
PE-Cy7
anti-mouse CD4 (Biolegend)
anti-mouse CD62L (Biolegend) PE
San Diego, CA, USA)
4
Micro beads
(BD
anti-mouse CD25
20
Pacific
(eBioscience,
MACS Buffer
anti-Biotin
4
15
deplete program
AutoMACS (Miltenyi Biotec GmbH)
selection
negative
FACS Aria(BD bioscience)
CD4+CD62L+CD25-
naïve T
FACS Aria
FACS
naïve T
X-VIVOTM 20 (Lonza)
naïve CD4+ T
4
µg/ml anti-CD3 (clone 145-2C11) 1 µg/ml anti-CD28 (clone 37.51; Biolegend)
96 well Flat Bottom(FALCON; BECTON DICKINSON
well
11B11)
(PeproTech)
)
10 µg/ml anti-IFN-γ (clone R4-6A2)
2x105 cells 250 µl/
10 µg/ml anti-IL-4 (clone
3 ng/ml CHO cell derived rhTGF-β1 (PeproTech), 40 ng/ml rmIL-6
10 ng/ml rmIL-1α (PeproTech)
12
10 ng/ml rmIL-1β (PeproTech)
100 ng/ml
rmIL-21 (PeproTech)
Laboratories
10 ng/ml rmIL-23(R&D systems)
)
50 ng/ml Rapamycin (LC
IL-21R Subunit/Fc Chimera
R&D systems (Minneapolis, MN, USA)
CFSE
T
(1x106 to 1x107 cells/ml)
5 µM CFSE (5- and
6-carboxyfluorescein diacetate succinimidyl ester; invitrogen; Carlsbad , CA, USA) / PBS
6
37
8x105
/ml
FACS
CantoIITM (BD Biosciences)
FlowJo software (TreeStar;
Ashland, OR, USA)
50 ng/ml PMA (Sigma) 500 ng/ml
ionomycin (Sigma) 2 µM monensin (Sigma) 37
5% CO2
anti-FcγRII/III receptor mAb (2.4G2)
5
4
10
2 µg/ml 7AAD (Sigma)
FACS Buffer
4% paraformaldehyde 200 µl
20
permeabilization buffer (0.1% saponin containing FACS
solution)
2
2
FACS Buffer
40
70 µm
13
/4
FACSCalibur™
FACSCantII
APC-
anti-CD4 (BD Bioscience)
PE-Cy7-
(eBiosciece) APC-Cy7PE-
anti-CD4 (Biolegend)
anti-B220 (Biolegend) FITC-
FITC-
anti-TCRβ (Biolegend)
anti-IL-1R1 (Biolegend)
Pacific Blue-
: FITC or PE or
anti-IFN-γ (Biolegend), FITC or APC or Pacific Blue-
(Biolegend), APC or PE(eBioscience)
anti-CD8a
anti-Foxp3 (BD Biosciences), APC-
Foxp3
anti-IL-17
RORγ(t)
Foxp3 staining Kit (eBioscience)
STAT3
Pacific Blue-
BD phosflow Technology
STAT3 (pY705)
(BD bioscience
)
PBS
1.5 cm
PBS
EDTA/10% FCS RPMI 20 ml
20
37
5 mM
50 ml
(
)
1~2
cm
5 mM EDTA/10% FCS RPMI 20 ml
EDTA
PBS
50 ml
20
10% FCS/2 mg/ml Collagenase/ 5 ml RPMI
37
Collagenase
Collagenase
15 ml
200 µm
1300 rpm 5
14
45%
GE healthcare
1:9
10% FBS RPMI1640
20
r.t
10xPBS
2200 rpm
10% FBS RPMI1640
2
lamina proprial lymphocytes; LPL
PMA/ionomycin
CD4+CD45RBhiCD25BALB/c
Il1r1-/-
BALB/c
Il17a-/CD4+CD45RBhiCD25-
naïve T
FACS Aria
2
2x106 cells/ml PBS
(BALB/c
200 µl
PBS
(4x105 cells/mouse) Rag2-/-
)
MCM
4% (w/v)
(Nissui; Tokyo, Japan)
2 ml
0.5 % EDTA
PBS
10 % FBS RPMI1640
Japana
4%
none treated 10
5x106 cells/ml
37
3
PBS
glass)
15
5% CO2
Asahi glass, Tokyo,
4
(Asahi
5x105 cells/ml
24 well-plate (Asahi glass)
10% FBS RPMI1640
1 ml
2
LPS 5 µg/ml Zymosan 50 µg/ml
2
MCM
LPS (Escherichia coli O55:B5)
cerevisiae )
T
Zymosan A (Saccharomyces
SIGMA (St Louis, MO, USA)
Real-Time RT-PCR
RNA
GenEluteTM Mammalian Total RNA Miniprep Kit (Sigma)
RNA
the High Capacity cDNA Reverse Transcription Kit
(Applied Biosystems, Inc.; CA, USA)
real-time RT PCRs
SYBER® Premix Ex TaqTM (TaKaRa; Shiga, Japan)
iCyclerTM System (Bio-Rad; Hercules,
CA, USA)
50
3:00
95
2:00
(95
0:15
60
1:00)x44
70
89.2
0.5
:
Gapdh 5’-TTCACCACCATGGAGAAGGC-3’ 5’-GGCATGGACTGTGGTCATGA-3’
Foxp3 5’-AGAAGCTGGGAGCTATGCAG-3’ 5’-TACTGGTGGCTACGATGCAG-3’;
Il17a 5’-CTCCAGAAGGCCCTCAGACTAC-3’ 5’-GGGTCTTCATTGCGGTGG-3’
Rorc 5’-AGCAGTGTAATGTGGCCTAC-3’ 5’-GCACTTCTGCATGTAGACTG-3’
Il1r1 5’-ACCTTCCCACAGCGGCTCCACATT-3’
5’-TTGTCAAGAAGCAGAGGTTTACAG-3’
Gata3 5’-CTTATCAAGCCCAAGCGAAG-3’ 5’-CATTAGCGTTCCTCCTCCAG-3’
Tbx21 5’-GGTGTCTGGGAAGCTGAGAG-3’ 5’-CCACATCCACAAACATCCTG-3’
16
Il21 5’-GCCAGATCGCCTCCTGATTA-3’ 5’-CATGCTCACAGTGCCCCTTT-3’
Il22 5’-TGACGACCAGAACATCCAGA-3’ 5’-AGCTTCTTCTCGCTCAGACG-3’
Batf 5’-CCAGAAGAGCCGACAGAGAC-3’ 5’-GAGCTGCGTTCTGTTTCTCC-3’
Nfkbiz 5’-CCTCCGATTTCTCCTCCACT-3’ 5’-GTTCTTCACGCGAACACCTT-3’
1
4
0;
1;
2;
t-
3;
P<0.05
17
Il1rn-/-
IL-6
Th17
Il1rn-/IL-1β IL-6
IL-17
TNF-α
Il1rn-/-
(Horai et al., 2004; Nakae et al., 2003)
Il1rn-/-Il6-/-
IL-6
Il1rn-/-Il6-/-
Il1rn-/-
(Figure 1a)
Il1rn-/-
IL-6
B220+
CD4
Il1rn-/-Il6-/-
CD8+
Il1rn-/-
(Figure 2) Il1rn-/-Il6-/CD4+
Th17
Th17
Il1rn-/-
Il1rn-/-
(Figure 1b 3)
Th17
IL-6
IL-1
IL-6
Il1rn-/-Il6-/IL-6
Th17
Th17
IL-1
Th17
Il6-/-
Th17
Il1rn-/-Il6-/-
CD4+
Il6-/-
IL-1
IL-1
IL-6
(Figure 4) (Kryczek et al., 2007)
18
CD4+IL-17+
in vitro
CD4+
IL-6
IL-21
TGF-β
Il1rn-/-Il6-/-
Th17
IL-6
Th17
IL-21
Il1rn-/- Il6-/- Il1rn-/-Il6-/IL-21 mRNA
Il1rn-/-
PCR
Il1rn-/-Il6-/-
Il6-/-
Il21
(Figure 5)
IL-1
IL-21
IL-1
IL-21
Th17
naïve CD4+ T
T
in vitro
naïve T
(CD4+CD62LhiCD25-
Th17
IL-1
CD4
/TGF-β
TGF-β+IL-6
/IL-1+TGF-β
TGF-β+IL-21
Th17
Th17
Th17
(Figure 6a c Figure 7)
Th17
IL-1
IL-17
Figure 6c
naïve CD4+ T
naïve CD4+
Th17
T
IL-1R1
FACS
IL-6
IL-1
IL-22
IL-21
IL-1
)
naïve T
PCR
IL-1R1
IL-21
IL-1R1
(Figure 6b c Figure 8)
Th17
IL-1
IL-6
IL-21
IL-1R1
naïve T
Th17
Th17
19
IL-1R
IL-1
Th17
in vitro
Th17
Th1
TGF-β
(Lee et al., 2009)
IL-1
IL-17
IFN-γ
IL-17A
IL-17F
Th17
Th17
(Chung et al., 2009)
Th17
IL-1
TGF-β+IL-21
round
naïve T
2nd
5
10
3rd round
(Lee et al., 2009) TGF-β
TGF-β
IL-1
(Figure 9)
IL-1
IL-1
TGF-β
Th17
IL-17
Th17
IL-21
IL-1
TGF-β
Th17
IL-17
IL-17
exogenous
Th17
IL-17
Th17
Th17
IL-1
Th17
TGF-β+IL-21
naïve T
Th17
IL-1
naïve T
IL-1
IL-1
IL-6
IL-6
IL-1
IL-17A
Th17
IL-6
IL-22
20
mRNA
Th17
T
(Figure 10a)
IL-1
IL-21
IL-1
Th17
Rorc
IL-1
IL-1
Foxp3
Nfkbiz
Batf
Foxp3
(Figure 10b)
Foxp3
IL-1
Foxp3
Th17
Th
IL-1
T
Tbx21
Th1 Th2 Th17 Treg
Figure 9
Gata3 Rorc
Il17a
TGF-β
Gata3
Rorc Foxp3
mRNA
2nd round
IL-1
Tbx21
IL-1
IL-1
Foxp3
(Figure 10c)
IL-1
Foxp3
FACS
Foxp3
(Figure 10d)
Foxp3
IL-1
IL-1
MyD88
naïve T
CD3
MyD88+/-
FACS
Th17
CD28
(TGF-β+IL-21)
TGF-β IL-1
2
MyD88
(Figure 10e)
IL-1
IL-1
MyD88
Th17
IL-17+
Foxp3
Foxp3
21
Foxp3
Foxp3
Foxp3
IL-1R1
(Figure 11)
Th17
4
FACS
T
MyD88-/-
IL-1
IL-1
T
(Sims and Smith, 2010)
IL-1
Foxp3-
Foxp3+
Foxp3
naïve T
Th17
IL-1
Foxp3
MFI
PE-
TGF-β/IL-1/IL-21
T
TGF-β/IL-21
Foxp3+
(Figure 12a b)
TGF-β
iTreg
96
Foxp3
Foxp3
CFSE
TGF-β+IL-21 TGF-β+IL-1+IL-21 TGF-β+IL-6
IL-1
Foxp3
IL-1
Foxp3
IL-1
Foxp3
IL-1
Foxp3
IL-6
(Figure 12c)
IL-21
et al., 2007)
IL-6
STAT3
IL-1
IL-21
Foxp3
Foxp3
(Huehn et al., 2009; Wei
IL-6
IL-6
naïve T
IL-1
(Figure 13a)
TGF-β+IL-21
IL-21R-Fc
Foxp3
IL-21
IL-6
Th17
Foxp3
IL-1
Foxp3-
IL-1
T
IL-21
22
IL-17
IL-1
Il6-/- naïve T
IL-21
IL-6
(Figure 13b)
IL-21
autocrine
IL-21
IL-1
Foxp3
IL-1
IL-21R-Fc (20 µg/ml)
(Figure 13c)
Foxp3
IL-21R-Fc
Foxp3
IL-21
STAT3(pY705)
IL-1
STAT3
IL-21R-Fc
STAT3
IL-21R-Fc
Foxp3
IL-1
IL-21
IL-21
IL-1
STAT3
Foxp3
(Figure 13d)
IL-1
Foxp3
IL-6
IL-21
IL-1
T
IL-6
IL-6
Foxp3
IL-1Ra IL-1
IL-1
Foxp3
(Horai et al., 2004; Li and He, 2006;
Il6-/-
Van Kooten et al., 1991)
CD4+T
T
Il1rn-/-Il6-/-
Th17
(TGF-β+IL-21)
anti-CD3/anti-CD28/IL-1
2
Foxp3
Foxp3
TGF-β
Foxp3
Il6-/-
TGF β
naïve T
Foxp3+
Th17
FACS
23
Il1ab-/IL-1
IL-6
IL-1
Il1ab-/-
IL-17
Il1rn-/-Il6-/-
Figure 13e
Foxp3
naïve
4
IL-1
Foxp3
IL-21
Il1ab-/-
Il1ab-/-
naïve T
T
IL-6
Figure 14
IL-1Ra
Th17
T
IL-1
IL-1
IL-6 IL-21
Foxp3
IL-6 IL-1
Foxp3
Th17
IL-1
IL-21
IL-21
Foxp3
IL-1
IL-21
IL-1
IL-21
/
IL-21
Il6-/-naïve T
TCR
IL-21R-Fc
IL-1
TGF-β/IL-21
2nd round
2nd round
24
mRNA
Real time PCR
Rorc
Il21
IL-1
IL-21
15
IL-1
Figure
IL-21
Foxp3 Batf Il17 Il22
IL-21
IL-1
Foxp3
IL-1
IL-21
IL-21
Nfkbiz
IL-1
Nfkbiz
IL-1
IL-21
IL-1R1
IL-21
IL-1
IL-21
IL-1R1
Th17
IL-1R1
24
IL-1
IL-1Ra
IL-21
Th17
Th17
Th17
IL-6
IL-6
Th17
(Ivanov et al., 2006)
Il1rn-/-
(Abdollahi-Roodsaz et al., 2008)
Il1rn-/-
Il1rn-/-Il6-/-
Th17
Il1rn-/Th1
Il6-/-
Il1rn-/-Il6-/-
Treg
Il6-/-
Th17
Th17
(Figure 16)
Il1rn-/-
Th17
Th17
Treg
IL-6
IL-1Ra
in vivo
In vivo
Il1rn-/-
Treg
IL-1
IL-1
Th17
Th17
Il1r1-/- CD4+CD45RBhiCD25CD4+CD45RBhiCD25-
Th17
iTreg
iTreg
Rag2-/Rag2-/-
naïve T
25
Th17
Foxp3+
iTreg
(Ahern et al., 2010)
の Il17a-/-
CD4+CD45RBhiCD25-
Il1r1-/-
CD4+CD45RBhiCD25-
(Figure
Il1r1-/-
17a)
(Figure 17b)
mesenteric lymph nodes
Th1
Th1 Th17 Treg
Treg
Th17
Figure 17c
Th17
T
IL-17
IL-1
IL-1
IL-6
Th17
LPS
zymosan
Il6-/-MCM
(MCM)
LPS
CD4+
Th17
Il1rn-/-
TLR4
(Abdollahi-Roodsaz et al., 2008)
zymosan
Th17
(LeibundGut-Landmann et al., 2007; Veldhoen et al., 2006)
CD3 CD28 IL-4 IFN-γ
MCM
Il6-/-
FACS
(Figure 18a)
CD4+
Th17
MCM
LPL
zymosan
Il1rn-/-Il6-/-
Th17
MCM
Il6-/-
Il6-/-
MCM
Th17
IL-1
IL 6
naïve T
26
TGF β
Th17
Il6-/-
MCM
naïve T
Th17
Il6-/-
(Figure 18b)
Th17
MCM
Th17
Foxp3
Th17
Th17
IL-6
27
HTLV-1 Tg
SKG
CIA
IL-6
human TNF-α Tg
(CAIA)
T
IL-6
(Iwakura et al., 2008)
IL-6
IL-17
IL-6
in vivo
in vitro
Il1rn-/-
Th17
IL-17
Il1rn-/-Il6-/-
T
Il1rn-/Il1rn-/-Il6-/-
(Horai et al., 2004; Nakajima et al., 2010)
Th17
Il1rn-/-
IL-6
IL-1
Foxp3
TGF-β
IL-1
Th17
Th17
Th17
Th17
Th17
IL-1
β
TGF
Th17
IL-6
1; IL-1R
IL-21
IL-1R1
naïve T
IL-1
Foxp3
naïve T
TGF β+IL-21
naïve T
Il1rn-/-
naïve T
IL-1R1
IL-21
Il1rn-/IL-1
IL-6
IL-21
IL-6
T
STAT3
28
IL-1Ra
IL-21
IL-1R1
IL-1R1
(Chung et al., 2009) IL-21
IL-21
IL-1R1
TGF β
STAT3
IL-1R1
IL-21
mRNA
CD4
FACS
IL-21
(data not shown)
CD4+
IL-21
CD4+
IL-21
NKT
CD4+Th17
CD4 Tfh
CD4+
(Monteleone et al., 2008)
IL-21
IL-21
IL-6
IL-12
IL-21
(Nakayamada et al., 2011; Schmitt et al., 2009)
IL-21
al., 2012)
(Puga et
IL-21
(Bubier et al.,
2009; Spolski and Leonard, 2010)
Th17
Th17
IL-1
IL-21
IL-1R1
IL-1
(Figure 6)
IL-17
IL-1R1
STAT3
IL-1R1
IL-1R1
Th17
IL-1
Nfkbiz
IL-1R1
29
Batf
IL-1R1
2; Foxp3
Th17
IL-6
STAT3
STAT3
IL-17
TGF β
Th17
RORγt
Th17
IL-21
IL-6
Foxp3
TGF β
et al., 2007)
IL-1
IL-6
IL-6
(Yang et al., 2008b)
(Korn
Th17
IL-1
IL-1
IL-1
T
RORα
Th17
IL-21
Foxp3
Foxp3
Th17
STAT3
IL-21
IL-6
Foxp3
IL-6
IL-21R-Fc
IL-21
IL-1
IL-21R-Fc
IL-21
IL-21
IL-21
IL-1
IL-21R-Fc
IL-21R-Fc
Fc
IL-6
IL-21
Foxp3
IL-1
Foxp3
IL-21
Foxp3
IL-1
Foxp3+
Foxp3
IL-1α/β
IL-6
Foxp3-
Foxp3
IL-1
Foxp3
Foxp3 EGFP
IL-1
Foxp3
30
IL-2
Th17
IL-1
(Huehn et al., 2009) IL-2
Th17
STAT5
Foxp3
IL-1
(Huehn et al., 2009)
IL-2
Foxp3
IL-1
Akt-mTOR
mTOR
(Delgoffe et al., 2011)
Smad2
IL-1
Smad7
Foxp3
TGF βR2
TGF β
(Bauge et al., 2007; Ishida et al., 2006)
IL-1
Foxp3
Rapamycin
mTOR
Foxp3+
Figure 19
IL-1
Foxp3
mTOR
MFI
IL-1
IL-1
Foxp3
IL-1
Foxp3
Foxp3
(Huehn et al., 2009)
IL-1
Nfkbiz
IL-1
Foxp3
Batf
Th17
Il17a
Il22
IL-6
IL-1
IL-21
Th17
(Chung et al., 2009; Guo et al., 2009)
Th17
IL-1
TGF-β
Th17
(Lee et al., 2009) ILStriteski et al.
31
TGF-β
Th17
IL-1
IL-23
Th17
(Stritesky et al., 2008)
IL-1
Th17
Th17
IL-1R1
Rag2-/-
naïve T
Th17
in vivo
Foxp3
(Figure 17)
IL-1
Th17
Foxp3+
Foxp3+
iTreg
Il1r1-/-
Foxp3+
T
IL-1
IL-6
IL-21
nTreg
Foxp3
IL-17
IL-1
IL-17
2008b)
Foxp3
(Chung et al., 2009; Yang et al.,
IL-1
Foxp3
Foxp3
IL-1
nTreg
naïve T
IL-1
IL-6
Foxp3+
iTreg
Th17
Foxp3+
Foxp3
IL-1R1
Foxp3+
Th17
IL-21
Th17
IL-1R1
Foxp3+
nTreg
Foxp3
(de Lafaille and Lafaille, 2009)
Foxp3
IL-1
32
Foxp3
T
(Zhou et al., 2009)
3; Il1rn-/-
IL-6
TNF-α
(Tak and Kalden, 2011)
al., 2009)
IL-1
IL-6R
(Jones et al., 2010; Nishimoto et
(Cohen et al., 2002; Furst, 2004)
IL-17
(Genovese et al., 2010; Hueber et al., 2010)
anti-IL-6R
anti-IL-6R
30%
Il1rn-/-
(Jones et al., 2010)
IL-6
Il1rn-/-
IL-6
IL-17
(Nakajima et al., 2010)
psoriatic arthritis
(Ramonda et al., 2011)
(Ramonda et al., 2011) Il1rn-/(Alexander et al., 2012)
IL-6
IL-17
IL-1
33
Il1rn-/TNF-α
4;
4-1; Il1rn-/Il1rn-/-
Il1rn-/Il1rn-/-
B
B
B
IL-21
anti-CD20
B
B
Il1rn-/-
IL-21
Th17
IL-21
Il1rn-/-
Il1rn-/-
Autoimmune disease
Auto inflammatory disease
(Dinarello, 2009) Human
IL-1Ra
Auto inflammatory disease
(Aksentijevich et al., 2009; Reddy et al., 2009) Auto inflammatory disease
TCR
Il1rn-/)/Rag2-/-
TCR Tg
Il1rn-/-Rag2-/-DO11.10 Tg
IL-6
MHC ClassII
F759
TCR
(Murakami et
al., 2011)
IL-17
DO11.10 (OVA
γδT
Il1rn-/34
Il1rn-/-
(unpublished observation)
T
4-2; IL-1
Th17
IL-1R1
IL-23R
Th17
IL-23R
(McGeachy et al., 2009)
IL-23
IL-7R
et al., 2010)
(Liu
Th17
γδ+T
IL-1R1
CD4+
unpublished observation
IL-23R
naïve T
IL-23
IL-23
IL-1R
IL-1R1
IL-1
IL-1R1
IL-1
IL-1
IL-1
Th17
Akt-mTOR
MAPK
NF-κB
IL-17
NF-kB
IL-17
c-Rel
(Ruan et al., 2011)
c-Rel
Rel-A
IL-17
Rel-A
35
IL-17
αβ+T
IL-17
(Powolny-Budnicka et al., 2011)
4-3; IL-6
IL-1
Th17
IL-6
Th17
al., 2011)
IL-1
IL-6
IL-1
IL-21
(Hu et
Th17
IL-6
IL-1
Th17
(Figure 18b)
IL-6
Th17
IL-6
Th17
(Manel et al., 2008)
Th17
Th17
Il1rn-/-
IL-23
IL-23
(Cho et al., 2006)
IL-6
Th17
IL-23
Th17
4 4; STAT3
STAT3
IL-6/IL-21/IL-23
Th17
STAT3
Th17
STAT3
IL-21
IL-6
Th17
Th17
(figure 6)
Th17
STAT
36
IL-21
2005)
STAT5
STAT3
STAT1 5
STAT3
(Yang et al., 2011) L-6
(Leonard and Spolski,
STAT3
IL-21
Th17
Th17
LIF(Leukemia inhibitory factor)
IL-6
IL-27
gp130
STAT3
(Batten et al., 2006; Cao et al., 2011; Stumhofer et al., 2006)
LIF
STAT3
ERK
IL-27
IL-27
IL-1
IL-1
SOCS3
STAT1
SOCS3
STAT1
STAT3
Th17
STAT3
Th17
IL-6
IL-1
Foxp3
IL-1
Th17
(Figure 20)
37
(Il1rn-/-)
IL-1
IL-1
Th17
Il1rn-/-
IL-6
Il1rn-/-Il6-/-
Il1rn-/-
IL-6
Il1rn-/-Il6-/-
Th17
IL-6
Th17
IL-1
IL-6
naïve CD4+T
Th17
Il6-/-
TCR
naïve T
TGF-β IL-1+IL-21
Th17
TGF-β
TGF-β
Nfkbiz
Batf
IL-1
Th17
naïve T
IL-1R1
Th17
Th17
IL-1
Th17
Th17
IL-21
Rag2-/-
IL-1
IL-1
Th17
Foxp3
IL-6
IL-21
TGF+IL-21
TGF-β
IL-1
IL-1
TGF-β IL-1
Th17
38
naïve T
(Abbreviations)
7AAD; 7-Amino-ActinomycinD
APC; Allophycocyanin
Ahr; Aryl Hydrocarbon Receptor
Batf; Basic leucine zipper transcription factor
Blimp-1; B lymphocyte induced maturation protein 1
CFSE; Carboxyfluorescein succinimidyl ester
CIA; collagen-induced arthritis
CTLA-4; Cytotoxic T-Lymphocyte Antigen 4
EDTA; ethylenediaminetetraacetic acid
FACS; Fluorescence-activated cell sorting
FBS; Fetal Bovine Serum
FITC; fluorescein isothiocyanate
Foxp3; the forkhead box P3
GATA3; GATA-binding protein 3
GITR; Glucocorticoid-induced TNF-receptor
HTLV; Human T-lymphotropic Virus
Hif1a; Hypoxia-inducible factor 1, alpha subunit
ICOS; inducible T-cell co-stimulator
IL-1RI; type I IL-1R
IL-1Ra; IL-1 receptor antagonist
IL; Interleukin
IRF4; Interferon regulatory factor 4
39
LIF; Leukemia inhibitory factor
LPL; lamina proprial lymphocytes
LPS; Lipopolysaccharide
MAPK; mitogen-actiated protein kinase
MCM; Macrophage conditioned media
MFI; Mean Fluorescent Intensity
MHC; major histocompatibility complex
Myd88; myeloid differentiation factor 88
NF-kB; nuclear factor kappa-light-chain-enhancer of activated B cells
Nfkbiz; NF-kappa-B inhibitor zeta
OVA; ovalbumin
PBS; Phosphate buffered saline
PE; Phycoerythrin
PI3K; Phosphoinositide 3-kinase
PKCθ; Protein kinase C θ
PMA; Phorbol 12-Myristate 13-acetate
R; receptor
RA; rheumatoid arthritis
ROR-γt; retinoic acid receptor-related orphan receptor
Rag2; recombination activating gene 2
Rorc; RORgamma
SIGIRR; Single Ig IL-1-related receptor
SPF; specific pathogen-free conditions
40
STAT; Signal Transduction and Activator of Transcription
T-bet; T box expressed in T cells
TCR; T cell receptor
TGF; transforming growth factor
Tg; transgenic
iTreg; induced regulatory T cell
mTOR; mammalian target of Rapamycin
rpm; rotation per minute
41
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51
a
b
IL#6R
&
Human&IL#1Ra&
TNF#α
Figure 0-1
a,
IL-6-TNF
b, IL-6 IL-1 TNF
Human IL-1Ra (
IL-6R
)
TNF
&
a
IL-1α
IL-1R1
NFkB AP-1
IL-1R AcP
IL-1β
IL-1Ra
Cell membrane
Il1rn-/-
Il1rn+/+
c
100!
Incidence of RA (%)!
b
Il1rn-/-Il17+/+!
50!
Il1rn-/-Il17-/-!
0!
0!
5!
10!
15!
Age (week)!
Horai et al., J. Exp. Med.,2000!
d


Figure 0-2 IL-1Ra
a, IL-1Ra
b, IL-1Ra
c,
d, IL-1Ra
IL-17 T
IL-1
IL-17
T
nude
Nakae et al., !
Proc. Natl. Acad. Sci. 2003
20!
β
Figure 0-3 Th17
T
IL-17
CD4+
CD4+
T
TGF-β IL-6
T
IL-17
T
Th17
Th17
TCR#
TGF-β#
IL-6/ IL-21!
IL-23
IL-1
IFN-γ#
IL-2 IL-27!
IL-4!
SOCS3
STAT3
Foxp3
STAT5
STAT1
STAT6
Irf4
AHR
Rorc
FoxP3
Ahr
RORγt
Rora
NFATc1
BATF
RUNX1
RORγt
STAT3
STAT5
RORα
Nfkbiz
IkBζ
Il17a!Promoter!&!
Enhancer
Il17a
Figure 0-4 Th17
IL-17
Th17
Th17
RORγt Th17
RORγt
STAT3 Th17
Th17
Foxp3
STAT3
Figure 0-5 Th17
Treg
Th17
Th17
TGF-β Foxp3 RORγ
IL-6
RORγt
Foxp3 RORγ
STAT3
Foxp3
TGF-β
RORγ
Foxp3+
Th17
a
6
100
*
5
Severity (Score)
Incidence (%)
80
60
40
Il1rn-/20
Il1rn-/-Il6-/-
b
Wt
12
week
4
20
Il6-/-
Il1rn-/-
0.18&
1.3&
8
12
16
20
week
Il1rn-/-Il6-/0.99&
IL-17
0.16&
16
2
0
0
8
3
1
Il1rn-/-Il6+/-
4
4
CD4
Figure 1. Il1rn-/-Il6-/(a) 
:
Il1rn-/:
Il6-/-,
(b WT,
in vitro
(%)
χ2
Il1rn-/-,
PMA/ionomycin
CD4+
Th17
n=15-22/
*, P < 0.05: Il1rn-/-
Il1rn-/-Il6-/-
Il6-/-Il1rn-/IL-17
LNs CD8+ cells !
(106 cells)
0"
4"
*
Il1rn-/80"
*
5"
0"
0"
*
3"
2"
1"
0"
Il6-/25"
*
*
60"
*
*
50"
40"
30"
20"
10"
0"
*
40"
20"
*
20"
15"
10"
5"
0"
3-4
70"
60"
50"
40"
20"
12"
10"
8"
6"
4"
2"
0"
Figure 2.
Il1rn-/-Il6-/-
± SD *P < 0.05. 2
B220+ cells !
(%)
30"
20"
10"
Spleen
cells (106 cells)
150"
100"
50"
Spleen/body weight (µg)
200"
*
15"
10"
5"
*
CD4+ cells !
(%)
0"
Spleen
5"
B220+ cells !
(106 cells)
10"
Spleen
5"
Spleen
15"
CD4+ cells !
(106 cells)
10"
60"
Spleen
20"
250"
CD8+ cells !
(%)
10"
*
p=0.09
Spleen
5"
*
*
CD8+ cells !
(106 cells)
15"
*
LNs B220+ cells !
(%)
LNs cells (106 cells)
25"
Spleen
15"
*
LNs CD4+ cells !
(%)
LNs B220+ cells !
(106 cells)
30"
LNs CD8+ cells !
(%)
LNs CD4+ cells !
(106 cells)
35"
[LNs]
[Spleen]
300"
12"
10"
8"
6"
4"
0"
2"
80"
0"
140"
120"
100"
0"
25"
0"
80"
60"
40"
20"
0"
50"
40"
30"
20"
10"
0"
20"
15"
10"
5"
0"
*
*
LN IFNg+ (TCRb+CD4+) cells!
LN Foxp3+ (TCRb+CD4+) cells!
2.5"
*
*
LN IL-17+ (TCRb+CD4+) cells!
n.s
2"
0.09"
0.08"
1.5"
1"
1.5"
1"
0.07"
(106 cells)
(106 cells)
(106 cells)
2"
*
0.06"
0.05"
0.04"
0.03"
0.5"
0.5"
0.02"
0.01"
0"
0"
Figure 3.
0"
Th
T
WT, Il6-/-, arthritic Il1rn-/-, Il6-/- , arthritic Il1rn-/-Il6-/PMA/ionomycin
IFN-γ, IL-17A, Foxp3
3-4
± SD 2
CD4+
Wt
0.6
2.7
Il6-/-
medium
2.3
6.9
6.5
9.4
IL-1
1.6
IL-17
3.5
IFN-γ#
Figure 4. IL-1 IL-6
CD4+
Th17
$/$
+
WT
Il6 &
CD4
IL-1
anti-CD3 (4 µg/
ml), anti-CD28 (1µg/ml), anti-IL-4 (10 µg/ml), anti-IFN-γ (10 µg/ml)
48well Flat
Bottom plate
5
5
PMA/ionomycine 5
FACS
CD4+
(%)
5
Il21
4
LNs
*
*
*
*
3
2
1
0
3
Spleen
*
*
2.5
Il21
2
1.5
1
0.5
0
None stimulation
PMA/Ionomycin
Figure 5. WT, Il1rn-/-, Il6-/-, Il1rn-/-Il6-/mRNA
real-time RT-PCR
IL-21
PMA/ionomycin
GAPDH mRNA
n=3 2
Il21 mRNA
4
5
0.1
IL-1
1.2
0.86
TGF/IL-6
17
1.01
TGF/IL-1,6
33
0.07
IL-17
TGF
0.03
TGF/IL-1
c
10
0.9
1.2
0.11
TGF/IL-21
8.2
0.11
0
TGF/IL-1,21
18
100
0.11
Rorc
medium
Foxp3
a
0.06
50
0
1.5
medium
IL-1
TGF
TGF/IL-1
Il17a
IFN-γ#
b
5
1
0.5
0
TGF/IL-6
TGF/IL-1,6
TGF/IL-21
TGF/IL-1,21
Il1r1
6
4
2
Il21
0
0.3
IL-1R1"
0.2
Il22
0.1
0
14
12
10
8
6
4
2
0
Figure 6. IL-1 IL-6
Th17
(a-b) In vitro Th17
BALB/cA
FACS
naïve
+
CD4 T
anti-CD3 (4 µg/ml) anti-CD28 (1 µg/ml) 10 ng/ml; anti-IFN-γ, 10 µg/ml; anti-IL-4,
10 µg/ml
(TGF-β, 3 ng/ml; IL-6, 40 ng/ml; IL-21, 100 ng/ml; IL-1, 10 ng/
ml)
IL-17A FN-γ
IL-1R1 PMA-ionomycin
FACS
(%)
(b) IL-1R
a
Anti-IL-1R1:
(
):
(c)
BALB/cA
FACS
naïve CD4+T
anti-CD3 (4 µg/ml)
anti-CD28 (1 µg/ml) 10 ng/ml; anti-IFN-γ, 10 µg/ml; anti-IL-4, 10 µg/ml
5
mRNA
mRNA
PCR
GAPDH RNA
0.04
IL-17
1.9
Foxp3
0.02
0.04
1.5
0.07
3.2
0.11
2.9
5.0
IL-1α,β 0.1-100ng/ml
Figure 7 naïve T
IL-1
FACS
naïve CD4+T
anti-CD3 (4 µg/ml)
ml; anti-IL-4, 10 µg/ml IL-1(0.1-100 ng/ml) 4
Foxp3
IL-17
anti-CD28 (1 µg/ml) anti-IFN-γ, 10 µg/
4
PMA/ionomycin
2
IL-1R1
TGF-β
TGF-β / IL-21 (20-1000ng/ml)
Figure 8 IL-21
IL-1R1
FACS
naïve CD4+T
anti-CD3 (4 µg/ml) anti-CD28 (1 µg/ml)
γ, 10 µg/ml; anti-IL-4, 10 µg/ml TGF-β (3 ng/ml) ; IL-21 (20, 100, 400, 1000 ng/ml) 4
PMA/ionomycin
IL-1R1
TGF-β
anti-IFN-
2
1st round
TGF/IL-21
2nd round
4.8"
9.9"
3nd round
3.1"
TGF
0.27"
IL-17
5.53"
1.3"
IFN-γ#
9.7"
3.5"
TGF/
IL-21
6.0"
0.24"
17.3"
11.5"
TGF/
IL-1,21
2.2"
1.34"
17.2"
12.2"
TGF/
IL-1
1.9"
0.24"
2.2"
1.4"
IL-1
0.79"
0.22"
0.83"
0.02"
med
3.9"
IL-17
3.9"
IFN-γ#
Figure 9 IL-1 TGF-β
IL-17
FACS
naïve CD4+T
Th17
anti-CD3 (4 µg/ml)
antiCD28 (1 µg/ml) 10 ng/ml; anti-IFN-γ, 10 µg/ml; anti-IL-4, 10 µg/ml; TGF-β, 3 ng/ml; IL-21, 100 ng/
ml
5
(1st round culture)
anti-IFN-γ, anti-IL-4
anti-CD3 anti-CD28
PMA/ionomycin
IL-17A
IFN-γ
FACS
3
3
d
c
Il17a
Il21
Il22
medium
2
0
0.3
1
24.4
14.4
7.9
24.9
IL-21
0.2
10
Foxp3
Nfkbiz
Batf
Rorc
1.6
0
1
Gata3
2
0.1
1.2
IL-1
0
6
0.4
4
Rorc
0.8
0.01
0.1
1
IL-1 (ng/ml)
2
10
12
Foxp3
0
13.4
0.5
IL-23
0
3.4
8
4
0
0
21.2
11.6
Foxp3
MyD88+/-
e
10
8
6
4
2
0
MyD88-/5
*
Foxp3+ cells (%)
0.01
0.1
1
IL-1 (ng/ml)
Tbx21
0
relative expression (fold)
+TGF
0.1
0
b
no TGF
Il17a
relative expression (fold)
4
Foxp3+ cells (%)
a
4
3
2
1
0
Figure 10. IL-1 in vitro Foxp3
(a), IL-1 Th17
Il6-/FACS
naïve CD4+T
Th17
anti-CD3 (4 µg/ml) antiCD28 (1 µg/ml) anti-IFN-γ, 10 µg/ml; anti-IL-4, 10 µg/ml; TGF-β, 3 ng/ml; IL-21, 100 ng/ml
4
IL-1 (0-10 ng/ml)
anti-CD3, anti-CD28 24
mRNA
Real time PCR
mRNA
(a)& IL#17A IL-22
IL-21 IL-1
(b) Nfkbiz
Batf
Rorc
Foxp3
GAPDH RNA
(c-d),
FACS
naïve CD4+T
Th17
anti-CD3 (4 µg/ml) anti-CD28 (1 µg/ml) anti-IFN-γ, 10 µg/ml; anti-IL-4, 10 µg/ml; TGF-β, 3
ng/ml; IL-21, 100 ng/ml
5
anti-CD3 anti-CD28 48
(c)Il17a Tbx21 Gata3 Rorc Foxp3 mRNA
GAPDH RNA
(d), PMA/ionomycin
Foxp3
FACS
;
TGF-β
;
TGF-β
(%)
(e) MyD88+/MyD88-/FACS
+
naïve CD4 T
Th17
anti-CD3 (4 µg/ml) anti-CD28 (1 µg/ml) anti-IFN-γ, 10
µg/ml; anti-IL-4, 10 µg/ml; TGF-β, 3 ng/ml; IL-21, 100 ng/ml
4
anti-CD3 anti-CD28 48
PMA/ionomycin
Foxp3
FACS
*P&<&0.05&(student&t#test)
IL-17+
Foxp3+
Il1r1-/Wt
2.7
42
54
IL-17
Foxp3
Figure 11. IL-1R
IL-17-Foxp3-
IL-1R1
Th17
IL-17+
Foxp3+
FACS
Il1r1-/- naïve CD4+T
Th17
antiCD3 (4 µg/ml) anti-CD28 (1 µg/ml) anti-IFN-γ, 10 µg/ml; anti-IL-4, 10 µg/ml; TGF-β, 3 ng/ml;
IL-21, 100 ng/ml
4
(1st round culture) 4
PMA/ionomycin
IL-1R1 Foxp3 IL-17A
WT T
-/IL-1R1
Il1r1 T
2
71
*
2000
TGF
1600
59
36
TGF/IL-21
3.7
67
26
Foxp3 MFI
(in Foxp3+ cell)
3.7
87
RORγt
TGF/IL-6
CFSE
6.1
Foxp3
Foxp3CFSE
800
400
0
5.7
Foxp3+
1200
TGF/IL-1,21
5.9
*
*
c
Foxp3
b
24
TGF"
Foxp3+ cell (%) in each division
a
90"
TGF/IL-21"
80"
TGF/IL-1,21"
70"
TGF/IL-6"
60"
50"
40"
30"
*
20"
10"
0"
0"
1"
2" 3" 4"
cell division
5"
6"
Figure 12. IL-1
Foxp3
-/(a) Il6
FACS
naïve CD4+T
CFSE
anti-CD3 (4 µg/
ml) anti-CD28 (1 µg/ml) anti-IFN-γ 10 µg/ml anti-IL-4 10 µg/ml
4
RORγt Foxp3
(%)
(b) Foxp3
(Mean fluorescence intensity; MIF)
+
(a)
(c)
Foxp3
CFSE
*P < 0.05 (student t-test) 3
b
d
0.53
TGF
27
3.5
Foxp3+ cell population (%)
"
a
TGF"
40&
TGF+IL-1"
medium
30&
20&
IL#1
10&
0&
TGF/IL-21
IL-21R-Fc "
TGF"
TGF+IL-1"
IIL#21R#Fc
20000&
9.7
15000&
10000&
5.8
IL#1&
IL#21R#Fc
5000&
0&
pSTAT3
IL-21R-Fc "
c
e
10
Il6-/-
Il1rn-/-Il6-/-
Il1ab-/-
20
15
10
0
IL-1
(0.01-10 ng/ml)
IL-21R-Fc 20 µg/ml
IL-1 10 ng/ml
5
IL-1 0 ng/ml
Foxp3+ cell (%)
25
Foxp3+ cells (%)
IL-17
TGF/IL-21
IL-1 10ng
Foxp3
Foxp3 MFI in Foxp3+ cells
"
13
8
6
4
2
0
med
TGF
TGF/IL-1
Foxp3
IL-6 IL-21
(a) Il6-/FACS
anti-CD3 (4 µg/ml) anti-CD28 (1 µg/ml) anti-IFN-γ, 10 µg/ml; anti-IL-4, 10 µg/ml;
4
PMA/ionomycin
Foxp3 IL-17
(%)
2
(b-c)
-/+
Il6
FACS
naïve CD4 T
Th17
anti-CD3 (4 µg/ml) antiCD28 (1 µg/ml) anti-IFN-γ, 10 µg/ml; anti-IL-4, 10 µg/ml; TGF-β, 3 ng/ml; IL-21, 100 ng/ml
4
IL-21R-Fc (1-15 µg/ml)
anti-CD3 anti-CD28
2
Foxp3
+
FACS
: Foxp3
,
: Foxp3 MFI (c-d) Th17
IL-1 (10 ng/ml) IL-21R-Fc (20 µg/ml)
anti-CD3 anti-CD28
Foxp3 pSTAT3
*P<0.05 (student t-test)(e) IL-1
Foxp3
IL-6 IL-1α/IL-1β,
-/-/-/-/IL-1Ra
Il6 , Il1rn Il6 , Il1αβ
FACS
naïve CD4+T
Th17
anti-CD3 (4 µg/ml) anti-CD28 (1 µg/ml) 10 ng/ml; anti-IFN-γ, 10 µg/ml; anti-IL-4, 10 µg/
ml; TGF-β, 3 ng/ml; IL-21, 100 ng/ml
4
anti-CD3 anti-CD28
Foxp3
2
Figure 13. IL-1
naïve CD4+T
Wt naïve T
1.2
Il1ab-/- naïve T
37
40
0.8
0.36
med
13
1.7
0.27
6.9
2.4
IL-6 0.8ng
11
IL-6 4ng
0.13
TGF/IL1
TGF
2.6
21
IL-6 20ng
27
IL-6 100ng
TGF/IL1
TGF
9.8
14
2.3
IL-6 0.8ng
2.7
4.2
IL-6 4ng
+ TGF
Foxp3"
34
0.37
IL-21 20ng
0.4
0.31
med
1.5
39
45
1.8
18
IL-6 100ng
13
IL-6 20ng
+ TGF
21
16
22
2.4
IL-21 100ng
2.9
IL-21 400ng
+ TGF
3.9
IL-21 1000ng
36
18
0.54
IL-21 20ng
23
17
1.7
IL-21 100ng
2.8
IL-21 400ng
+ TGF
2.7
IL-21 1000ng
IL-17A"
Figure 14. IL-6 IL-21
Foxp3
IL-17
IL-1
-/+
FACS
Il1ab naïve CD4 T
TGF-β: 3 ng/ml, IL-6: 40 ng/ml
or IL-21: 100 ng/ml, anti-IFN-γ: 10 µg/ml, anti-IL-4: 10 µg/ml; anti-CD3 (4 µg/ml); anti-CD28 (1 µg/ml)
4
PMA/ionomycin
IL-17A, Foxp3
(%)
4.5"
4"
3.5"
3"
2.5"
2"
1.5"
1"
0.5"
0"
2"
1.8"
1.6"
1.4"
1.2"
1"
0.8"
0.6"
0.4"
0.2"
0"
Nfkbiz/Gapdh
2"
1.5"
1"
1.5"
1"
0.5"
0"
2"
1.8"
1.6"
1.4"
1.2"
1"
0.8"
0.6"
0.4"
0.2"
0"
2.5"
2"
Il21/Gapdh
Il17a/Gapdh
0.5"
2"
Il22/Gapdh
Foxp3/Gapdh
2.5"
2.5"
Rorc/Gapdh
0"
2"
1.8"
1.6"
1.4"
1.2"
1"
0.8"
0.6"
0.4"
0.2"
0"
Batf/Gapdh
3"
1.5"
1"
0.5"
0"
1.8"
1.6"
IL#1
IL#21
Rorc Il21
1.4"
&
1"
0.8"
IL-21
Il1r1
1.2"
IL#21
&
Foxp3 Ba4 5
Il17 Il22
IL#1
0.6"
IL#1
IL$1
IL#21
IL$21
N9biz
5
0.4"
0.2"
0"
med" IL-1"
Fc"
Fc
IL-1"
IL-1
Figure 15. IL-1 IL-21
Il6-/FACS
naïve CD4+T
Th17
anti-CD3 (4 µg/ml)
(1 µg/ml) anti-IFN-γ, 10 µg/ml; anti-IL-4, 10 µg/ml; TGF-β, 3 ng/ml; IL-21, 100 ng/ml
4
IL-1 (10 ng/ml) IL-21R-Fc (20 µg/ml)
anti-CD3, anti-CD28 24
mRNA
PCR
mRNA
anti-CD28
Real time
LPL&
LPL!Foxp3+!cell!
(gated!on!CD4+TCR9b+)
LPL!IFN9g+!!
(gated!on!CD4+TCR9b+)!
35&
25&
LPL!IL917+!%!!
(gated!on!CD4+TCR9b+)!
14&
25&
10&
12&
20&
10&
(%)
15&
(%)
(%)!
20&
15&
5&
0&
0&
①
②
③
④
8&
6&
10&
5&
*
16&
30&
4&
2&
①
②
③
0&
④
①
②
③
Wild type"
Il1rn-/-!
Il6-/-!
Il1rn-/-Il6-/-
Figure 16. IL-1 IL-21
Il1rn-/- Il6-/- Il1rn-/-Il6-/Foxp3 IFN-γ
LPL)
IL-17
)
LPL
FACS
PMA/ionomycin
5
④
a
b
*
130"
Body weight (%)
120"
110"
R1"
90"
17"
80"
70"
0"
1"
2"
3"
4"
weeks after transfer
6"
W"
Il1r1-/-"
17"
*
c
12&
MLN!IL917+!cell!(%)!!
15&
10&
5&
0&
10&
1.5&
8&
6&
4&
W
0&
Il1r1-/- Il17a-/-
0&
LPL!IL917+!cell!(%)!!
10&
5&
Il1r1-/- Il17a-/-
W
20&
5&
4&
3&
2&
1&
0&
0&
W
Il1r1-/-
Figure 17. IL-1 in vivo
Il17a-/- Il1r1-/(a) 
test)
(b)
6
(c)
6
MLN
Il17a-/-
Foxp3 IFN-γ
15&
10&
5&
0&
W
Il1r1-/- Il17a-/-
Th17
IL-17
CD4+CD45RBhiCD25Rag2-/1
:Il1r1-/LPL)
IL-17
W Il1r1-/- Il17a-/-
*
6&
15&
1&
0.5&
2&
20&
LPL!IFN9g+!cell!(%)!!
MLN!Foxp3+!cell!(%)!!
20&
2&
LPL!Foxp3+!cell!(%)!!
MLN!IFN9g+!cell!(%)!!
25&
test)
5"
cm
W"
100"
*
10"
9"
8"
7"
6"
5"
4"
3"
2"
1"
0"
FACS
W Il1r1-/- Il17a-/-
:Il1r1-/-
p<0.05 (student t-test)
)
PMA/ionomycin
:Il1r1-/-
p<0.05 (student t-
5
p<0.05 (student t-
medium
a
LPS
5.9
zymosan
7.4
3.5
Wt
MCM
3.4
3.7
4.8
6.8
0.4
2.8
Il6-/MCM
3.9
0.19
200012.2.800 Workspace.jo
1.1
Layout
16
11
Il1rn-/-Il6-/MCM
2.5
3.9
2.5
IL-17
7.9
IFN-γ#
TGF
b
10 2
0
0
1.25
10 4
47.7
10 5
10
10 3
10 2
0
Foxp3
10 5
10 2
10 3
IL-17
10 4
29
0
10 5
1.13
10 5
Foxp3
10 2
10 2
0
10 2
3.12
10 3
IL-17
10 4
10 5
10 3
IL-17
10 4
26.7
10 4
21.1
10 5
0.626
10 3
10 5
69.1
0
0.749
10 5
10 2
9.22
10 3
IL-17
10 4
16.9
10 5
0.822
10 4
10 3
10 3
10 2
IL-6
64.9
Th1767.4
0
TGC
anti-CD3
anti-CD3 anti-CD28
PMA/ionomycin
27.9
10 3
IL-17
4
0
9.07
10 2
0
10 2
10 2
55.8
10 4
10 3
66.8
0
10
0
IL-17
Foxp3
10 4
10 3
10 5
0.831
10 3
24.6
10 3
IL-17
34
10 5
0.235
Figure 18. Il1rn-/67.6
-/-/-I
(a)
Il6
Il1rn l6-/(MCM)
Il6-/-CD4+
5
PMA/ionomycin
(b) naïve T
5
10 2
4
0
1.75
10 4
Il1rn-/-Il6-/MCM
71.1
10 2
50.3
4.46
4
10 2
0
4
0
10
10 5
0.278
10 5
Foxp3
10 3
IL-17
Foxp3
Foxp3
10
10 2
0.257
10 3
10 2
46.3
3.97
4
Foxp3
10 3
10 5
MCM
10 5
10
0
Il6-/Il6-/MCM
0.203
Foxp3
Wt Wt
MCM
MCM
Foxp3
10
52.2
4
Foxp3
10 5
TGF
zymosan
Zym
MCM
TGF
LPS
LPL
MCM
medium
0
10 2
10 3
IL-17
10 4
anti-CD28
anti-IFN-γ
0
7.64
10 5
10 2
14.8
10 3
IL-17
10 4
10 5
LPS zymosan
anti-IFN-γ atni-IL-4
FACS
atni-IL-4
0
TGF-β (3 ng/ml) CM
FACS
5
5
12000"
25"
*
15"
10"
5"
0"
*
*
MFI (Foxp3+ cell)
Foxp3+ cell (%)
20"
*
10000"
8000"
6000"
4000"
2000"
0"
Figure 19. IL-1 mTOR
Foxp3
-/Il6
FACS
naïve CD4+T
Th17
anti-CD3 (4 µg/ml)
anti-CD28 (1 µg/ml) anti-IFN-γ, 10 µg/ml; anti-IL-4, 10 µg/ml; TGF-β, 3 ng/ml; IL-21, 100 ng/ml
4
Rapamycin 50 ng/ml
anti-CD3 anti-CD28
2
+
Foxp3
FACS
: Foxp3
,
: Foxp3 MFI *P<0.05 (student
t-test)
!IL91
IL-21!
IL-6
IL-21!
IL-6
Th17
IL-1
IL-1
TGF
NFκB"
MAPK"
mTOR"
RORγ
IL-1R1
Batf!
Ikbiz
STAT3
!
STAT3
Foxp3
naïve T
IL-21R
IL-21R
IL-17
IL-22
IL-1R
Foxp3+
Foxp3
IL#1
IL#1
IL#21
TGF
+IL-21
Foxp3+
RORγt+
naïve T
IL#1
Foxp3RORγt+
IL-17++
IL#1
Foxp3+
RORγt+
Foxp3RORγt+
IL-17++
Nfkbiz↑
Batf↑
TGF
Foxp3RORγt+
IL-17++
Foxp3
downregulation
Th17 cells
Foxp3+
RORγt-
iTreg
Figure 20
Th17
Foxp3 RORγt
IL-1
IL-21 IL-1R1
IL-1,IL-6,IL-21
naïve T
Foxp3
TGF-β
Th17
52