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Regulatory T cells and the induction of IL-17.
LiLi Xu, Atsushi Kitani
Mucosal Immunity Section, National Institute of Allergy and Infectious Diseases
Bethesda, MD, USA
Differentiation of CD4 T cells
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Anti-LAP+Anti-Foxp3
CD4+CD25+ CD4+CD25-ActivatedCD4+CD25+ CD4+CD25+
Isotype ctrl
Freshly Isolated or Activated CD4/CD25+Foxp3+ Cells Express Surface TGF-β
Confocal fluorescent microscope view of intracellular staining of Foxp3 (green) and cell surface LAP (red).
Naïve CD4 cells + TGF-β/IL-6 Th17 differentiation
Treg cells with surface TGF-β/IL-6 Th17 differentiation ?
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101
102
103
104
26.8
100 101 102 103 104100
101
102
103
104
19.2
100 101 102 103 104100
101
102
103
104
2.41
ActCD25+/CD25-/IL-6 CD25-/TGF-β/IL-6ActCD25-/CD25-/IL-6
CFSE
IL-1
7
CD4+CD25- T cells were pre-labeled with CFSE, and cultured in a 1:1 mixture with activated CD4+CD25+ or CD4+CD25- cells with IL-6. Dot plots were gated on CFSE+ cells.
Activated CD25+ Treg Cells Can Induce IL-17-producing Cellswith IL-6 Alone.
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101
102
103
104
10.1 3.23
63
23.7
100 101 102 103 104100
101
102
103
104
24.8 1.03
72.2 2.01
ALK5 Inhibitor(-) ALK5 inhibitor(+)
IL-17
IFN
-γ
The Induction of IL-17 Producing Cells by ActivatedCD4+CD25+ Treg Cells Is TGF-β Dependent.
CD4+CD25- were pre-labeled with CFSE and stimulated with IL-6 in the presence of ALK5 inhibitor in a 1:1 mixture with preactivated CD4+CD25+ or CD4+CD25- cells.
9 10 11 12 13
TGA Stop codon
IRES-GFP PolyA8
Targeted FoxP3 locus
FoxP3-GFP Knock-in Mice
Provided by Dr. Mohamed Oukka (Harvard Medical School)
Green Fluorescent Protein (GFP)+ cells from FoxP3-GFP knock-in mice are a source of highly purified Treg cells.
ActGFP+/GFP-/IL-6
10 0 10 1 10 2 10 3 10 410 0
10 1
10 2
10 3
10 4
21
57.4 16.7
4.88
100 101 102 103 104100
101
102
103
104
0.47
99.2
0.061
0.26
ActGFP-/GFP-/IL-6
10 0 10 1 10 2 10 3 10 410 0
10 1
10 2
10 3
10 4
1.65
87
0.15
11.2100 101 102 103 104
100
101
102
103
104
5.41
80.8
0.35
13.5
GFP+/GFP-(1:1)/IL-6 GFP+/GFP-(2:1)/IL-6
GFP (Foxp3)
IL-1
7GFP-/TGF-β/IL-6
100 101 102 103 104100
101
102
103
104
3.64
36.1
8.82
50.5
IL-1
7
GFP (Foxp3)
Activated CD4+CD25+Foxp3+ T cells are Strong Inducers of IL-17-producing Cells with IL-6 Alone.
CD4+GFP-(FoxP3-) cells were stimulated with IL-6 in a mixture with CD4+GFP+ cells or activated CD4+GFP+ or CD4+GFP- cells. Cells were collected at day 4 for intracellular Cytokine staining. Act, activated.
IL-1
7 (p
g/m
l)
0
500
1000
1500
2000
2500
3000
3500
GFP+ +GFP-+IL-6
ActGFP+
GFP- + IL-6
ActGFP- GFP-
TGF-β +IL-6 1:1 2:1
* ** **
CD4GFP-(FoxP3-) T cells Cocultured with CD4+GFP+(FoxP3+)Cells Produce Large Amounts of IL-17.
Produced amounts of IL-17 were assayed by ELISA. *, p < 0.05; **, p < 0.01; compared with activated CD4+CD25- cells.
GFP+/IL-6 GFP-/IL-6
GFP(FoxP3)
IL-1
7
CD4+CD25+T Cells Themselves Differentiate into IL-17-producing Cells in the Absence of Exogenous TGF-β.
GFP+ (FoxP3+) or GFP- (FoxP3-) T cells each alone were stimulated with IL-6. Cells were collected at day 6 for intracellular cytokine and Foxp3 staining.
100 101 102 103 104100
101
102
103
104
7.18 12.6
73.36.88
100 101 102 103 104100
101
102
103
104
0.37 0.019
99.5 0.077
Isot
ype
ctrl
Isot
ype
ctrl
100 101 102 103 104100
101
102
103
104
0 0
14.2 85.8
100 101 102 103 104100
101
102
103
104
6.67
93.3 0.045
0
GFP+
GFP (Foxp3)
TGF-
β
DC (coculture with GFP+ cells)
0.12
TGF-
β
GFP-
100 101 102 103 104100
101
102
103
104
26.1
70.9 1.47
1.57
100 101 102 103 104100
101
102
103
104
11.9
3.72 26.4
58
GFP (Foxp3)
GFP+
DC (coculture with GFP- cells)
DC (coculture with GFP+ cells)
100 101 102 103 104100
101
102
103
104
54.3 0.66
44.8 0.2100 101 102 103 104
100
101
102
103
104
83.1
16.8 0
GFP (Foxp3)GFP (Foxp3)
FoxP3+ Treg CellsInduce DCs to Produce Increased Amounts of TGF-β.
CD4+GFP+ or CD4+GFP- cells were cultured with DC and stimulated with anti-CD3 and LPS. On day 5, cells were collected and intracellular TGF-β staining. Dot plots were either gated onCD4+ cells (T cells, top panels) or CD4- cells (DCs, bottom panels).
0
0.5
1
1.5
2
2.5
3
3.5
4
GFP+/TNBS TNBS
His
tolo
gy s
core
0
10
20
30
40
50
60
GFP+/TNBS TNBS
IFN
-γ(n
g/m
l)
0
500
1000
1500
2000
2500
GFP+/TNBS TNBS
IL-1
7 (
pg
/m
l)
P<0.05
Transfer of GFP+(FoxP3+) Cells After Onset of TNBS-Colitis Results in Increased IL-17 Production.
After 2 days induction of TNBS-Colitis, activated CD4+GFP+(FoxP3+) cells were transferred to the mice and sacrificed on day 5. Mesenteric lymphnode cells were stimulated with
anti-CD3/CD28 and produced cytokines were assayed by ELISA.
T cells are stimulated with plate bound anti-CD3(10ug/ml), soluble anti-CD28(2ug/ml) in the presence of
IL-1β(50ng/ml) and IL-6(100ng/ml)
Human Treg Cells also Differentiate into Th17 cells in the Presence of IL-1β and IL-6
IL-17100 101 102 103 104
100
101
102
103
104
73.1 7.44
17.9 1.54
100 101 102 103 104100
101
102
103
104
2.76 0.66
80.3
16.2100 101 102 103 104
100
101
102
103
104
46.1
36.9
8.42
8.53
CD4+CD25high CD4+CD25-
Donor 1
Donor 2
Foxp
3
Conclusions
1. Treg cells induce CD4+CD25-Foxp3- T cells to differentiate intoTh17 cells in the presence of IL-6 without exogenous TGF-β.
2. Treg cells themselves differentiate into Th17 cells in the presence of IL-6 without exogenous TGF-β.
3. Treg cells induce DCs to produce high amounts of soluble TGF-β.
Hypotheses:
• IL-17 producing cells arising from Treg cells in the absence of inflammation may have homeostatic function
• Under some circumstances Tregs may contribute to inflammation.
Acknowledgement
Mucosal Immunity SectionNational Institute of Allergy and Infectious Diseases
LiLi Xu
Ivan Fuss
Warren Strober
Center for Neurologic DiseasesHarvard Medical School
Mohamed Oukka
Note that1. Inflamed IBD mucosa contains an increased number of CD4+CD25high
FOXP3+ T cells, compared with noninflamed mucosa. Maul J, Zeitz M, Duchmann R et al. Gastroenterology. 2005
2. Lamina Propria (LP) CD4(+)CD25(bright) T cells with regulatory T activity were significantly increased in patients with active inflammatory bowel disease (IBD).
Makita-S, Watanabe-M et al. J Immunol 20043. Accumulation of Foxp3 cells were observed in inflamed LP in
patients with Crohn or ulcerative colitis.Uhllig-HH, Powrie-F et al. J Immunol 2006
Treg cells may differentiate into Th17 cells by the exposure of IL-6 & IL-1β in the inflamed mucosa in patients with IBD.
Additional work will be necessary to determine the conditionsunder which this occurs.
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1.81
22.2
7.99
68100 101 102 103 104
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101
102
103
104
2.78 0.57
2.37
94.3
CD4+CD25+ CD4+CD25-
CD25
TGF-
β
Purified CD4+CD25+ and CD4+CD25- cells were stained with FITC anti-CD25 and Biotin anti TGF- β mAb (from BD) followed by streptavidin PE-Cy5
CD4+CD25+ Treg cells express surface TGF-β
Act iv
e f o
rm o
f TG
F-β
(pg /
ml )
0
100
200
300
400
500
600
700
Tota
l TG
F-β
(pg/
ml)
ActCD4+
GFP+ActCD4+
GFP-ActCD4+
GFP+ medium
CD4+GFP-
0
500
1000
1500
2000
**
****
TGF-β Production in the Supernatant by the Coculture of CD4+GFP-(FoxP3-) T cells and CD4+GFP+(FoxP3+) Cells.
Supernatants collected at day 4 were assayed for TGF- β1. *, p < 0.05; ** , p < 0.01; compared with activated CD4+GFP+cells.Active form TGF-β1 (without acid treatment) or total TGF- β1 (with acid treatment)were measured using TGF- β1 ELISA.
GFP+
+IL-6
GFP-
+
IL-6
GFP (Foxp3)
IL-1
7
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101
102
103
104
0.37 0.019
99.5 0.077
Day 6
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102
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104
0.16 0.53
3.36 95.9100 101 102 103 104
100
101
102
103
104
9.26 7.91
9.44 73.4100 101 102 103 104
100
101
102
103
104
1.52 1.52
8.31 88.6100 101 102 103 104
100
101
102
103
104
7.18 12.6
73.36.88
Day 2 Day 3 Day 4 Day 6
Th1polarization
GFP (Foxp3)
IL-1
7
100 101 102 103 104100
101
102
103
104
0.059 0.059
7.7692.1
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101
102
103
104
75.7 0.095
24.1 0.14
GFP+ GFP-
GFP (Foxp3)
IFN
-γ
Both IL-17 Single-positive and GFP/IL-17 Double-positive Cells Increased During the Culture of GFP+(FoxP3+) T cells with IL-6.
CD4+GFP+ were stimulated with IL-6 for 2, 4, and 6 days or polarized under Th1 condition.Foxp3+ cells can co-express IL-17 and that IL-17 single-positive cells pass through an Foxp3/IL-17 double-positive stage. Thus, CD4+Foxp3 cells can undergo self-induced Th17 differentiation, but resist Th1 differentiation.
GFP-
TGF-β+IL-6
IL-1
7 (p
g/m
l)
GFP+ GFP-
IL-6
0
500
1000
1500
2000
2500
3000
3500
4000
*
**
IL-17 produced in the Supernatants from Cultures of CD4+CD25+ or CD4 +CD25- T cells Stimulated with IL-6
Produced amounts of IL-17 for 6 days were assayed by ELISA.* p < 0.05; **, p < 0.01; compared with GFP- cells.
GFP+ GFP-
GFP(Foxp3)
IL-1
7
100 101 102 103 104100
101
102
103
104
10.5
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101
102
103
104
0.3
Differentiation of IL-17-producing Cells from CD4+CD25+Foxp3+T cells in the Coculture of DCs with TLR Ligation
CD4+GFP+ or CD4+GFP- cells were cultured with bone marrow-derived DC, Stimulated with anti-CD3 and LPS which induces IL-6. On day 5, cells were collected for surface CD4 and intracellular IL-17 staining.Dot plots were gated on CD4+ cells.
100 101 102 103 104100
101
102
103
104
4.04 23.8
14.7 57.5
100 101 102 103 104100
101
102
103
104
0.022 0.15
15.7 84.1
100 101 102 103 104100
101
102
103
104
1.97 0.03
97.6 0.39
100 101 102 103 104100
101
102
103
104
0.24
99.5
0.03
0.21
GFP(Foxp3)
IL-1
7
GFP (Foxp3)
IFN
-γ
GFP+ GFP-
Differentiated Th-17 Cells from GFP+(FoxP3+) Cells AreStable After a Long Term Culture.
GFP+ or GFP- cells were stimulated with anti-CD3/anti-CD28/IL-2 and IL-6, and
were re-stimulated after 7days, then analyzed on 14 days.