jci suppl figs 171121 rm · 2018. 2. 22. · supplemental figure 4. requirement of zap70 in γδt...
TRANSCRIPT
Supplemental Figure 1. TCR-induced tyrosine phosphorylation of Syk as well as Zap70 in γδT cells
γδT cells isolated from neonatal WT mice were stimulated with anti-CD3ε antibody
(2C11) for 2 min. Cell lysates were immunoprecipitated with antiphosphotyrosine
antibody (4G10) followed by immunoblotting with the indicated antibodies.
Representative immunoblotting from two independent experiments was shown.
INPUT
CD3 � � � �IP:4G10
IB: Syk
IB: Zap70
IB:
75 kDa
75 kDa
37 kDa
IB: -actin
Supplemental Figure 1 Muro et al
37 kDa
Lat
Supplemental Figure 2. IL-17 production of Syk-deficient γδT cell in the periphery
of adult mice (A) Absolute numbers (per mouse) of total γδT cells in thymus (Thy), spleen (Spl), and
lung from indicated fetal liver (FL) chimeric mice are shown (lower, n = 3). The mice
were analyzed 8 weeks after FL cell transplantation. (B) Representative flow cytometry profiles for cell surface Vγ4 and intracellular IL-17A expression in γδT cells from the
indicated tissues after stimulation with PMA and ionomycin. Symbols and lines in graphs indicate individual mice and mean, respectively. Error bars indicate SEM. ∗p <
0.05; ∗∗p < 0.01; n.s., not significant, by unpaired t-test. (C-E) FL chimeric mice were
treated daily for 5 days with IMQ cream on the ear (WT FL n = 2, Sykb−/− FL n = 3). Kinetics of IMQ-induced ear swelling (C), flow cytometry analysis of IL-17+ cells in γδT cells from ear-draining lymph nodes at day 5 (D), and the number of IL-17+ γδT cells from ear-draining lymph nodes at day 5 (E). Data represent single experiment.
01234
012345
01234
Cel
l num
ber (
x105
)To
tal abT
Thy Spl Lung
0 102 103 104 105
0
102
103
104
105 52.4 2.95
0.59
0 102 103 104 105
0
102
103
104
105 23.8 0.17
0.51
0 102 103 104 105
0
102
103
104
105 25.8 1.9
0.08
0 102 103 104 105
0
102
103
104
105 23.9 0.1
0.29
0 102 103 104 105
0
102
103
104
105 24 11.2
0.8
0 102 103 104 105
0
102
103
104
105 20.6 0.55
0.09
IL-17A
Va4
Thy Spl Lung
�� n.s. n.s.
:7�)/�ĺ�Tcrb Tcrd < <
Sykb���)/�ĺ�Tcrb Tcrd < << <
< <
< <
WT FL�ĺ�Tcrb Tcrd < < < <
Sykb FLĺ�Tcrb Tcrd < <
< <
< <
Supplemental Figure 2 Muro et al
A B
C D E
Ear
thic
knes
s (m
m)
:7�)/�ĺ�Tcrb Tcrd < <
Sykb���)/�ĺ�Tcrb Tcrd < << <
< <
< <
0 102 103 104 105
0
102
103
104
105 28.1 28.6
0.260 102 103 104 105
0
102
103
104
105 27.7 0.06
0.27IL-17A
Va4
01234
01234
Cel
l num
ber (
x104
)
Total abTIL-17A
Va4IL-17A+ +
�ĺ�Tcrb Tcrd < < < <�ĺ�Tcrb Tcrd < < < <
WT FL Sykb FL< <
:7�)/�ĺ�Tcrb Tcrd < <
Sykb���)/�ĺ�Tcrb Tcrd < << <
< <
< <
Day (Time)0 3 4 5
0.2
0.3
0.4
Supplemental Figure 3. IFNγ-producing potential of immature γδT cells and DN
thymocytes
Thymocytes from indicated mice were stimulated with PMA plus ionomycin for 4 h. Contour plots show intracellular staining for IFNγ production in γδT cells from
Zap70−/−, Sykb−/−, Zap70−/− Sykb−/−, Lat−/− or Rhoh−/− mice (n = 4-26), or CD11b−
CD11c− CD49b− TER119− B220− Gr-1− (lineage-negative) cells from Rag2−/− mice (n = 4). Data represent the combined result of six independent experiments.
0102 103 104 1050
50K
100K
150K
200K
250K
7.7
0102 103 104 105
11.8
0102 103 104 105
22.9
0102 103 104 105
43.3
0102 103 104 1050102 103 104 105
32.2
SS
C-A
IFNa
WT Zap70 Syk Zap70 Syk< < < < < < < <
Lat< <
TCRb���CD3¡
Rag2< <
Lineage <
Supplemental Figure 3 Muro et al
20.013.3
0102 103 104 105
RhoH< <
Supplemental Figure 4. Requirement of Zap70 in γδT cell development
(A and B) Flow cytometry analysis of CD5 expression in thymic Vγ6+ γδT cells from
WT or Zap70−/− mice at E15.5 (n = 5–10) or day 0 (n = 3). Relative MFI of CD5 expression is shown. (C) Zap70 protein (top) and mRNA (bottom) expression in thymic γδT cell subsets from neonatal WT mice. The numbers beside the flow cytometry
histograms indicate MFI (mean ± SE) of Zap70 protein expression (n = 4). Zap70 mRNA expression levels were normalized to β-actin mRNA (n = 3). (D) Representative
flow cytometry profiles for Vγ chains in γδT cells from indicated tissues (upper).
Absolute numbers (per mouse) of indicated γδT cell subsets and total γδT cells are
shown (lower, n = 4–6). (E) Intracellular staining for IL-17A and IFNγ production in
indicated spleen or lung γδT cells from 5-week-old WT or Zap70−/− mice (n = 3–4).
Graphs show the absolute number of IL-17A+ or IFNγ+ γδT cells. All graphs indicate the mean ± SEM. ∗p < 0.05; ∗∗p < 0.01; n.s., not significant, by unpaired t-test. Data
represent more than two independent experiments (D and E) or single experiment
(A-C).
Va4
17D
1
27.9
6.3
19.1
0.8
34.4
12.7
0 102 103 104 105
0102
103
104
105 11.2
2.5
IL-17A
IFNa
0 102 103 104 105
0102
103
104
105
0 102 103 104 105
0102
103
104
105
0 102 103 104 105
0102
103
104
105
Total
Va4
abT
WT Zap70
Cel
l num
ber (
x10
)4C
ell n
umbe
r (x1
0 )5
Spleen
0 102 103 104 105
0
102
103
104
105 7.0
27.2
0 102 103 104 105
0
102
103
104
105 51.8
25.8
0 102 103 104 105
0
102
103
104
105 0.9
18.4
0 102 103 104 105
0
102
103
104
105 60
17.0
0 102 103 104 105
0
102
103
104
105 2.2
16.1
0 102 103 104 105
0
102
103
104
105 18.5
30.2
0 102 103 104 105
0
102
103
104
105 64.8
15.1
0 102 103 104 105
0
102
103
104
105 38.6
28.5
Va4 Va6 Va1
Spleen Lung
Cel
l num
ber (
x10
)5V a
1
Va4
17D
1V a
1
Spleen LungWT Zap70
IFNa
IL-17A
00.51.01.52.02.5
0
0.5
1.0
1.5
Total abT Va4
Total abT Va4
Zap70
Va5
Va6
Va4
Va1 0102 103 104 105
0 102 103 104 1050
20
40
60
80
100
0 102 103 104 1050
20
40
60
80
100
CD5
E15.5 Day 0
WT Zap70Isotype
A C
B
E
D
00.51.01.52.0
E15 Day 0Rel
ativ
e C
D5
MFI
< <
< <
< <WT Zap70
< <
10793
2840
1521
2532
Va6
WT Zap70< <
WT Zap70< <
Supplemental Figure 4 Muro et al
��
��
����
n.s.
� ��
�
� �
�
�
00.51.01.52.0
01234
Total Va4 Va6 Va1Total
n.s.n.s.
Cel
l num
ber (
x10
)5
Va6
Va5
Va4
Va1
0.00.51.01.52.0
Zap70
mR
NA
Exp
ress
ion
(Rel
ativ
e to
`-a
ctin
)
��
��
8.2
14.1
0.7
6.0
8.0
20.0
0
4.6
0 102 103 104 105101 0 102 103 104 105101
0 102 103 104 105101 0 102 103 104 105101
0
103
104
105
0
103
104
105
0
103
104
105
0
103
104
105
Total
Va4
abT
IFNa
IL-17A
0
2
4
6
8
00.20.40.60.81.0
Cel
l num
ber (
x10
)4C
ell n
umbe
r (x1
0 )5
Total abT Va4
Total abT Va4
IL-17A
IFNa
WT Zap70< <
Lung
�� ��
n.s.p = 0.05
± 799
± 155
± 49
± 97
WT Zap70< < WT Zap70
< <
Supplemental Figure 5. TCR-induced Akt phosphorylation in γδT cells is
dependent on PI3K activity
(A and B) Representative flow cytometry profiles of TCR-induced phosphorylation of Erk (A) or Akt (B). Cells were pretreated with IC87114 at a final concentration of 5 µM
for 1 h. Graphs show relative MFI of phospho-Erk or phospho-Akt (n = 3). All graphs indicate the mean ± SEM. ∗p < 0.05; ∗∗p < 0.01; n.s., not significant, by 2-way
ANOVA. Data represent single experiment.
0
2
4
6
8
0 1 2
% o
f Max
pErk
pAkt
Rel
ativ
e pE
rk M
FIR
elat
ive
pAkt
MFI
1 min 2 min
Time after Stimulation (min)
Time after Stimulation (min)
anti-CD3 + DMSO
anti-CD3 + IC87114 5 +M
non-stim anti-CD3 + DMSO
anti-CD3 + IC87114 5 +M
Supplemental Figure 5 Muro et al
A
B
0 102 103 104 1050
20
40
60
80
100
0 102 103 104 1050
20
40
60
80
100
0 102 103 104 1050
20
40
60
80
100
0 102 103 104 1050
20
40
60
80
100
% o
f Max
1 min 2 min
�n.s.
0.8
1.2
1.6
2.0
2.4
0 1 2
�� ��
0
Supplemental Figure 6. γδT cell development in RhoH-deficient mice
(A) Frequency of total γδT cells (% of total thymocytes) and indicated γδT cell subset
(% of total γδT cells) from indicated age of WT or Rhoh−/− mice (n = 5–9). (B) Absolute number of indicated γδT cells (per mouse), as described in A. Graphs indicate
mean ± SE. (C) Representative flow cytometry profiles for Vγ expression in γδT cells
from indicated tissues of 6-week-old WT or Rhoh−/− mice. Graphs indicate the cell number (per mouse) of indicated γδT cell subsets in individual mice (circles) and their
mean ± SE (n = 5–7). All graphs indicate the mean ± SEM. ∗p < 0.05; ∗∗p < 0.01; n.s.,
not significant, by unpaired t-test. Data represent two independent experiments (C) or
the combined result of five independent experiments (A and B).
Skin
0
1
2
3
02468
Va4 Va5�� n. s.25.1
26.32.5
69.1
Supplemental Figure 6 Muro et al
012345
0123
Cel
l num
ber (
x10
)5 Va1 Va4
01234
0123
Cel
l num
ber (
x10
)4 Va4 Va6
17D
1
WT Rhoh
Va1
Va4
Va4
��
��
��
n. s.
A
WT Rhoh
48.9
28.3
65.7
13.8
28.0
38.0
1.4
8.9
0 103
104
105
0 103
104
105
0 103
104
105
0 103
104
105
010
3
104
105
010
3
104
105
010
3
104
105
010
3
104
105
Spleen
Lung
Cel
l num
ber (
x10
)3
Va4
Va5
0
1
2
3
0 10 20 30 40 50
0
20
40
60
80
0 0.5 1.0 1.5 2.0 2.5
0 5 10 15 20 25
0 12345
0
20
40
60
80
0 5 10 15 20 25
0
20
40
60
0 5 10 15 20 25
Freq
uenc
y (%
)C
ell n
umbe
r(10
)4
Va5 Va6 Va4Va1Total abT E
15.5
E18
.50
do1
wo
6 w
o
E15
.5E
18.5
0 do
1 w
o6
wo
E15
.5E
18.5
0 do
1 w
o6
wo
E15
.5E
18.5
0 do
1 w
o6
wo
E15
.5E
18.5
0 do
1 w
o6
wo
WT Rhoh
B
C< <
< <
E15
.5E
18.5
0 do
1 w
o6
wo
E15
.5E
18.5
0 do
1 w
o6
wo
E15
.5E
18.5
0 do
1 w
o6
wo
E15
.5E
18.5
0 do
1 w
o6
wo
E15
.5E
18.5
0 do
1 w
o6
wo
< <
0 103
104
105
102
0 103
104
105
102
0
103
104
105
102
0
103
104
105
102
56.730.0
12.7
50.329.9
18.90 10
310
410
510
20 10
310
410
510
2
0
103
104
105
0
103
104
105
Va1
Va7
00.51.01.52.02.5
0
2
4
6
Cel
l num
ber (
x10
)5 Va1 Va7
WT Rhoh< <
WT Rhoh< <Small intestineWT Rhoh< <
n. s. n. s.
Supplemental Figure 7. A schematic model of γδT17 cell differentiation regulated
by Syk-dependent TCR signals
In response to γδTCR stimulation, Syk activates LAT-dependent canonical pathway,
including the MAPK cascade, and Lat-independent non-canonical pathway mediated by the PI3K-Akt axis. The former acts as a mainstream signal, which is required for γδT
cell maturation and differentiation toward IFNγ-producing cells, while the latter induces
alternative differentiation program toward γδT17 through the expression of transcription
factors Sox13 and RORγt.
T cell
IFN
TCR
Canonical pathways (MAPK-Erk,
NF- B & NFAT)
Non-canonical (Syk-dependent) pathway
Zap70
PI3K
Akt
Syk
IL-17
LAT
Supplemental Figure 7 Muro et al
Supplemental Table 1. Summary of mouse γδT cell subsets
Summary of mouse abT cell subsets
V chain
Heilig and Tonegawa’s nomenclature
Garman’s nomenclature Tissue distribution Cytokine
production
V 1
V 4
V 5
V 6
V 7
V 1.1
V 2
V 3
V 4
V 5
lymphoid tissue, liver
lymphoid tissue, liver, lung, dermis
dermis
lung, uterus, tongue
intestine
IFN
IFN or IL-17
IFN
IFN
IL-17
Supplemental Table 1 Muro et al