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