Immunity, Volume 44

Supplemental Information

Transforming Growth Factor-b Signaling

Guides the Differentiation of Innate

Lymphoid Cells in Salivary Glands

Victor S. Cortez, Luisa Cervantes-Barragan, Michelle L. Robinette, Jennifer K.Bando, Yaming Wang, Theresa L. Geiger, Susan Gilfillan, Anja Fuchs, Eric Vivier, Joe C.Sun, Marina Cella, and Marco Colonna

TRAIL

7.33

9.35

17.6

24.9

6.34

7.75NK1

.1

NKp46102

103

104

105

106

Liver

siLP siI

E

Liver siLP siIE

# N

K1.1

+ NKp

46+ C

D3- c

ells

2.43

2.23

NK1.1

CD

49b

2.01

0.3331.3

1.92

0.3127.3

NK1.1

CD

3

105

106

107

Spl

n N

K ce

lls

0

10

20

30

40

50

60

% IF

N-γ

+ Cel

ls

46.1

45

SplnIL-12 + IL-18

NK1

.1

IFN-γ

% C

D10

7α+ C

ells

0

10

20

30

40

50

60

SplnP/I

NK1

.1

CD107α

47.2

46.9

WT

Tgfbr2f/f Ncr1Cre

Liver

siLP siIE

Spln

CD73

Liver

WT

Tgfbr2f/f Ncr1Cre

1.13

41.6

0.50

42

82.6

NK1.1

GAT

A3

CD127

KLR

G1 86.3

NK1.1

RO

Rgt

Thy1

.2

CCR6

NKp

46

NK1.1 0.00.20.61.0

203040

10

NK1.1+

ILC2

ILC3

SG ILC2 SG ILC3

0 0

64.935

% C

ells

A

B C D E

F G

H I

WT Tgfbr2f/f Ncr1Cre

CD49a

CD69

CD62L

Liver siLP siIEWT Tgfbr2f/f Ncr1Cre

WT Tgfbr2f/f Ncr1Cre

WT Tgfbr2f/f Ncr1Cre

NS

NSNS

NS

NSNS

69

35.4 4.4

3426

70.7

37.8 4.6

18 39.7

RO

Rγt

CD45

CC

R6

NKp4602

468

10

1214

# SI

LP

NKp

46+ I

LC3

(x10

4 )

WT Tgfbr2f/f Ncr1Cre

NS

Figure S1

siLP

Spln Spln

Figure S1. Impact of TGF-β on other ILC populations (related to Figure 1). (A) Relative frequency of SG ILCs (NK1.1+,CD3-,CD19-), SG ILC2 (GATA3+, KLRG1+, CD127+, NK1.1-CD3-,CD9-) and SG ILC3 (RORgt+, Thy1.2+, NKp46+/-, CCR6+/-, NK1.1-, CD3-, CD19-). (B) Frequency and numbers of splenic NK cells from WT and Tgfbr2f/f x Ncr1Cre mice. (C) Frequen-cy of splenic NK1.1+CD49b+ NK cells from WT and Tgfbr2f/f x Ncr1Cre mice. (D-E) Expression of (D) intracellular IFN-γ and (E) surface CD107a after stimulation with IL-12 + IL-18 or PMA/ionomycin of splenic NK cells from WT and Tgfbr2f/f x Ncr1Cre mice. (F) Frequency and numbers of NK1.1+NKp46+CD3- cells from liver, small intestine lamina propria (siLP), and small intestine epithelium (siIE) from WT and Tgfbr2f/f x Ncr1Cre mice. (G) Expression of CD49a, CD69, and CD62L on NK1.1+CD3- cells from liver, siLP, and siIE from WT and Tgfbr2f/f x Ncr1Cre mice. (H) Frequency and numbers of ILC3 (RORgt+, NKp46+, CD3-,CD19-) from the siLP of WT and Tgfbr2f/f x Ncr1Cre mice. (I) Expression of TRAIL on liver ILC1 (CD49a+NK1.1+CD3-) and CD73 on NK1.1+NKp46+CD3-CD19- cells from the spleen, liver, siLP, and siIE of WT and Tgfbr2f/f x Ncr1Cre mice. All cells from SG, liver, siLP, and siIE are gated on as CD45+. Data are presented as ± SD and are representative of at least 3 mice per group.

Figure S1

76.2

64.9

NK1.1

CD

3WT

Tgfbr2f/f Ncr1Cre

CD49b NKp46

IL-2

IL-2 +TGF-β1 Ctrl

WT Tgfbr2f/f Ncr1Cre

A B

Figure S2

Figure S2. NK cells maintain CD49b and NKp46 expression after in vitro culture with TGF-β1 (related to Figure 2). (A) Frequencies of NK1.1+CD3- NK cells from spleen of WT and Tgfbr2f/f x Ncr1Cre mice after magnetic enrichment using CD49b microbeads. (B) Expression of CD49b and NKp46 after 7 days of culture with either IL-2 + TGF-β1 or IL-2 alone on enriched splenic NK cells from WT or Tgfbr2f/f x Ncr1Cre mice.

84.1 14

84.1 74.4

60.8 13

Liver

SG

WT Nfil3-/-A

Figure S3

3.6

24

0.2

29.7

0.6

26.8

WT Tbx21-/-Eomesf/f Ncr1Cre

Spleen

C

Spleen

93.5 21.7

98.9 98.3

98.5 67.6

WT Nfil3-/-

T-betEomes

0

0.5

1.0

1.5

2.0Sp

leni

c N

K ce

lls (x

106 )

***

NK1.1

CD

3

WT

Tbx21-/-

Eomesf/f Ncr1Cre

Liver

SG

Spleen

WT Tbx21-/-

SG ILCsCD103

SG ILCsNKp46

WT

SG-NK1.1

Eomesf/f Ncr1Cre

SG

D ESG ILCsTRAIL

WT Eomesf/f Ncr1Cre

WT Spln NK

0.95 92.4

6.03CD49b

CD

49a

Tbx21-/- SG ILCsF G

B

Figure S3. SG ILCs have differential requirements for Eomes and T-bet (related to Figure 3). (A-B) Expression of (A) Eomes and (B) T-bet within NK1.1+CD3-CD45+ cells from the SG, liver, and spleen of WT and Nfil3-/- mice. (C) Frequencies and numbers of splenic NK cells from WT, Eomesf/f x Ncr1Cre, and Tbx21-/- mice. (D) Expression of NK1.1 within the SG (gated on CD45+CD3-) of WT and Eomesf/f x Ncr1Cre mice. (E) Expression of TRAIL by SG ILCs from WT and Eomesf/f x Ncr1Cre mice, and WT splenic NK cells. (F) Expression of CD49a and CD49b by SG ILC from Tbx21-/- mice. (G) Expression of CD103 and NKp46 by SG ILCs from WT and Tbx21-/- mice. (H) Expres-sion of tissue and pan NK cell makers on SG ILCs from WT and Tbx21-/- mice. Data are presented as ± SD and are representative of at least 3 mice per group. Student’s t test, *p <0.05, **p < 0.01, for comparisons between mice.

.

CD49a

CD103

CD69CD62

L

TRAILCD73

0

20

40

60

80

100

Figure S4

A

% H

igh

SG IL

Cs

WT Smad4f/f Ncr1Cre

The phenotype of SG ILCs is Smad4 independent. (related to Figure 4). (A) Expression of indicated markers by SG ILCs from WT and Smad4f/f x Ncr1Cre mice. Data are presented as ± SD and are representative of at least 3 mice per group.

Figure S5

Cxcr6

Tcrg-V2Tcrg-V3

Cd3gTcrg-C

SG CD49

a+

ILCs

Spleen

NK

Liver

NK

Spleen

ILC1

Liver

ILC1

IEL ILC

1

CD127

WT SG ILCs

Eomes- siLP NK1.1+CD3- (ILC1)WT Spln NK1.1+CD3-

A B

Figure S5. SG ILCs express some ILC core signature genes but not IL-7ra (related to Figure 5). (A) Heat map showing expression of the ILC signature genes Tcrg-V2, Tcrg-V3, Tcrg-C, Cd3g, and Cxcr6 by CD49a+ SG ILCs, liver ILC1, IEL ILC1, spleen ILC1, liver NK, and spleen NK. (B) Expression of CD127 (IL-7ra) by SG ILCs (NK1.1+CD3-), splenic NK1.1+CD3- cells, and Eomes-NK1.1+CD3- ILC1 from the siLP (Black bar indicates negative staining).

.

Row Min Row Max

Expression (Relative)

Figure S6

Figure S6. Chimerism of splenic cNK cells after 4 weeks of parabiosis (related to Figure 6). (A) Frequencies of splenic NK cells (NK1.1+CD3-CD45+) which are host or donor derived after 4 weeks of parabiosis between WT (CD45.1) and WT (CD45.2) mice. (B) Frequencies of splenic NK cells (NK1.1+CD3-CD45+) which are host or donor-derived after 4 weeks of parabiosis between WT (CD45.1) and Tgfbr2f/f x Ncr1Cre(CD45.2) mice. Data are presented as mean ± SD and are representative of at least two independent experiments.

A

01020304050607080

WT

(CD45

.1)

Par

abion

tW

T (C

D45.2)

Par

abion

t

%Po

sitiv

e C

ells

CD45.1CD45.2

0102030405060708090

100

%Po

sitiv

e C

ells

CD45.1CD45.2

WT (CD45

.1)

Parabio

nt

Tgfbr

2f/f

x Ncr

1Cre

(CD45

.2) P

arab

iont

B

Figure S7

A10.4

44

1.17

SG

B

NK1.1

CD

3

Germ-free

0.0

0.5

1.0

1.5

2.0

D

AdultYoung

Eom

es rM

FI

*

Eomes

8.53 79.4

11.4CD49b

CD

49a

Germ-free

CD103

Germ-free

6 weeks

40.3 55.7

NK1

.1

CD49a

E F

Tgfbr1Tgfbr2

Smad7SkilSki

SG CD49

a+ ILC

s

Spleen

NK

SG CD49

a- ILC

s G

3.8

15.4

1.3

6.9

25.8

0.4

NK1.1

CD

3

Young

1

5

10

15

# SG

ILC

(x10

4 ) **AdultYoung

Adult

C

Figure S7. SG ILC expression of TGF-b dependent markers is microbiota independent but age dependent (related to Figure 7). (A) Frequencies of NK1.1+CD3- cells from the SG of germ-free mice. (B) Expression of CD49a and CD49b, and CD103 on SG ILCs from germ-free mice. (C) Frequencies and absolute numbers of SG ILCs (NK1.1+CD3-CD45+) from young and adult mice. (D) Expression of Eomes by SG ILCs from young and adult mice. (E) Sorting gates for CD49a-(green) and CD49a+ (red) SG ILCs used for microarray analysis. (F) Heat map generated from microarray analysis showing expression of Tgfbr1, Tgfbr2, and the TGF-β induced genes Smad7, Skil, and Ski from CD49a- SG ILCs, CD49a+ SG ILCs, and splenic NK cells. (G) BrdU incorpo-ration by CD49a- and CD49a+ SG ILCs from adult WT mice. Mice were injected with 1 mg BrdU i.p. every 24 hours for three consecutive days. Data are presented as mean ± SD and are representative of at least two independent experiments of at least 4 mice. Student’s t test, *p <0.05, **p < 0.01, for comparisons between mice.

.

CD49a-

BrdU

7.5

19.8

CD49a+

SG ILCs

0

5

10

15

20

25

CD49a-

CD49a+

%Br

dU+

**

Supplemental Experimental Procedures

Antibodies

Fluorochrome- and biotin-conjugated antibodies to mouse NK1.1 (PK136), CD3 (145-2C11), CD45 (30-F11), CD103 (2E7), CD49b (HMa2), CD27 (LG.7F9), TRAIL (N2B2), CD11b (M1/70), CD45.1 (104), CD45.2 (A20), CD11c (N418), KLRG1 (2F1), Eomes (Dan11mag), T-bet (eBIO 4B10), and IFN-γ (xma1.2) were purchased from eBioscience. Fluorochrome- and biotin-conjugated antibodies to mouse CD49a (Ha31/8), CD69 (H1.2F3) CD62L (MEL-14), CD43 (S7), IL-21r (4A9), and CD107a (1D4B) were purchased from BD Biosci-ences. Biotin-conjugated GITR (DTA-1) was purchased from Biolegend. Streptavidin-APC, Streptavidin-Per-CP/Cy5.5, and Streptavidin-PE-Cy7 were purchased from Invitrogen, Biolegend, and eBioscience, respectively.

Primer Pairs for qRT-PCR

Primers were used as follows:

Gapdh forward-ACGGCAAATTCAACGGCACAGTCA; Gapdh reverse-TGGGGGCATCGGCAGAAGG; Tgfb1 forward-TTGCTTCAGCTCCACAGAGA;Tgfb1 reverse- TGGTTGTAGAGGGCAAGGAC; Tgfb2 forward- CCGGAGGTGATTTCCATCTA; Tgfb2 reverse- GCGGACGATTCTGAAGTAGG; Tgfb3 forward- ATTCGACATGATCCAGGGAC; Tgfb3 reverse- TCTCCACTGAGGACACATTGA.Inpp4b forward- AGAACCTCAGATGGTGGCAAAInpp4b reverse- CCCGCTCAGACTTTCTGGTGTsc22d1 forward- CCAGTGGCGATGGATCTAGGATsc22d1 reverse- CTTGCACCAGAGCTATTGTCA

Gut and liver lymphocyte isolation

To isolated intestinal lymphocytes, small intestine mesentery and peyers patches were excised followed by length wise cut and gently washing to remove luminal contents. To isolate the intestinal epithelial fraction tissue was incubated in 1x HBSS, 5%BCS, HEPES, and 5mM EDTA for 20 min (performed twice) at RT on a shaker. After removal of epithelial fraction the intestine was washed, minced, and incubated with RPMI1640, 5%BCS, HEPES, and 1mM Collagenase (II or IV) shaking at 37○C for up to 90min. Both the intestinal epithelial fraction and lamina propria fraction were passed through a 100uM filter and layered on a 70%/40% percoll gradient to enrich for lymphocytes in a manner similar to SG. To isolate hepatic lymphocytes, livers were passed through a 70uM filter, followed by red blood cell lysis and layered on a 70%/40% percoll gradient in a manner similar to SG.

Microarray and data analysis

In accordance with ImmGen project standards, 6-week-old male C57BL/6J mice obtained from Jackson Laboratories were used for cell sorting experiments. One to three replicates of RNA were obtained from each sample that passed quality control. RNA amplification and hybridization to the Affymetrix Mouse Gene 1.0 ST array were carried out by ImmGen with a standardized TRIzol extraction protocol.

Data generation and quality-control documentation also followed the ImmGen protocol. Data were analyzed with GenePattern software (Broad Institute). Raw data were normalized with RMA. Differences in gene expression were identified with the Multiplot Studio function of GenePattern, from a filtered subset of genes with coefficients of variation less than 0.2 in all samples and expression of at least 120 relative units in one subset by the class mean functions, a value that corresponds to 95% confidence of true expression across the ImmGen data set.

Hierarchical clustering of sample replicates was carried out in Gene-E from the same dataset, with the Pearson correlation used as a metric. For PCA, the top 15% of the most variable probe sets was calculated with the Popula-tionDistances PCA program (S. Davis, Harvard Medical School). This dataset was log2-transformed in MATLAB and used to generate a PCA with the functions pca and scatter3. Volcano plots comparing change in expression (fold) versus change in expression (fold) were produced in Multiplot Studio. Heat maps were generated with Gene-E. Data were log2-transformed and visualized by 'relative' expression per row.

MiceAll mice were bred at a pathogen free facility at Washington University. All animal experiments were conducted according to USA Public Health Service Policy of Humane Care and Use of Laboratory Animals. All protocols were approved by the Institutional Animal Care and Use Committee (School of Medicine, Washington University, St. Louis, MO).