Supporting information

SI materials and Methods

Mice

All mice were backcrossed with C57B6/J mice more than eight times before use. Ticam1-

/- 1 and Mavs-/- 2 mice were generated in our laboratory. Irf3-/- and Ifnar-/- mice were

generously provided by Dr. T. Taniguchi (The University of Tokyo). Batf3-/- mice were

purchased from the Jackson laboratory (Maine, USA). C57B6/J WT female and male

mice were purchased from CLEA Japan (Tokyo, Japan). For preparation of standard

serum and NW, we used Balb/c mice purchased from CLEA Japan because IgA

production by polyI:C was more efficient in Balb/c mice than in C57B6/J mice. All mice

were maintained under specific pathogen-free conditions in the Animal Facility at

Hokkaido University Graduate School of Medicine (Sapporo, Japan) and used when they

were 8 to 12 weeks of age. This study was carried out in strict accordance with the

recommendations in the Guide for the Care and Use of Laboratory Animals of the

National Institutes of Health. The protocol was approved by the Committee on the Ethics

of Animal Experiments in the Animal Safety Center, Hokkaido University, Japan. All

mice were used according to the guidelines of the Institutional Animal Care and Use

Committee of Hokkaido University, who approved this study as no.13-0049.

In vivo infection

Influenza strain A/Puerto Rico/8/34 (H1N1) (PR8) virus was propagated in 10-day-old

embryonated chicken eggs at 37 °C. Fourteen days after the last immunization, the mice

were infected with 250 plaque-forming unit (pfu) in 40 l of PBS instilled into the nasal

cavity. After infection, body weight and survival of the mice were monitored daily, and

mice were sacrificed when they reached the ethical end point of 30% loss of their initial

body weight.

FACS analysis and cell sorting

Cells prepared from NALT, LN and spleen were blocked with anti-CD16/CD32 Ab (93)

and stained with the fluorescence-labeled following Abs; anti-B220 (RA3-6B2), anti-

CD45 (30-F11), anti-IgA (mA-6E1), anti-CD4 (GK1.5), anti-CD8 (53-6.7), anti-CD11c

(N418), anti-CD103 (2E7), anti-TLR3 (11F8), anti-CD11b (M1/70), anti-CD80 (16-

10A1), anti-CD86 (GL1), anti-CD40 (3/23), anti-CD40L (MR1), anti-CD69 (H1.2F3),

anti-CD3 (145-2C11), anti-PD-1 (RMP1-30), anti-CXCR5 (L138D7), anti-Fas (Jo2),

anti-GL7 (GL7), anti-BAFF (121808), anti-APRIL (A3D8) and anti-pSmad2 (D27F4) .

For intracellular staining, surface makers were stained and cells were fixed and

permeabilized using BD Cytofix/Cytoperm Fixation/Permeabilization Solution Kit (BD

Biosciences), then stained with anti-IFN- (XMG1.2). All Abs were purchased from

BioLegend Stained cells were subjected to flow cytometry (BD FACSCalibur and BD

Aria II).

For isolation of CD103+DCs from NALT, cells were collected from twelve

NALTs and CD11c+ cells were enriched using anti-CD11c MACS beads (Miltenyi). Cells

were stained with allophycocyanin (APC)-labeled anti-CD45, phycoerythrin (PE)-

cyanine dye (Cy)7-labeled anti-CD11c, PE-labeled anti-CD103, isothiocyanate (FITC)-

labeled anti-CD8 Abs and 7-Amino-Actinomycin D (7AAD) (BD Biosciences) and

sorted by Aria II (BD Biosciences).

To isolate B cells from NALT, we stained cells with 7AAD, APC-labeled anti-

CD45 and FITC-labeled anti-B220 Abs and sorted them by Aria II (BD Biosciences). The

purity of isolated B cells was >95 %. To isolate CD103+DCs from spleen, NALT and LNs,

we prepared cell suspensions from spleen, NALT and LNs, and treated the cells with anti-

CD16/32 Ab and then labeled them with biotin-conjugated anti-B220, anti-F4/80, anti-

CD3 and anti-DX5 Abs at 4 °C for 10 min. Cells were incubated with streptavidin beads

(Miltenyi) at 4 °C for 15 min and passed LD column (Miltenyi). Flow-through cells were

labeled with biotin-conjugated anti-CD103 Ab (Miltenyi) and incubated with streptavidin

beads. Labeled cells were passed MS column (Miltenyi) twice to increase purity. The

purity of isolated cells was more than 90 %.

In vitro IgA induction

For IgA class switch B-DC coculture experiments, splenic B cells (5 x 104 cells) isolated

using anti-CD19 MACS beads (Miltenyi) were cocultured with 25 g/ml anti-CD40

(BioLegend) and 10 g/ml anti-IgM (Jackson ImmunoResearch) in the presence or

absence of DCs (5 x 104 cells) prepared from MLN using anti-CD11c MACS beads

(Miltenyi) for 5 days. Cells were stained with PE-labeled anti-CD19, FITC-labeled anti-

IgA and APC-labeled anti-CD45 Abs and then expression levels of IgA were measured

by flow cytometry.

Immunostaining

NALTs were fixed with 4% paraformaldehyde (PFA)/PBS for 30 min at 4 °C. Fixed

tissues were impregnated with 15% sucrose/PBS for 2 h following 30% sucrose/PBS for

overnight at 4 °C. Tissues were then embedded in O.T.C. compound (Sakura Finetek

Japan) and the frozen tissue blocks were sectioned by using cryotome (LEICA CM1850).

NALT sections were fixed with acetone on ice for 30 min, and after three washes in PBS,

the sections were blocked with mouse serum IgG in 5% BSA/PBS for 1 hr at R.T.

Sections were stained with FITC-labeled anti-CD11c, APC-labeled anti-TLR3, FITC-

labeled anti-CD103 or APC-labeled B220 Abs and monitored at x 63 or 40 magnification

using an LSM510 META microscopy (Zeiss). For in vivo polyI:C inoculation, mice were

anesthetized and 5 l of solution containing 500 ng of R-PIC (Invivogen) or PBS was

instilled into each nostril. After 2 hrs, NALTs were collected and fixed with 4% PFA/PBS

for 30 min at 4 °C.

Analysis of Ag-specific T cell response

Two weeks after the second vaccination, 1 x 106 splecnoytes were seeded in 96-well U

bottom plate and restimulated by 0-10 g/ml of split-HA vaccine for 3 days. Cells were

incubated with 10 g/ml of brefeldin A (Sigma) during the last 6 hrs of culture to inhibit

protein transport. Cells were stained with FITC-conjugated anti-CD4, PE-conjugated

anti-CD8 and PE-Cy7-conjugtaed anti-CD3 Abs and then fixed, permeabilized, and

stained with anti-IFN- Ab Stained cells were subjected to flow cytometric analysis. The

IFN- production in the culture supernatants was quantified with mouse IFN- ELISA kit

(eBioscience) or Cytometric Bead Array Flex Set system (BD Biosciences) following the

manufacturer’s instructions.

In vitro R-PIC inoculation

Isolated CD103+CD11c+ DCs were suspended in chilled RPMI medium (Invitrogen)

containing 10% heat-inactivated fetal bovine serum, penicillin, streptomycin and 2-

mercaptoethanol and added with 5 g/ml R-PIC for 30 min on ice. After incubation, cells

were washed with cold medium twice and incubated with pre-warmed medium at 37 °C

for 30 min. Cells were washed with PBS twice, fixed with 4% PFA/PBS and stained with

FITC-conjugated anti-TLR3Ab. Cells were monitored at x 63 or 40 magnification using

an LSM510 META microscopy (Zeiss).

RT-PCR

Total RNA was purified from NALT B cells using TRIzol (Invitrogen) following the

manufacturer’s instructions. Total RNA was subjected to DNase I treatment (Takara BIO

INC). Reverse transcription-PCR was performed by a High Capacity cDNA Reverse

Transcription kit (Applied Biosystems) according to the manufacturer’s instructions.

Real-time PCR was performed using a Step One real-time PCR system (Applied

Biosystems). Primers used in this study are listed in S1 Table. Levels of target mRNAs

were normalized to -actin and fold-induction of transcripts was calculated using the

ddCT method relative to unstimulated samples.

S1 Table. Primers’ sequences in this study

Supplementary Figure S1. The number of IgA+ B cells and B220+ B cells

Cells were prepared from NALT after vaccination, and counted numbers and proportions

of IgA+ B cells and B220+ B cells by flow cytometry. (a) The proportion of IgA+ B cells.

(b) The proportion of B cells. (c) The number of IgA+ B cells. (d) The number of B220+

B cells. The values are presented as the mean ± SD of 4 samples for each group. *, p <

0.0 5; **, p < 0.01 in Student's t-test.

Supplementary Figure S2. TLR3 expression in LNs and DC subsets in NALT

(a) Cells prepared from LNs and NALT were stained with 7AAD, APC-labeled anti-

TLR3, PE-labeled anti-CD11c, PE-labeled anti-CD11b and FITC-labeled anti-CD45 and

subjected to flow cytometric analysis. (b) The isolated cells from MLN and NALT were

stained with 7AAD, Alexa700-labeled anti-CD45, PE-labeled anti-CD103, PE-Cy7-

labeled anti-CD11c and APC-labeled anti-CD8 Abs and subjected to flow cytometric

analysis. The indicated numbers show the proportion of the gated populations.

Supplementary Figure S3. Cholera toxin enhanced Ig production in Ticam1-/- and Batf3-

/- mice

Mice were vaccinated with vaccine alone or vaccine and cholera toxin (CT) twice. After

second vaccination, NW and serum were collected to measure IgA and IgG production

by ELISA. Cells were isolated from vaccinated mice and proportions of IgA+ B cells and

B220+ B cells were quantified by flow cytometry. (a) The proportion of IgA+ B cells. (b)

The proportion of B cells. (c) IgG production in serum. (d) IgA production in NW. The

values are presented as the mean ± SD of 4 samples for each group. *, p < 0.0 5; **, p <

0.01; ***, p < 0.001 in Student's t-test.

References

1. Akazawa, T. et al. Antitumor NK activation induced by the Toll-like receptor 3-

TICAM-1 (TRIF) pathway in myeloid dendritic cells. Proc Natl Acad Sci U S A

104, 252-257 (2007).

2. Oshiumi, H. et al. The TLR3/TICAM-1 pathway is mandatory for innate immune

responses to poliovirus infection. J Immunol 187, 5320-5327 (2011).

Supplementary Table S1. Primers' sequences

Primers' name sequences (5'-3')

Tlr3 nested PCR Fw1 gagccagaactgtgccaaatac

Tlr3 nested PCR Rv1 aagcttctctgtgaggtggggg

Tlr3 nested PCR Fw2 ctgcacgaacctgacagaactc

Tlr3 nested PCR Rv2 tgttcaagaggagggcgaataac

Aicda Fw caagcgcccagatccaaag

Aicda Rv ggtccgtctcaggcactatg

Mmp9 Fw caagtgggaccatcataacatca

Mmp9 Rv gatcatgtctcgcggcaagt

Serpine1 Fw tagcacaggcactgcaaaaggtc

Serpine1 Rv tgtgccgaaccacaaagagaaag

Tgfbi Fw tccaacaaagacatcctggcc

Tgfbi Rv aggatgtcaatggcagtggag

Il4 Fw ggtctcaacccccagctagt

Il4 Rv gccgatgatctctctcaagtg

Il6 Fw gttctctgggaaatcgtgga

Il6 Rv tccagtttggtagcatccatc

Il10 Fw ggcgctgtcatcgatttctc

Il10 Rv tgctccactgccttgctctta

Ifnb Fw ccagctccaagaaaggacga

Ifnb Rv cgccctgtaggtgaggttgat

aGLT Fw caagaaggagaaggtgattcag

aGLT Rv gagctggtgggagtgtcagtg

bactin Fw tttgcagctccttcgttgc

bactin Rv tcgtcatccatggcgaact

Tgf-b1 Fw gctgaaccaaggagacggaat

Tgf-b1 Rv caagagcagtgagcgctgaa

Tgf-b2 Fw tcccgaataaaagcgaagagc

Tgf-b2 Rv ggtgccatcaatacctgcaaa

Tgf-b3 Fw ccagatacttcgaccggatga

Tgf-b3 Rv tgacatcgaaagacagccattc

Il21 Fw gccagatcgcctcctgattag

Il21 Rv atgctcacagtgcccctttac

0500

10001500200025003000350040004500

# of

IgA+ B

cel

ls

0

1

2

3

4

5

6

7

# of

B22

0+ cel

ls (x

104 )

Takaki et. al.,

Supplementary Figure S1

VaccinePolyI:C

+-

++

+-

++

+-

++

WT Batf3-/- Ticam1-/-

VaccinePolyI:C

+-

++

+-

++

+-

++

WT Batf3-/- Ticam1-/-

* ***

a b

0

10

20

30

40

50

60

70

80

90

VaccinePolyI:C

+-

++

+-

++

+-

++

WT Batf3-/- Ticam1-/-

B220

+ /CD

45+ c

ells

(%)

0

0.25

0.5

0.75

1.0

1.25

1.5

IgA+ /B

220+ c

ells

(%)

VaccinePolyI:C

+-

++

+-

++

+-

++

WT Batf3-/- Ticam1-/-

c d

* ***

n.s.

n.s.

0 50K 100K 150K 200K 250K0

50K

100K

150K

200K

250K

58.80 102 103 104 105

0

50K

100K

150K

200K

250K 99.4

0 102 103 104 1050

50K

100K

150K

200K

250K 81.1

0 102 103 104 1050

1000

2000

3000

4000

5000

54.2

0 102 103 104 105

0

102

103

104

105

9.4722.2

2.53

0 50K 100K 150K 200K 250K0

50K

100K

150K

200K

250K

77.10 102 103 104 105

0

50K

100K

150K

200K

250K 96.1

0 102 103 104 1050

50K

100K

150K

200K

250K 0.359

0 102 103 104 1050

50

100

150

200

15.3

0 102 103 104 105

0

102

103

104

105

0.06260.0313

9.52

FCS

SSC

7AAD

SSC

CD45

SSC

CD11c CD103C

D8α

CD45+ CD11c+

MLN

NALT

Takaki et. al.,

Supplementary Figure S2

100 101 102 103 104100

101

102

103

104

0.0256

0.0909

100 101 102 103 104100

101

102

103

104

0.0224

1.57e-3

100 101 102 103 104100

101

102

103

104

0.102

0.397

100 101 102 103 104100

101

102

103

104

0.0646

0.0646

LNs

NALT

TLR3

CD

11c/

11b

WT Tlr3-/-a

b

0

0.25

0.5

0.75

1.0

1.25

1.5

0

10

20

30

40

50

60

70

80

VaccineCT

+-

++

+-

++

+-

++

WT Tlr3-/- Batf3-/-

VaccineCT

+-

++

+-

++

+-

++

WT Batf3-/-Tlr3-/-

B220

+ /CD

45+ c

ells

(%)

IgA+ /B

220+ c

ells

(%)

n.s.***

0

400

800

1200

1600

2000Ig

A in

NW

(Uni

t)

VaccineCT

+-

++

+-

++

+-

++

WT Batf3-/-Tlr3-/-

IgG

in s

erum

(x10

3 Uni

t)

VaccineCT

+-

++

+-

++

+-

++

WT Batf3-/-Tlr3-/- Takaki et. al.,Supplementary Figure S3

a b

c d

*** *****

**

*

05

1015202530354045

* ****