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Supporting Information Ito et al. 10.1073/pnas.1311022111 SI Materials and Methods Induction of Lymph Node Hypertrophy. Equal volumes of complete

Freund adjuvant (CFA) containing 1 mg/mL heat-killed Mycobacterium tuberculosis H37RA (Difco Laboratories) and sterile PBS were emulsified and 100 μL of emulsified CFA was injected subcutaneously. Three or 6 days later, lymph nodes (LNs) were harvested. Where indicated, 500 μg of 55A2C or hamster IgG [control (cont) Ig] was injected intraperitoneally the day before CFA injection. Purified Peptides. Proteolipid protein (PLP)139–151 (HSLGKWLGHPDKF) and myelin oligodendrocyte glycoprotein (MOG)35–55 (MEVGWYRSPFSRVVHLYRNGK) were purchased from Sigma-Aldrich. The purity of peptides used in the current study was >80%. Experimental Autoimmune Encephalomyelitis Induction. Experimental autoimmune encephalomyelitis (EAE) was induced in 6-wk-old female SJL/J mice by subcutaneous injection on day 0 with 50 μg of PLP139–151 peptide emulsified with CFA and intraperitoneally with 400 ng of pertussis toxin (PT) (List Biological Laboratories) on days 0 and 2. For prophylactic treatment, mice were treated with 500 μg of anti-α9 integrin antibody (55A2C) or normal hamster IgG (cont Ig) the day before PLP139–151/CFA immunization. Clinical scores of EAE were assessed daily by previously described criteria (1). Production of Anti-Mouse α9 Integrin Antibodies. Anti-mouse α9 integrin antibodies, designated 55A2C, 18R18D, and 12C4′58, were generated as described previously (2). Embryo-Derived Lymphatic Endothelial Cell Isolation. Embryo-derived lymphatic endothelial cells (ED-LECs) were isolated as described previously (3) with some modification. In brief, day 13–15 embryos were harvested. After removal of spleen and liver, embryos were digested with type II collagenase (Worthington) with stirring at 37 °C for 30 min and centrifuged at 400 × g. After washing, CD3+, CD11b+, and B220+ cells were depleted by using IMagnet (BD Biosciences), and then lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1)+ cells were sorted by the MACS procedure (Miltenyi Biotec). IMagnet and MACS were performed according to the manufacturers’ instructions. Isolated cells were seeded on 0.1% gelatin (Millipore)-coated dishes. Lymphocyte Egress Analysis. Recipient mice received 5 × 10 2D2 6

CD4+ T cells and were immunized or not with MOG35–55 (100 μg)/CFA, MOG, or CFA alone the following day. To inhibit LN lymphocyte entry, mice were treated with 100 μg of anti-α4 (PS2) and -αL (M17/4) antibodies at the indicated times. These antibodies were purified from hybridomas from the American Type Culture Collection. Transferred cells (Vα3.2+, Vβ11+) within inguinal and mesenteric LNs were analyzed by flow cytometry at the indicated times. To test the effect of blocking α9 integrin, mice were treated with 500 μg of 55A2C or cont Ig at the indicated times. As a control, some mice were treated intraperitoneally with FTY720 (Cayman Chemical) at 1 mg·kg−1·d−1 on days 1, 2, 3, 4, and 5 (Fig. 2 and Figs. S5 and S7) or days 1, 2, and 3 (Fig. S6). Preparation of Recombinant Proteins. Recombinant proteins of the third fibronectin type III repeat domain of human TN-C (R797–P896) in which RGD was mutated to RAA (rTN-C) were generated as described previously (2). Ito et al. www.pnas.org/cgi/content/short/1311022111

Sphingosine 1-Phosphate Release Assay. Sphingosine 1-phosphate (S1P) production by LECs was measured as described previously (4). In brief, six-well plates were coated with 20 μg/mL rTN-C or gelatin, and then 1 × 106 ED-LECs were seeded in each well. After 22 h, the supernatant was discarded and cells were labeled with [3H]sphingosine (American Radiolabeled Chemicals) in 10 mg/mL fatty acid-free BSA (Sigma-Aldrich)/TIL media (Immuno-Biological Laboratories) for 1 h. Subsequently, media and cells were collected for lipid extraction. Total cellular lipids were extracted from cells as described previously (5), whereas lipids were extracted from media by the method designed for S1P extraction (6). Lipids were analyzed by TLC and detected by autoradiography. Flow Cytometric Analysis. LN cells were stained with phycoerythrin (PE)-anti-podoplanin (BioLegend; clone 8.1.1), fluorescein isothianate (FITC)-anti-CD45 (BioLegend; clone 30-F11), and PE-Cy7-anti-CD31 (BioLegend; clone 390) antibodies and biotinylated anti-α9 integrin antibody (clone 18R18D), followed by allophycocyanin (APC)-streptavidin (BD Biosciences). Isolated ED-LECs were stained with anti-vascular endothelial growth factor receptor 3 (VEGFR3) antibody, followed by FITCconjugated anti-goat IgG (Jackson ImmunoResearch). For intracellular cytokine staining, cells that had been stained with anti-CD3 (clone 145-2C11) and anti-CD4 (clone GK1.5) antibodies (BioLegend) were fixed and permeabilized by Cytofix/ Cytoperm buffer (BD Biosciences), and then cells were stained with anti–IL-17 (BD Biosciences; clone TC11-18H10) and anti– IFN-γ (BioLegend; clone XMG1.2) antibodies. For analysis of the integrin activation state, cells were stained with biotinylated anti-α9 integrin antibody (clone 18R18D), anti-activated form of β1 integrin (BD Biosciences; clone 9EG7), or anti–pan-β1 integrin (BD Pharmingen), followed by streptavidin-APC (for α9 integrin; BD Biosciences) or PE-anti-rat IgG (Jackson ImmunoResearch). For analysis of CD169+ cells, LNs were digested with 2 mg/mL collagenase D and minced. Then, cells were stained with Alexa647-anti-CD169 (Serotec), PE-anti-CD11b, and FITC-antiF4/80 (BioLegend) antibodies and anti-α9 integrin antibody (clone 18R18D), followed by streptavidin-APC-Cy7. All analyses were performed on FACSCalibur and FACSCanto II (BD Biosciences) systems with FlowJo software (Tree Star). Histology. Spinal cords were removed after perfusion with PBS, fixed with 10% (vol/vol) formaldehyde, and embedded in paraffin. Transverse sections (10-μm) were stained with hematoxylin/eosin (HE) and Luxol fast blue (LFB). Pathological scores of the spinal cords were evaluated by previously described criteria (7). Cellular infiltration: 0, normal; 1, weak perivascular infiltration; 2, mild perivascular infiltration; 3, mild parenchymal infiltration; 4, severe parenchymal infiltration. Demyelination: 0, normal; 1, reduction of LFB stain; 2, mild generation of demyelination plaque; 3, severe generation of demyelination plaque. For immunohistochemistry, frozen sections were made from OCT (Sakura)-embedded LNs. After drying, sections were fixed with acetone for 7 min at −20 °C and blocked with 2% normal goat serum in 0.05% Tween-TBS for 30 min, followed by primary antibodies for α9 integrin (clone 12C4′58), LYVE-1 (Abcam), TN-C (Abcam; clone MTn-12), FN-EDA (Abcam; clone IST9), CD3 (BioLegend; clone 145-2C11), PNAd (BioLegend; clone MECA-79), or the activated form of β1 integrin (BD Pharmingen; clone 9EG7) for 2 h at room temperature and by AlexaFluor546-conjugated anti-rabbit IgG (for LYVE-1), 1 of 11

biotinylated anti-rat IgG, streptavidin-FITC (for TN-C), FITCanti-goat IgG (for FN-EDA), DyLight649-anti-Syrian hamster IgG (for α9 integrin), or streptavidin-FITC (for CD3 and PNAd). Sections were analyzed by laser confocal microscopy (FluoView FV1000; Olympus). Staining area was measured by Scion Image software. At least three or four different fields per section were examined using at least three sections. Measurement of Activated β1 Integrin on ED-LECs. ED-LECs (2 × 105 cells) were cultured in 20% FCS/TIL medium for 1 d. After washing the cells to remove FCS, medium was replaced by FCS-free TIL, and then TGF-β (1–10 ng/mL) or other cytokines (IL-6, IL-17, IL-23, or IL-1β) were added to the culture. After 16 h, cells were harvested by using EDTA-free 0.05% trypsin/ PBS, fixed with 2% formaldehyde, and then stained with antibodies to 9EG7 epitope, α9 integrin, or pan-β1 integrin as described above. All analyses were performed on a FACSCanto II (BD Biosciences) system with FlowJo software (Tree Star).

suspension (2 × 104 cells per well) in DMEM containing 0.25% BSA was applied and incubated for 1 h at 37 °C. The medium was removed and all wells were washed twice. Adherent cells were fixed and stained for 30 min with 0.5% crystal violet in 20% methanol. All wells were rinsed three times with water, and adherent cells were then lysed with 20% acetic acid. The resulting supernatants were analyzed using an immunoreader (Nalge Nunc), and the absorbance at 590 nm was measured to determine the relative number of cells adhered to the wells. Evaluation of Lymphatic Flow. Mice that had been treated with 55A2C or cont Ig on the day before CFA injection into the footpad received an Evans blue injection into the footpad 3 d after CFA injection. Sixty minutes after Evans blue injection, popliteal and inguinal LNs were harvested and immersed in a 6:3 mixture of acetone and 0.5% aqueous sodium sulfate solution, and then OD620 was measured.

were coated at 37 °C for 1 h with the indicated concentration of proteins. Plates were blocked with 0.5% BSA/PBS for 1 h at room temperature. After washing with PBS, 200 μL of cell

Statistical Analysis. All data are presented as mean ± SEM. Significant differences between two groups were determined using the Mann–Whitney U test (Fig. 6 A and E) and Student t test. *P < 0.05 and **P < 0.01 against control. N.S., not significant.

1. Stromnes IM, Goverman JM (2006) Active induction of experimental allergic encephalomyelitis. Nat Protoc 1(4):1810–1819. 2. Kanayama M, et al. (2009) Alpha9 integrin and its ligands constitute critical joint microenvironments for development of autoimmune arthritis. J Immunol 182(12): 8015–8025. 3. Morisada T, et al. (2005) Angiopoietin-1 promotes LYVE-1-positive lymphatic vessel formation. Blood 105(12):4649–4656. 4. Kawahara A, et al. (2009) The sphingolipid transporter spns2 functions in migration of zebrafish myocardial precursors. Science 323(5913):524–527.

5. Kihara A, et al. (2003) Sphingosine-1-phosphate lyase is involved in the differentiation of F9 embryonal carcinoma cells to primitive endoderm. J Biol Chem 278(16): 14578–14585. 6. Yatomi Y, et al. (2000) Sphingosine 1-phosphate as a major bioactive lysophospholipid that is released from platelets and interacts with endothelial cells. Blood 96(10): 3431–3438. 7. Kroenke MA, Carlson TJ, Andjelkovic AV, Segal BM (2008) IL-12- and IL-23-modulated T cells induce distinct types of EAE based on histology, CNS chemokine profile, and response to cytokine inhibition. J Exp Med 205(7):1535–1541.

Adhesion Assay. Wells of 96-well flat-bottom plates (Nalge Nunc)

T

T

T

Fig. S1. Anatomical location and definition of medullary and cortical sinuses in the LN. Serial LN sections were stained with LYVE-1 (shown as red) and CD169, CD3, or PNAd (shown as green). Medullary sinuses (arrows) are located close to the LN hilus and are associated with CD169+ macrophages. In contrast, cortical sinuses (arrowheads) were located within CD3+ T-cell areas and adjacent to PNAd+ high endothelial venules. (Scale bars, 200 μm.) The dotted lines indicate the T-cell zone (T).

Ito et al. www.pnas.org/cgi/content/short/1311022111

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a

c dLN weight

b

0

3 6 day

* mm2

naïve (day 0)

4

** **

mm2

2 1 0

0

3 6 day

** **

0.2

**

0.1 0

dLN area 3

staining area

LYVE-1 CD169

0.3

0

3 6 day

integrin LYVE-1

Immunized (day 6)

mg

8

0

LYVE-1

** **

12

Medullary sinus Corcal sinus

d LYVE-1 FN EDA

TN-C KO

naïve (day 0)

Immunized (day 6)

WT

naïve (day 0)

LYVE-1 TN-C

Fig. S2. Distribution of α9 integrin and its ligand within draining (d)LNs after CFA injection. Draining LNs were removed on the indicated days after mice were injected with CFA. (A and B) Draining LN weight (A) and area (B). (C) Representative immunohistochemistry for LYVE-1 (red) and α9 or CD169 (green) of dLNs from naïve mice and mice at 6 d after injection of CFA. As shown in Fig. S1, according to the association of CD169+ macrophages with lymphatic sinuses, medullary and cortical sinuses were distinguished. Arrows and arrowheads indicate medullary and cortical sinuses, respectively (MSs and CSs, respectively). Yellow indicates areas of colocalization of LYVE-1 and α9 on MS and CS LECs. The graph shows the area of staining for LYVE-1 and α9. Error bars represent means (±SEM) (n > 3 per group). *P < 0.05 and **P < 0.01 denote values significantly different between the indicated pairs. (D) Representative immunohistochemistry for LYVE-1 (red) and TN-C or FN-EDA (green) of dLNs from naïve mice and mice at 6 d after injection of CFA (n > 3 per group). Yellow indicate areas of colocalization of LYVE-1 and TN-C or FN-EDA on MS and CS LECs. (Bottom) Negative controls (LNs of TN-C KO mice) are shown. (Scale bars, 100 μm.)


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c mLN area cont Ig

8

N.S.

0 55A2C

0 cont Ig

2 55A2C

4

5

cont Ig

mm2

mg

mm2

6 10

cont Ig 55A2C

55A2C

15

LYVE-1+ area 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0

N.S.

55A2C

b mLN weight N.S.

cont Ig

a

d

0.6 0.5 0.4 0.3 0.2 0.1 0

55A2C

N.S.

cont Ig

Ratio

CD169

Vacancy

cont Ig55A2C

cell number (x105)

e

CD4 T cells 12 10 8 6 4 2 0

N.S.

CD8 T cells 8

N.S.

6

6

4

4

2

2

0 cont Ig 55A2C

CD19 B cells 8

N.S.

0 cont Ig 55A2C

cont Ig 55A2C

Fig. S3. Effect of 55A2C on control LNs. Mice were treated with anti-α9 (55A2C) or control (cont Ig) antibody 1 d before CFA injection. Six days after CFA injection, mesenteric LNs (mLNs; nondraining LNs) were analyzed. (A and B) The mean weight (A) and area (B) of mLNs (n = 4 per group) are shown. (C) Mesenteric LN sections stained for LYVE-1. (Scale bars, 100 μm.) Areas of LYVE-1–positive regions were calculated by Scion Image (n = 4 per group). (D) Representative three-color staining of mLN sections with LYVE-1 (red), CD169 (green), and DAPI (blue) (n = 4 per group). (Scale bars, 200 μm.) Vacancy was calculated according to the formula vacant sinus number/total sinus number. (E) Total number of mLN CD4+, CD8+, and CD19+ cells per mouse for each group (n = 6), determined by FACS. Bars indicate means (±SEM).


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a

-2

-1

0 (days)

55A2C or cont Ig

2D2 CD4 transfer

analyze

mLN

N.S.

cell number (x104)

cell number (x104)

iLN 6 4 2 0

N.S.

6 4 2 0

b

55A2C or cont Ig

-1 CFSE2D2 CD4 transfer

cont Ig 54 6 32 1 0

3

1

0 MOG35-55 /CFA

55A2C or cont Ig

55A2C 5 6 4 32 1 0

CFSE

Cell number (x105)

-2

(days)

analyze

12 10 8 6 4 2 0

55A2C

0

1 2 3

4 5 6

Fig. S4. α9 integrin is not involved in T-cell homing and T-cell proliferation. (A) 2D2 CD4+ T cells (5 × 106 cells) were injected i.v. into recipient mice that had been treated with anti-α9 integrin (55A2C) or cont Ig. Twenty-four hours after T-cell transfer, 2D2 cells, defined as T-cell receptor (TCR) Vα3.2+Vβ11+, in inguinal (i)LNs and mLNs were counted by flow cytometric analysis. The graphs show 2D2 CD4+ T-cell number per LN. (B) Carboxyfluorescein succinimidyl ester (CFSE)-labeled 2D2 CD4+ T cells (5 × 106 cells) were transferred into recipient mice that had been treated with 55A2C or cont Ig. Twenty-four hours after transfer, mice were immunized with MOG35–55/CFA. Four days after cell transfer, CFSE fluorescence intensity of 2D2 cells obtained from iLNs of recipient mice was analyzed. Numbers indicate the 2D2 cell division number. Bars indicate means (±SEM) (n = 4 mice per group).


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2 1.5

N.S.

0.5 FTY720

cont Ig

55A2C

0 cont Ig55A2C FTY720

FTY720

N.S. N.S. N.S. N.S. N.S. 55A2C

0.16 0.14 0.12 0.1 0.08 0.06 0.04 0.02 0

cont Ig

FTY720

N.S. N.S. ** **

1

FTY720

55A2C

N.S. N.S. N.S. N.S. N.S.

55A2C

Cell number (x104)

N.S. N.S.

cont Ig

0.16 0.14 0.12 0.1 0.08 0.06 0.04 0.02 0

N.S. N.S.

2D2 / CD4

*

cont Ig

Cell number (x104)

60 50 40 30 20 10 0

b

2D2 / CD4

mLN

iLN

a

Fig. S5. Absolute number and 2D2 cell ratio in lymphocyte egress experiments using MOG/CFA. Absolute number and 2D2 cell ratio in Fig. 2 are shown. (A) Absolute numbers in iLNs and mLNs were counted by flow cytometric analysis. (B) The ratio of 2D2 cells to CD4 cells using the formula 2D2 CD4+ T cells/total CD4+ cells. White and black bars represent the absence or presence of anti-α4αL antibodies, respectively. *P < 0.05 and **P < 0.01 denote values significantly different between the indicated groups. N.S., no significant difference.


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a

FTY720 -1

0

3

4 (days)

anti 4 L 55A2C or non-treatment cont Ig

55A2C 2D2 CD4 or transfer cont Ig b

2

mLN

N.S.

N.S. N.S. ** **

2 1.5

4

1

2

0.5

0

0

** **

N.S.

N.S. N.S.

0

cont Ig

0.04

N.S.

N.S.

N.S.

N.S. N.S.

0.12 0.08 0.04

FTY720

N.S.

2D2 / CD4

0.12 0.08

**

0.16

55A2C

2D2 / CD4

0.16

200 160 120 80 40 0

N.S. N.S.

0 cont Ig

160 120 80 40 0

LN cell ratio

LN cell ratio

**

N.S.

N.S.

FTY720

6

N.S. N.S. ** **

4)

4)

iLN 8

analyze

55A2C

-2

1

non-treatment anti 4 L Fig. S6. Involvement of α9 integrin is not evident in lymphocyte egress in naïve mice. (A) Experimental protocol for flow cytometric analysis of lymphocyte egress. Antiα4αL, treatment with anti-α4 and -αL antibodies. (B) The number of transferred 2D2 CD4+ T cells in iLNs and mLNs, defined as TCR Vα3.2+Vβ11+. (Top) Absolute numbers. (Middle) A ratio using the formula (2D2 CD4+ T-cell number in antiα4αL-treated group/2D2 CD4+ T-cell number in nontreated group) × 100. (Bottom) The ratio of 2D2 cells to CD4 cells using the formula 2D2 CD4+ T cells/total CD4+ cells. Bars indicate means (±SEM) (n = 4 per group). White and black bars represent the absence or presence of anti-α4αL antibodies, respectively. **P < 0.01 denotes values significantly different between the indicated groups.


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a

FTY720 1 2 3 4 5

0

6

anti 4 L 55A2C 2D2 CD4 MOG35-55 55A2C or or transfer non-treatment cont Ig cont Ig iLN

55A2C

0

*

c

N.S. N.S. N.S. N.S. N.S.

cont Ig

*

0.16 0.14 0.12 0.1 0.08 0.06 0.04 0.02 0

2D2 / CD4

N.S.

**

160 140 120 100 80 60 40 20 0

FTY720

**

FTY720

0.5

iLN

55A2C

*. N.S.

LN ratio

1.5

cont Ig

Cell number (x104)

2

1

non-treatment anti 4 L

iLN

cont Ig

b

(days)

analyze

FTY720

-1

55A2C

-2

FTY720 -2

-1

1 2 3 4 5

0

55A2C 2D2 CD4 or transfer cont Ig

CFA

6

anti 4 L 55A2C or non-treatment cont Ig

(days)

analyze

non-treatment anti 4 L

**

300 250

1.5 LN ratio

**

1 0.5

**

200 150 100

FTY720

55A2C

0

cont Ig

FTY720

55A2C

50 0

0.16 0.14 0.12 0.1 0.08 0.06 0.04 0.02 0

N.S. N.S. N.S. N.S.

N.S.

FTY720

N.S.

cont Ig

N.S. **

iLN

2D2 / CD4

2

iLN

55A2C

iLN N.S.

cont Ig

Cell number (x104)

d

Fig. S7. Lymphocyte egress in either CFA- or MOG-immunized mice. (A and C) Experimental protocols for flow cytometric analysis of lymphocyte egress. (B and D) The number of transferred 2D2 CD4+ T cells in iLNs (dLNs), defined as TCR Vα3.2+Vβ11+. (Left) Absolute numbers. (Center) A ratio using the formula (2D2 CD4+ T-cell number in antiα4αL-treated group/2D2 CD4+ T-cell number in nontreated group) × 100. (Right) The ratio of 2D2 cells to CD4 cells using the formula 2D2 CD4+ T cells/total CD4+ cells. Bars indicate means (±SEM) [n = 6 per group (cont Ig and 55A2C), n = 3 (FTY720)]. White and black bars represent the absence or presence of anti-α4αL antibodies, respectively. *P < 0.05 and **P < 0.01 denote values significantly different between the indicated groups.


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100 80 60 40 20 0

b

0.4 OD590

% of max

a

VEGFR3

0.3 0.2 0.1 0 2.5

BSA rTN-C 9 integrin

c

* N.S.

GelaƟn rTN-C

d 4000

* C.P.M.

0.2 0.16 0.12 0.08 0.04 0

N.S.

N.S.

20

10

3000 2000

55A2C

cont Ig

1000

BSA

OD590

5

proteins ( g/ml)

-

% of max

10 100 80 60 40 20 0

0

( g/ml)

rTN-C Fig. S8. Characteristics of isolated ED-LECs. (A) ED-LECs were isolated as described in SI Materials and Methods and stained with anti-VEGFR3 and anti-α9 integrin antibodies. Fluorescence intensity is shown as open histograms, whereas the control (without first antibody) is shown as filled histograms. (B) Assay of ED-LEC adhesion to α9 integrin ligands. (C) Inhibition of adhesion by anti-α9 integrin (55A2C) treatment. Before cell seeding, cells were incubated with 55A2C or cont Ig (50 μg/mL). The cell-adhesion assay was performed as described in SI Materials and Methods. Bars indicate the means (±SEM) of triplicate wells. (D) Proliferation assay. ED-LECs (1 × 106 cells) were cultured for 48 h on plates coated with proteins as indicated and labeled with [3H]thymidine for the last 16 h of culture, and incorporated counts per min (C.P.M.) was measured. *P < 0.05 denotes values significantly different between the indicated groups.


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CD45

60

20 0 LEC 100

20 0 100

LEC

BEC

podoplanin

inflamed

100

60

% of max

CD31

naive

100

N.S.

50 MFI

BEC

30 10 0 N.S.

800 60

60

20 0 100 101 102 103

20 0

MFI

CD31

% of max

a

400 0

naive inflamed

100 101 102 103 9

day 6

TN-C FN-EDA G3PDH

100

100

80

80

% of max

% of max

d

60 40 20 0 100 101 102 103

9

MFI

100

3 6 day

20

1

80

1

N.S. N.S. N.S.

100

80

80

60 40 20 0 100 101 102 103

0

60 40 20 0 100 101 102 103

9EG7

3 6 day

unstained none 1 ng/ml 5 ng/ml 10 ng/ml

0 100 101 102 103

N.S. N.S. N.S.

50

0

60 40

9

unstained none IL-6 IL-1

e

9EG7 unstained none IL-17 IL-23

TGF** 0.15 0.1 0.05 0 naive inflamed

60 MFI

150

160 120 80 40 0

4 3 2 1 0

FN-EDA **

relative value

day 3

fold increase

naive

TN-C * **

100

% of max

c % of max

b

0 0 1 5 10

40 20 0

0 1 5 10

TGF(ng/ml)

Fig. S9. TGF-β did not affect α9 and pan-β1 integrin expression. (A) Flow cytometric analysis of α9 integrin expression on LECs. Cells obtained from iLNs of naïve mice or mice on day 3 after CFA injection were divided into LECs (CD45−CD31+podoplanin+) and blood vascular endothelial cells (BECs) (CD45+CD31+ podoplanin−) as previously reported, respectively (1, 2). Fluorescence intensity of α9 integrin is shown by open histograms, whereas isotype control is shown by filled histograms. The graphs show the mean fluorescence intensity (MFI) for α9 integrin. (B) Western blot analysis of TN-C and FN-EDA in the dLN. The graphs show the fold increase (set at 1 on day 0, normalized to G3PDH) (n = 3). (C) Effect of various cytokines on 9EG7 expression. Expression is indicated by open histograms, whereas the control is shown as filled histograms. (D) The expression of α9 and pan-β1 integrin when ED-LECs were treated with TGF-β as described in Fig. 4B. The graphs show MFI (n = 3). (E) Gene expression of TGF-β in the dLN at day 3 after CFA injection (n = 4). 1. Grigorova IL, et al. (2009) Cortical sinus probing, S1P1-dependent entry and flow-based capture of egressing T cells. Nat Immunol 10(1):58–65. 2. Pham TH, et al. (2010) Lymphatic endothelial cell sphingosine kinase activity is required for lymphocyte egress and lymphatic patterning. J Exp Med 207(1):17–27.


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Fig. S10. Schematic diagram of the possible role of α9 integrin in lymphocyte egress from dLNs. Under naïve, noninflamed conditions, (i) T cells migrate to the lymphatic sinus based on the S1P gradient, and lymphocyte egress is α9 integrin-independent. Therefore, (ii) anti-α9 antibody (55A2C) does not affect lymphocyte egress. (iii) FTY720 induces down-modulation of the S1P receptor on lymphocytes, thus inhibiting lymphocyte egress. Under conditions of tissue inflammation, lymphangiogenesis is induced in the dLN, increasing the interaction of α9 integrin and TN-C due to integrin activation. (iv) This leads to augmentation of lymphocyte egress, possibly by induction of S1P secretion. (v) Anti-α9 integrin antibody interferes with the interaction between α9 integrin and TN-C in the medullary and cortical sinuses and inhibits lymphocyte egress. (vi) FTY720 induces down-modulation of S1P receptors on lymphocytes, inhibiting lymphocyte egress.


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