European Journal of Immunology

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European Journal of. Immunology. Supporting Information for. DOI 10.1002/eji.201343429. Christina Hesse, Wiebke Ginter, Theresa F¨org, Christian T. Mayer,.
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European Journal of Immunology Supporting Information for DOI 10.1002/eji.201343429 Christina Hesse, Wiebke Ginter, Theresa Forg, Christian T. Mayer, ¨ Abdul Mannan Baru, Catharina Arnold-Schrauf, Wendy W. J. Unger, Hakan Kalay, Yvette van Kooyk, Luciana Berod and Tim Sparwasser In vivo targeting of human DC-SIGN drastically enhances CD8+ T-cell-mediated protective immunity

C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.eji-journal.eu

Supplemental Information In vivo targeting of human DC-SIGN drastically enhances CD8+ T cellmediated protective immunity Christina Hesse, Wiebke Ginter, Theresa Förg, Christian T. Mayer, Abdul Mannan Baru, Catharina Arnold-Schrauf1, Wendy W. J. Unger, Hakan Kalay, Yvette van Kooyk, Luciana Berod and Tim Sparwasser

OVA -DTT

1500

1000

Conjugate

250 150 100 75

500

50

VA O

25

SSC-Lin

15 +AZN-D1

Lymphocytes 60K

40K

40K

59.45 20K

66.63

20K

0

0 0

20K

40K

60K

0

FSC-Lin +AZN-D1

0.5

5

15

soluble OVA (µg/mL)

AZN-D1:OVA

+AZN-D1

10

4

10

3

10

2

20K

40K

10

1

10

0

60K

10

4

10

3

10

2

VA A

-D ZN

O 1:

VA

Live Cells

95.34

10

0

SSC Area

10

1

10

2

3

10

10

4

CFSE 400 300

DAPI

5 0

Single Cells

60K

DAPI

WT hSIGN

Medium

# CFSElow OT-II+ / Well (x103)

D

10

1

10

0

93.86

# Cells

C

:O pe y ot Is

SSC-Lin

OVA

10

0

OVA

37

20

Raji WT Raji DC-SIGN

MFI

Conjugate IgG1

B

AZN-D1 +DTT

AZN-D1

Isotype

AZN-D1:OVA

Isotype:OVA

OVA 1 µg

10 µg

AZN-D1:OVA -DTT

A

AZN-D1:OVA +DTT

Suppl.Fig1

200

69.56

30.44

100

100

101

CD4

102

103

104

0 100

101

102

103

104

CFSE

Suppl.Fig.1 (A) Chemical conjugation of OVA to the AZN-D1 and isotype control antibodies was verified using SDS-gel electrophoresis and silverstaining (left panel) or Western blot (right panel) using anti-OVA (mouse, Ig2a) primary antibody and rabbit-anti-mouse Ig2a secondary antibody (B) Binding of αDCSIGN:OVA (AZN-D1:OVA), isotype:OVA and soluble OVA protein to human DC-SIGN (black bars) or mock transfected (grey bars) Raji cells. Transfected Raji cells were incubate with 10µg/mL of AZN-D1:OVA, isotype:OVA or soluble OVA. Cells were washed and stained with a fluorescent labelled secondary goat-αmouse IgG1 antibody. The mean fluorescence intensity is compared (C) WT or hSIGN BMDCs were loaded with different concentrations of soluble OVA protein with or without 0.5µg/mL unconjugated αDC-SIGN antibody (AZN-D1), medium or 0.5µg/mL αDC-SIGN antibody conjugated to OVA (AZN-D1:OVA). Proliferation of CD4+OT-II cells was assessed using CFSE dilution. The number of CFSElowCD4+OT-II T cells is displayed. (D) Progressive gating strategy for the identification of proliferating CD4+OT-II and CD8+OT-I T cells (Suppl.Fig.1C)

Suppl.Fig 2 Supplementary to figure 1 A Lymphocytes Single Cells 30K

Live Cells

Proliferating cells

30K

30K

SSC-Lin

10K

0

10K

20K

0

10K

20K

30K

96.18 0

SSC Area

10

1

10 OT-II 8 6 4 2

10

3

0 10

1

10

2

10

3

0 0

10

CD4 / CD8

10

1

10

2

10

3

10

4

CFSE

14 OT-I 12 10 8 6 4 2 0

M

Concentration in µg/mL

2

101

0

1

0. 5

0. 1

1

5 0. 0

0. 0

0. 0

05

0

M ed i

2

10

eFl450

C

um

# CFSElow OT-II+ / Well (x104)

FSC-Lin

B

0

0

30K

10

10K

# CFSElow OT-I+ / Well (x104)

92.18

0

2

101

Aqua

10K

10

84.54

103 20K

SSC-Area

20K

SSC-Lin

20K

CD4 / CD8

103

m 05 .01 .05 iu .0 0 0 d 0 e

1 0.

5 0.

1

Concentration in µg/mL

WT + isotype:OVA + CD40 WT + DC-SIGN:OVA + CD40 hSIGN + isotype:OVA + CD40 hSIGN + DC-SIGN:OVA + CD40

Suppl.Fig.2 (A) Progressive gating strategy for the identification of proliferating CD4+OT-II T cells (Fig.1C and Suppl.Fig.2B,C). (B,C) Total numbers of (B) CFSElowCD4+OT-II and (C) CFSElowCD8+OT-I T cells cocultured with pulsed WT or hSIGN DCs receiving increasing doses of αDC-SIGN:OVA or isotype:OVA antibody plus αCD40. Mean of duplicate wells from one out of three experiments is depicted.

Suppl.Fig.3 A

C Counts

WT hSIGN 92.8%

200K 150K

SSC-A

8.4%

250K

100K

cDC

pDC

50K

74.70

0 0

hDC - SIGN

B

50K 100K 150K 200K 250K

FSC-A 250K

10

5

10

4

10

3

200K

CD8

80.75

100K

Aqua

96.0%

SSC-A

150K

10

50K

2

0 0 0

99.9%

SiglecH

85.9%

10

5

10

4

10

3

10

2

103

104

105

Linage neg

pDC cDC CD8

Counts

SSC-W

CD4

18.08 0 102

50K 100K 150K 200K 250K

0

10

5

10

4

10

3

10

2

0 0 10

2

CD11c

10

3

10

4

10

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0 10

2

10

3

10

4

10

5

CD4

hDC - SIGN

Suppl.Fig.3 (A) Expression of human DC-SIGN on cDCs (CD11chighSiglecH-, left) and pDCs (CD11cintSiglecH+, right). Human DC-SIGN is expressed on hSIGN (black line), but not on WT (grey) DCs. (B) Expression of human DC-SIGN on cDC subsets (CD4+, CD8+ and double negative CD11c+ cDCs), One representative of three experiments. (C) Progressive gating strategy for the identification of different DC subsets. Linage positive cells are defined by NK1.1, CD19 and CD3 expression (Suppl.Fig.3A,B).

Suppl. 4 Supplementary to Fig.2 Lymphocytes

20K

20K

30K

10K

23.13

0 0

FSC-Lin

20K

30K

84.33 10K

SSC Area

2 1 3 5 7 9 Days post-immunisation

20K

10

1

30K

C

3

1

2

99.03

0 0

4 OT-II

0

10

0

% OT-I+ of total CD8+

% OT-II+ of total CD4+

B

10K

10K

3

PI

SSC-Lin

SSC-Lin

10

11

0

10

PE

1

10

2

10

3

CD45.1

30K

Transferred Cells

Live Cells

Single Cells

30K

SSC-Area

A

20K

10K

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3

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0 0 0 10

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CD4 / CD8

10

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CD4 / CD8

10

3

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4

30 OT-I 20 hSIGN+ Isotype:OVA

10

hSIGN+ Isotype:OVA + CD40 hSIGN+ DC-SIGN:OVA

0

hSIGN+ DC-SIGN:OVA + CD40

1

3 5 7 9 11 Days post-immunisation

Suppl.Fig.4 (A) Progressive gating strategy for the identification of adoptively transferred CD45.1+CD4+OT‑II or CD45.1+CD8+OT‑I T cells (Fig.2 and Suppl.Fig.4B,C). (B,C) Expansion of adoptively transferred (B) CD45.1+CD4+OT‑II or (C) CD45.1+CD8+OT‑I in the blood of WT or hSIGN mice treated with αDC-SIGN:OVA or isotype:OVA with or without αCD40 on day 1 to 11 post-immunisation. 3x105 enriched CD4+OT-II or CD8+OT-I T cells were transferred. Mean of 2 mice/group. One of two experiments performed.

Supp.Fig. 5 Supplementary to Fig. 3 + CD40

***

0.1%

0.3%

hSIGN 0.04%

0.04%

100 n.s.

80

MOG35-55

60 40

72.0%

50.1%

72.2%

99.2%

20

DC-SIGN:OVA CD40

C

B

WT

+ -

+ -

+ +

WT

hSIGN

Lymphocytes

SIINFEKL

+ +

IFN

% IFN

+

of CD45.1+CD8+

A

hSIGN

WT

CD8

Single Cells

Live Cells

FSC-Lin

# Cells

PI

SSC-Area

80.51

SSC-Lin

SSC-Lin

72.90

81.52

SSC Area

CFSE

46.88

53.12

6.65

CFSE

CFSE

Suppl.Fig.5 (A) Frequency of IFNγ-producing CD45.1+CD8+OT‑I T cells in immunised WT and hSIGN mice after ex vivo re-stimulation with SIINFEKL peptide. Bar graphs represent frequencies from pooled data from two experiments. (B) Representative dot plots from data shown in Fig.3 and Suppl.Fig.5A re-stimulated with SIINFEKL as well as re-stimulation with an irrelevant peptide (MOG35-55). (C) Progressive gating strategy used to calculate specific killing of SIINFEKL-loaded spleen cells (Fig.3B,C).

SUPP 6 A

Lymphocytes

Single Cells

Live Cells

EMA

SSC-A

SSC-A

92.90

52.64

98.97

FSC-A

SSC-H

PE

8.13

CD8

SIINFEKL

11.60

SIINFEKL

CD62L

Suppl.Fig.6 (A) Progressive gating strategy for the identification of CD62Llowpentamer+CD8+ T cells of immunised WT and hSIGN after infection (Fig.4B,C and Fig.5E).

Supplementary to Figure 5

1.0

IgG2c

OD405 nm

0.8 0.6 0.4 0.2 0.0

*

B

10

CFU / Liver (log10)

A

1:500

1:2000 1:4000 1:8000 Dilution

WT non-vaccinated WT vaccinated hSIGN non-vaccinated hSIGN vaccinated

8 6 4 n.s.

2

DC-SIGN:OVA CD40 OVA:IFA boost

n.s.

WT

+

+ + +

+ + +

+ + -

hSIGN

Suppl.Fig.7 (A) OVA-specific IgG2c titration in the serum of vaccinated WT and hSIGN mice measured by ELISA. (B) Bacterial burden in the spleen of vaccinated WT and hSIGN mice 4 days after infection with LMOVA (2x105 CFU) and 39 days after initial immunisation.

Supplementary Methods Binding Assay Stable Raji transfectants expressing the human DC-SIGN receptor (Raji DC-SIGN) were generated as previously described in (Geijtenbeek et al., 2000). Raji or Raji- DC-SIGN cells were then incubated with 10µg/ml soluble OVA, AZN-D1:OVA or isotype:OVA at 4°C for 30min. After washing, cells were incubated with APC-labelled goat-anti-mouse IgG1 antibody (GaM-IgG-AF647, Molecular Probes) for 30min on ice. The mean fluorescence intensity was determined by flow cytometry (Facs Calibur, BD Biosciences). Western Blot and Silverstain Samples were boiled in Laemmli sample buffer before proteins were separated on a 7.5-8% sodium dodecyl sulfate (SDS)-gel and transferred by electroblotting onto PVDF membranes (Immobilon-P, Millipore). Membranes were blocked with 5% dry milk and incubated with primary antibodies detecting OVA (IgG2a, clone: 6G2, AntibodyShop). Membranes were washed in 0.1% Tween/Tris(hydroxymethyl)aminomethane– buffered saline and incubated with the respective horseradish peroxidase–conjugated secondary antibody: rabbit-anti–mouse-immunoglobulin (anti-IgG2a, Invitrogen). Detection was performed using enhanced chemiluminescence reagent (Pierce). Silverstaining was performed using the BioRad silverstain kit according to manufacturer’s instructions.

Publication Geijtenbeek, T.B., Kwon, D.S., Torensma, R., van Vliet, S.J., van Duijnhoven, G.C., Middel, J., Cornelissen, I.L., Nottet, H.S., KewalRamani, V.N., Littman, D.R., et al. (2000). DC-SIGN, a dendritic cell-specific HIV-1binding protein that enhances trans-infection of T cells. Cell 100, 587-597.