Supporting Online Material for - Sciencescience.sciencemag.org/content/suppl/2007/01/02/315.5808.107.DC… · Supporting Online Material for Differential Antigen Processing by Dendritic

www.sciencemag.org/cgi/content/full/315/5808/107/DC1

Supporting Online Material for

Differential Antigen Processing by Dendritic Cell Subsets in Vivo

Diana Dudziak, Alice O. Kamphorst, Gordon F. Heidkamp, Veit Buchholz, Christine Trumpfheller, Sayuri Yamazaki, Cheolho Cheong, Kang Liu, Han-Woong Lee, Chae

Gyu Park, Ralph M. Steinman, Michel C. Nussenzweig*

*To whom correspondence should be addressed. E-mail: [email protected]

Published 5 January 2007, Science 315, 107 (2007) DOI: 10.1126/science.1136080

This PDF file includes:

Materials and Methods Figs. S1 to S11 References

Dudziak et al (SOM) Supporting Online Material

Materials and Methods

Mice. Specific pathogen-free, adult (6-8 weeks) C57BL/6, CD45.1/B6.SJL (B6.SJL-

Ptprc), C3H/HeJ, B10.BR and C57BL/6 OT-II transgenic mice were purchased from

Jackson Laboratory. C57BL/6 OT-I transgenic mice were bred at the Rockefeller

University. Human DEC205 cDNA (GenBank Accession number AY682091) was

cloned into a synthetic CD11c promoter (GenBank Accession number DQ658851) (S1,

S2) The linearized construct was injected into C57BL/6 fertilized pronuclei, and

transgene positive mice were backcrossed to C57BL/6 or B10.BR. All experiments with

mice were performed in accordance with NIH guidelines and approved by the

Rockefeller University animal care and use committee.

Flow Cytometry. Antibodies were biotin-, PE- or FITC- conjugated anti CD1d (1B1),

CD3 (145-2C11), CD4 (L3T4), CD8 (53-6.7), CD11b (M1/70), CD11c (N418, HL3),

CD22 (Cy34.1), CD24 (HSA, M1/69), CD40 (3/23), CD44 (IM7), CD45.1 (A20),

CD45.2 (104), CD69 (H1.2F3), CD80 (B7.1, 16.10A1), CD86 (B7.2, GL1) Vα2 (B20.1),

MHCII (AF6-120.1), αH2-M (kindly provided by L. Denzin), mouse IgG1 (A85-1), goat

anti mouse IgG, control rat IgG2a and rat IgG2b (all from eBioscience, BD Pharmingen

or Jackson Immunotech). αDEC205 (NLDC-145), 33D1 (DC) and Aw3.18.14 (αI-Ak-

HEL, ATCC) (17), were purified and labeled with biotin, Alexa Fluor 488 or Alexa Fluor

647 (MSKCC, Rockefeller Monoclonal Antibody Core Facility). Isotype controls rat

IgG2a and rat IgG2b A488 and A647 were from Molecular Probes. Intracellular staining

was performed according to the manufacturer’s protocol (BD Pharmingen). Data was

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Dudziak et al (SOM) acquired on a FACSCaliburTM. Analysis was performed using FlowJo (Treestar) and

WinMDI.

Cloning of 33D1 and hDEC205 antibodies. RNA was prepared from 33D1 (rat IgG2b,

kappa) or αhDEC205 (mouse IgG2b, lambda, clone MG38.2 (18)) hybridoma cells,

respectively using RNeasy Midi Kit (Qiagen). For cloning the variable regions of the rat

33D1 antibody 5’RACE PCR was performed according to the manufacturer’s instructions

(Invitrogen) using the following oligonucleotides for light chain (LC) and heavy chain

(HC): 3.1 33D1-LC: 5’-GGGTGAGGATGA-3’, 3.2 33D1-HC: 5’-CTTGGTGCTGC

TG-3’. In the second amplification round the following nested primers were used: 3.3

33D1-LC 5’-CAACCTCACAGGTATAGAG-3’ and 3.3 33D1-HC 5’-GGGCTACGTTG

CAGGTGAC-3’. The variable regions were subcloned by overlapping PCR into

DEC205-OVA HC and DEC-kappa LC (S3) with 5.1 33D1-HC 5’-GCGGGGGAATTC

GCCACCATGGACATCAGGCTCAGCTTG-3’, 5.12 33D1-HC 5’-CATGGTCACAG

TCTCCTCAGCCAAAACGACA-3’, 3.27 33D1-HC 5’- ATGGGGGTGTCGTTTT

GGCTGAGGAGACTGT-3’, 3.28 33D1-HC 5’-CCCCGGGCTAGCTTTACCAGGA

GAGTGGGAG-3’and 5.1 33D1-LC 5’-GC GGGGGAATTCGCCACCATGGCCGTG

CCCACTCAG-3’, 5.2 33D1-LC 5’-CCAAGCTGGAATTGAAACGGGCTGATGCT

GCAC-3’, 3.7 33D1-LC 5’-GTGCAGCATCAGCCCGTTTCAATTCCAGCTTGG-3’,

3.8 33D1-LC 5’-CCCCGGGCGGCCGCTCAACACTCATTCC-3’.

The variable regions of the αhDEC205 antibody were produced with 5’-RACE PCR

(Invitrogen) using primers specific for 3’-ends of mouse IgG2b and Ig lambda. Specific

primers for cloning of mouse IgG2b and Ig lambda V region: 2bI GSP1-hDEC-HC 5’-

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Dudziak et al (SOM) TACTTGTGGAGCTCTGACTAG-3’, 2bI GSPII-hDEC-HC 5’-TTAATTTTTGA

GATGGTTCTCTCG-3’; Lambda 2+3 GSP1-hDEC-LC 5’-ACTCTTCTCCACAGTGT

CC-3’; Lambda 2+3 GSP2-hDEC-LC 5’-AACTGTTGTGAGATCTCCACTGGTC-3’.

To obtain full-length heavy Ig cDNA, the V region encoding for heavy chain was cloned

in frame with a signal peptide and IgG1 constant domain expressing the HEL peptide at

its C-terminus (14) using the following primers: 5’leader-SP 5’-TGCCAAGAGT

GACGTAAGTACCGC-3’, V1 heavy-hDEC-HC 5’-GCAACTGGAGTACATTCAGA

GGTCCAGCTGCAACAGTCTGGAC-3’, V2 heavy-hDEC-HC 5’-TGGGCCCTTGG

TGGTGGCTGAGGAGACTGTGAGAGTGGTGCCTTG-3’, C1 heavy-hDEC-HC 5’-

ACTCTCACAGTCTCCTCAGCCACCACCAAGGGCCCATCTGTC-3’, 3’end-hDEC-

HC 5’-CAAACCACAACTAGAATGCAG-3’. Full-length lambda Ig cDNA was

obtained by cloning the lambda V region in frame with a signal peptide (14) and mouse

Ig lambda constant domain using 5’leader-SP 5’-TGCCAAGAGTGACGTAAGTACC

GC -3’, V1 lambda Dec 5’-GCAACTGGAGTACATTCACAGGCTGTTGTGACTC

AGGAATCAG-3’, V2 lambda Dec 5’-GGGAGTGGACTTGGGCTGACCTAGGACA

GTGACCTTGGTTCC-3’C1 lambda Dec 5’-AAGGTCACTGTCCTAGGTCAGCCCAA

GTCCACTCCCACACTC-3’, 3’end lambda Dec 5’-ATAGTTTAGCGGCCGCTTA

GAGACATTCTGCAGGAGACAGAC T-3’.

Production of chimeric antibodies and targeting. Chimeric antibodies were expressed

by transient transfection as described (14). All antibodies were tested for LPS

contamination (Fisher-Cambrex) and decontaminated when necessary (Pierce). Each

batch was tested for binding to splenic DCs by FACS analysis. Mice were injected

4

Dudziak et al (SOM) intraperitoneally (i.p.) or intravenously (i.v.) with antibodies with or without 50 µg IC10

agonistic αCD40 monoclonal antibody (MSKCC, Rockefeller Monoclonal Antibody

Core Facility), or 30 µg LPS (E.coli Serotype 0111:B4, Sigma).

Adoptive transfer and T cell proliferation responses. CD4+ OT-II T cells were

enriched and labeled with CFSE (Molecular Probes, 5-(6)-carboxyfluorescein diacetate

succinimidyl diester) as described (14, 16) and 1-2 x 106 OT-I or 3-4 x 106 OT-II T cells

were injected i.v. into B6.SJL mice; targeting antibodies were injected i.p. 24 hours later

with or without maturation stimuli. In the experiments to address antigen persistence

antibodies were injected 10, 7, 5, 3 or 1 day before T cell transfer. In vitro proliferation

assays with CD4+ OT-II or CD8+ OT-I T cells were performed as described (14, 16). For

subset purification, DCs from C57BL/6 or CD11c-hDEC205 transgenic mice were

negatively enriched with a mixture of CD19, DX5, CD90.2 beads (Milteny) and sorted

into CD11cHighCD8- and CD11cHighCD8+ DCs. FACS sorting resulted in 99% pure

populations. CD4+ OT-II or CD8+ OT-I T cells were as described above. In control

experiments either H-2Kb-restricted OT-I peptide (SIINFEKL, 1.0 µM) or I-Ab-restricted

OT-II peptide (LSQAVHAAHAEINEAGR, 2.0 µM) was added to the cultures. T cell

proliferation was determined by [3H]-thymidine incorporation.

Internalization Assay. DCs were purified from collagenase treated spleens by negative

enrichment with CD19, DX5 and CD90.2 microbeads (Milteny) and incubated with 3

µg/ml purified rat 33D1, αDEC205 (NLDC-145), rat IgG2a, and rat IgG2b antibodies for

30 min on ice. Cells were further incubated on ice (0 min) or at 37°C for 30 to 60 min in

5

Dudziak et al (SOM) a heating block in 100 µl RPMI medium supplemented with 5% FCS, 100 U/ml

penicillin, 100 µg/ml streptomycin, 1 mM sodium pyruvate, 2 mM L-glutamine. The

reaction was terminated by fixation with paraformaldehyde before staining with mouse

anti rat Cy5 labelled secondary antibody (Jackson ImmunoResearch), CD11c PE and

CD8 FITC (BD Pharmingen).

Retroviral infection of BMDCs. DCIR2 was cloned into the vector pMX-PIE carrying

an IRES-GFP. BOSC 23 cells were cotransfected with pMX-PIE::DCIR2 and pCL-ECO

plasmids and supernatants harvested after 48 hours. BMDCs were prepared with GM-

CSF (S4) and spin-infected with supernatants containing retrovirus carrying DCIR2-

IRES-GFP mixed with 10 µg/ml polybrene and 20 mM HEPES on day 2, 3 and 4. Cells

were assayed for expression of DCIR2, DEC205 and GFP on day 6, and sorted into

CD11c+GFP+ and CD11c+GFP- BMDCs. The purified cells were incubated for 16 hours

with 0.5 µg/ml 33D1-OVA, αDEC205-OVA, Iso-OVA or PBS and further 12 hours in

the presence of 100 ng/ml LPS, then washed and cocultured with 1x105 OT-II T cells. T

cell proliferation was determined by [3H]-thymidine incorporation 48 hours later.

Immunoblots. Purified DCs (see above) were lysed in RIPA-buffer (Triton-X 100,

NP40) containing protease inhibitors (PMSF, Na-vanadate, EDTA-free protease-inhibitor

cocktail (Roche)), incubated on ice for 30 min, and debris spun out at 14,000 rpm, 4°C.

Samples (50 µg) were separated on 4-20% acrylamide Tris/Glycine/SDS gels (Fisher),

transferred to PVDF membranes (Millipore) and blotted with antibodies in TBST/3%

milk as described (S5, 21) to Cathepsin H (N-18), Gilt (T-18), TAP1 (M-18), Tapasin (D-

6

Dudziak et al (SOM) 16), Cystatin C (P-14), Calnexin (H-70), Calreticulin (T-19) (all from Santa Cruz),

LAMP-1 (1D4B, BD-Pharmingen), AEP (kindly provided by Collin Watts, UK), beta-

Actin (AC15, Sigma), and incubated with secondary goat anti mouse, donkey anti sheep,

donkey anti rat or donkey anti rabbit antibodies respectively (Jackson Immunotech).

Western blots were developed using enhanced ECL (Pierce). Immunoblots were

quantified using Scion Image analysis software.

Immunofluorescence. Spleens from C57BL/6 mice were embedded in optimum cutting

temperature compound and frozen at -80°C. Frozen tissue was sectioned 20 µm in

thickness on a microtome and was fixed in acetone. All incubations were done in a

humidified chamber. Sections were blocked in 5% BSA in PBS and were sequentially

blocked with excess streptavidin and biotin (Vector Laboratories). The primary

antibodies were B220 A647 (CalTag), purified 33D1 and biotin-anti-mouse DEC205 (the

Rockefeller University Monoclonal Antibody Core Facility, New York, New York). The

33D1 signal was then amplified by incubation with FITC-anti-rat IgG followed by Alexa

488-anti-FITC (Jackson ImmunoResearch). The DEC205 signal was amplified by

sequential incubation with PE-streptavidin (BD Pharmingen), purified rabbit-anti-PE

(Acris) followed by Cy3-anti-rabbit IgG (Jackson ImmunoResearch). Sections were

mounted in Fluoromount-G (Southern Biotech) and were stored at 4°C until microscopic

examination.

Confocal microscopy. Confocal images were acquired on a Zeiss LSM 510 system with

488-, 543- and 633-nm excitation lines at the Rockefeller University Bio-Imaging

7

Dudziak et al (SOM) Facility. Tiled images were obtained with the motorized stage using a 40x objective.

Microarray. CD11c cells were enriched from spleens of WT or B16 melanoma cells

secreting Flt3L injected mice (S6) by negative depletion with DX5, CD19 and CD90.2

MACS beads. Cells were stained with DEC205-bio, then with CD11c-PE, 33D1-A647

and SAPECy7, and purified by cell sorting into CD11cHigh33D1 and CD11cHighDEC205.

B cells (enriched with CD43 beads) were sorted into B220+CD19+ B cells. T cells were

enriched with Thy1.2 beads (Milteny) and FACS sorted into CD3+CD4+ or CD3+CD8+ T

cells. All cell samples were purified to more than 99% homogeneity. Total RNA was

prepared using Qiagen RNeasy Mini kit (Qiagen). DNA microarray analysis of gene

expression was performed at the gene array facility (MSKCC, New York). Fluorescent

images of hybridized microarrays (Affymetrix, MOE-430 2.0) were obtained using an

Affymetrix Genechip Scanner. Microarray data were analyzed using Affymetrix

GeneSpring 7.0 software. All samples were repeated at least three times with individually

sorted cells and averaged. The data discussed in this publication have been deposited in

NCBIs Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) and are

accessible through GEO Series accession number GSE6259.

Cloning of Lectins. Total RNA of mouse splenocytes was extracted using the RNeasy

Midi kit (Qiagen). Single-stranded cDNA was synthesized from 5 µg of total RNA by

reverse transcription using Superscript II reverse transcriptase (Invitrogen Life

Technologies) and an oligo-dT primer (Amersham Biosciences) at 42°C. cDNA was

amplified with primers designed to amplify the entire coding sequence of DCIR1 5’-

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http://www.ncbi.nlm.nih.gov/geo/

Dudziak et al (SOM) GCGGGGGAATTCGCCACCATGGCTTCAGAAATCACTTATG-3’,5’-CCCCGG GC

GGCCGCTCATAAGTTTATTTTCTTCATCTG-3’ DCIR3 5’-GCGGGGGAATTC

GCCACCATGTTTTCAGAAAACATTTATGTTAAC-3’, CCCCGGGCGGCCGCTCA

TAAGTATATTTTTTTCACATGGC-3’, Dectin-2 5’-CCCCGGGCGGCCGCTCATAG

GTAAATCTTCTTCAT-3’, 5’-GCGGGGGAATTCGCCACCATGGTGCAGGAAA

GACAATCC-3’ DC-Sign 5’-GCGGGGGAATTCGCCACCATGAGTGATTCTAAG

GAAATG-3’, 5’-CCCCGGGCGGCCGCTCACTTGCTAGGGCAGGAAG-3’, DCAR

5’-GCGGGGGAATTCGCCACCATGGTTCAGGAAAGACAGCTACAAG-3’, 5’-

CCCCGGGCGGCCGCTCATAAGTTTATTTTCTTCATCTGAC-3’, Clec4g 5’-

GCGGGGGAATTCGCCACCATGAACACTGGTGAATACAACAAGC-3’, 5’-CCCC

GGGCGGCCGCTTAGTAGCAACTGCTCCTCTTCTCAC-3’, as well as candidate

genes DCIR2 5’-GCGGGGGAATTCGCCACCATGGCTTCAGAAATCACTTATGC

AG-3’, 5’-CCCCGGGCGGCCGCTCATAAGTATATTTTCTTCACCTGAC-3’ and

DCIR4 5’-GCGGGGGAATTCGCCACCATGGCATTACCAAACATTTATACTGAC

GTG-3’, 5’-CCCCGGGCGGCCGCTCATACATAGAGCTGCCTCATCTCACAAA

TC-3’ (Note: DCIR2 and DCIR4 were not included in MOE-430 2.0 micro array

Afffymetrix chip). PCRs were performed using PFU DNA Polymerase (Promega) as

follows: 94°C for 4 min, than 35 cycles of 94°C for 1 min, 54°C for 1 min, and 72°C for

1-3 min, followed by a final extension step at 72°C for 10 min (PerkinElmer). The

amplified fragments were cloned into the pcDNA3.1 vector and sequenced. Candidate

cDNAs were transfected into 293T cells using Lipofectamine (Invitrogen) and 2 days

later transfected cells were FACS analyzed for binding of 33D1-A647 antibody.

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Dudziak et al (SOM) Supporting Figures and Legends

Fig. S1

Fig. S1. Surface features of 33D1 expressing DCs in the spleen. (A) Dot plots show

comparison of spleen cells gated on CD11cHigh, stained with 33D1 A647 and DEC205

bio/SAPerCP (upper panel) vs. CD4 PerCP and CD8 APC (lower panel). (B) Dot plots

show expression of CD1d, CD4, CD8, CD11b, CD22, CD24, CD44, CD80, CD86 and

MHCII on 33D1 (upper row) or DEC205 (lower row) expressing cells (C) Isotype

control staining with IgG2b A647 and IgG2a bio/SAPerCP (upper panel) or IgG2a A488

and IgG2b A647 (lower panel).

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Dudziak et al (SOM) Fig. S2

11

Dudziak et al (SOM)

Fig. S2. 33D1 antibody recognizes the C-type lectin DCIR2. (A) Splenocytes were

incubated with 33D1-A647 antibody in the presence of different concentrations of EDTA

for 30 min. Cells were fixed in 2% PFA and further incubated with anti CD11c-PE and

DEC205-A488. FACS analysis shows gated CD11cHigh cells. 33D1 staining was

diminished at a concentration of 0.05 mM EDTA. A representative experiment of three is

shown. (B) List of candidate genes of Affymetrix microarray showing differences

between CD8+DEC205+ and CD8-33D1+ subsets in WT and Flt3 injected mice (Fold

difference). (C) Sequence of DCIR2. Shown are the nucleotide sequence and the

corresponding amino acid sequence. DCIR2 is a type II transmembrane C-type lectin

(transmembrane domain is underlined) of 236 aa consisting of 6 exons. The molecular

weight is 27.25. The intracellular N-terminus contains an potential ITIM (ITYAEV, S7)

and an endocytosis (YAEV) motif as well as an additional Tyr at position 22. The

extracellular domain codes for a C-type lectin domain. DCIR2 belongs to the mannose

binding lectins as it includes an EPN motif. The Poly A tail of the mRNA contains rapid

RNA degradation motifs (ATTTA, underlined).

12


Fig. S3. Cloning the 33D1 antibody. (A) Diagrammatic representation of chimeric

antibodies. (B) A Coomassie stained 12% SDS-PAGE reducing gel comparing chimeric

33D1-OVA, and 33D1-rat antibodies with molecular weights in kDa indicated. (C) Dot

plot shows splenocytes stained with chimeric antibody 33D1-OVA and anti-mouse IgG

PE secondary, αDEC205-A647 and αCD11c-FITC after gating on CD11cHigh DCs. (B-C)

Antibody purity and binding was tested for every newly produced batch of antibody.

13


Fig. S4. Internalization of 33D1-OVA and DEC-OVA. Histogram shows intracellular

αDEC205 or 33D1 antibodies in CD8+DEC205+ and CD8-33D1+ DCs 30 minutes after

intravenous injection of 10 µg of αDEC205-OVA or 33D1-OVA or Iso-OVA control,

visualized with anti-mouse IgG-FITC. Arrows indicate significant staining when

compared to controls.

14


Fig. S5. Chimeric antibody injection does not induce DC maturation in vivo.

Histograms show expression of the indicated maturation markers by CD8+DEC205+ and

CD8-33D1+ spleen DCs 12 hours after mice received OT-II T cells (4x106) and 6 hours

after injection with 3 µg 33D1-OVA, αDEC-OVA, Iso-OVA or PBS (A) or in (B) with

additional 30 µg LPS. Data represent four independent experiments.

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Fig. S6. CD8+DEC205+ and CD8-33D1+ DCs present processed MHC-I and MHC-II

peptides. Bar graphs show [3H]-thymidine incorporation by (A) OT-I or (B) OT-II T

cells cultured with CD8+DEC205+ and CD8-33D1+ DCs purified from C57BL/6 mice

injected with either 3 µg 33D1-OVA or αDEC-OVA and pulsed in vitro with the

appropriate cognate peptide at a concentration of (A) 1 µM OT-I or (B) 2 µM OT-II. The

panels are representative of two experiments.

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Fig. S7. Cloning the hDEC205 antibody. (A) A Coomassie stained 4-20% SDS-PAGE

reducing gel comparing hDEC205-HEL chimeric, and hDEC205 (MG38.2) antibodies

with molecular weights in kDa indicated. (B) Histogram shows staining of hDEC205

transfected CHO and untransfected controls stained with 1 µg/ml hDEC205-HEL or Iso-

HEL antibody followed by anti-mouse IgG PE secondary antibody. (C, D) Dot plot

shows CD11cHigh splenocytes from (C) CD11c-hDEC205+ transgenic and (D) or B10.BR

WT mice stained with biotinylated αhDEC205 (MG38.2) followed by incubation with

SAPE, αCD8 APC and αCD11c FITC.

17


Fig. S8. Both CD8+DEC205+ and CD8-33D1+ DCs in CD11c-hDEC205+ transgenic

mice are targeted by αhDEC205-HEL in vivo. Histograms show extracellular IgG1 on

CD8+DEC205+ and CD8-33D1+ DCs 30 minutes after intravenous injection of 30 µg of

αhDEC205-HEL (black) or PBS (grey) on CD11c-hDEC205+ transgenic (left) and

CD11c-hDEC205- (right) control WT mice. Arrows indicate significant staining when

compared to controls.

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Fig. S9. CD4 T cell response to antigen targeted to DCs in CD11c-hDEC205

transgenic mice in vitro. Graphs show [3H]-thymidine incorporation by 1x105 OT-II T

cells cultured with the indicated number of irradiated CD8-33D1+hDEC205+ and

CD8+DEC205+hDEC205+ DCs purified from CD11c-hDEC205 transgenic mice injected

with 10µg 33D1-OVA (top panel), αDEC-OVA (middle panel) or αhDEC-OVA (lower

panel) 12 hours earlier. Control CD11c cells were purified from Iso-OVA and PBS

injected mice. Panels are representative of experiments repeated 2 times. The experiment

shows that only CD8-33D1+hDEC205+ DCs efficiently present on MHCII when both

subsets are targeted in the hDEC205 transgenic mice.

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Fig. S10. Comparative analysis of DCIR2 and DEC205 expressing BMDCs. BMDCs

were prepared and retrovirally transduced with DCIR2. (A) Histograms show 33D1 and

IgG2b (left) and αDEC205 and IgG2a (right) staining on day 6 gated on CD11c+GFP-

positive (GFP pos) and CD11c+GFP negative (GFP neg) DCs. (B) Retrovirally

transduced BMDCs were sorted into CD11c+GFP+ (DCIR2+, left) and CD11c+GFP-

(DCIR2-, right) DCs. BMDCs were incubated o/n with 1 µg/ml 33D1-OVA, αDEC-

OVA, Iso-OVA or PBS and for further 12 hours in the presence of 100 ng LPS to

stimulate DC maturation and antigen presentation (22), washed and cocultured with

1x105 OT-II T cells. Graph shows [3H]-thymidine incorporation. Data represent three

independent experiments. This experiment cannot be done with spleen or LN DCs since

they do not divide in vitro and die rapidly.

20


21

Dudziak et al (SOM) Fig. S11. Analysis of expression pattern of MHC class I and MHC class II associated

molecules. (A) List of MHCII (upper part) and MHCI (lower part) processing pathway

associated molecules on Affymetrix microarray showing expression differences between

CD8+DEC205+ and CD8-33D1+ DC subsets in WT and Flt3L injected mice (Fold

difference). (B) Ratio of the expression of MHCII (upper part) and MHCI (lower part)

associated proteins in CD8+DEC205+ and CD8-33D1+ DC subsets of WT mice measured

by immunoblot data as analyzed by Scion Image software (Fold difference). LAMP1 was

used as a loading control and all data were corrected based on LAMP1 expression before

calculating the ratios.

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Dudziak et al (SOM) Supporting References

S1. T. Brocker, M. Riedinger, K. Karjalainen, J Exp Med 185, 541 (1997). S2. R. L. Lindquist et al., Nat Immunol 5, 1243 (2004). S3. S. Boscardin et al., J Exp Med 203, 599 (2006) S4. K. Inaba et al., J Exp Med 176, 1693 (1992). S5. D. Dudziak et al., J Virol 77, 8290 (2003). S6. J. R. Mora et al., Nature 424, 88 (2003). S7. E.E. Bates et al., J Immunol 164, 1973 (1999)

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Documents

Supporting Online Material for - Sciencescience.sciencemag.org/content/suppl/2007/01/02/315.5808.107.DC… · Supporting Online Material for Differential Antigen Processing by Dendritic