Tlr4SIINFEKL versusLive EG7 versus X-rays EG7 WT versus

DC

SIINFEKLPBSIn vitro

restimulation

0

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**

Supplemental Figure 1[ H

]T x

10

c.p

.m3

3 IF

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S upplem ental F ig . 1 . Requirem ent for D C to express TLR 4 in the im m unogenicity o f irrad iated tum or ce lls. DC from WT or Tlr4-/- mice were first loaded with antigen (SIINFEKL peptide, live or irradiated EG7 cells) and then injected into the footpad of Tlr4-/- recipients. Five days later, the local immune response was measured either as IFN-γ secretion (as in a) or as proliferation as described in Fig. 2.

a

Supplemental Figure 2Ph

agoc

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is in

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x 1

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.p.m

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RATIO BM-DC / Allogenic T cells

S upplem ental F ig . 2. Uptake and m aturation of TLR 4 -defic ient D C . (a) P hagocytos is index of irrad ia ted C T26 (le ft pane l) and EL4 ce lls (righ t pane l) by syngen ic W T or T lr4 -/- o r T rif-/- o r M yd88-/- D C . In 12-well plates, tumor cells were labeled with Celltracker Green (Calbiochem) and cultured with DC for 2 hours at a ratio of 1:1. At the end of the incubation, adherent cells were harvested with versene, pooled with non-adherent cells, washed and stained with CD11c-APC antibody. Phagocytosis was assessed by FACS analysis on double positive cells. Phagocytic indexes refer to the ratio between values obtained at 37°C versus 4°C. (b ). M ouse dendritic ce ll m atura tion induced by dy ing tum or ce lls is H M G B 1 independent. The release of the indicated cytokines was measured in specific ELISA in the supernatants of BM-DC (WT or T lr4 -/-) loaded for 24 hrs with oxaliplatin-treated EG7 cells or their corresponding 24hrs supernatants or rHMGB1 (1µg/ml, five times superior to HMGB1 concentrations released by dying tumor cells supernatants) in the presence of neutralizing anti-HMGB1 Ab (or isotype control). (c). Allostim u la tory capacities o f m ouse B M -D C in the presence o f LP S or H M G B 1. WT and T lr4 -/- mouse C57BL/6 BM-DC were incubated with 2x105 allogenic T cells (from BALB/c mice) at the indicated ratios in the presence of 1 µg/ml of LPS or rHMGB1. T-cell proliferation was assessed after 4 days by 3H-thymidine incorporation. The results represent the mean + SEM of triplicate wells of two independent experiments.

Supplemental Figure 3

c

b

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Chloroquine (µM)

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WTTlr4 -/-

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+ C

D11

c ce

lls

*

*

* **

89(± 6)82(± 9) 85(± 5)

84(± 10)83(± 7)91(± 9)

MFI K (±SD)b

WT Tlr4 -/-

0 120 min 300 min

* *

Time: 120 min

10 µm

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IL-2

(pg/

ml)

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25D1.16

CD

11c

WT DCDC Tlr4 -/-

0

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7.5%7.5%7.5%1.2%1.2%

CoAb

Co Chloroquine Anti-HMGB1

CoAb

S upplem enta l F ig . 3 . E nhanced phagosom e-endosom es/lysosom es fus ion in TLR 4 defic ien t D C . (a ) The acqu is ition o f K b/S IIN KE K L com p lexes on D C p lasm a m em brane is severe ly im pa ired in T lr4 -/- D C . H-2b DC (T lr4 -/- or T lr4 +/+) were incubated with either dying OVA-transfected TS/A (H-2d) (at a 1:5 ratio) or with saturated amounts of free SIINFEKL peptides (10 µg/ml) for the indicated periods of time. We monitored the percentages of CD11c+ cells staining with anti-Kb Ab or with a specific antibody recognizing the Kb/SIINKEKL complexes (25D1.16 Ab directly labeled with fluorochrome Alexa 647) by flow cytometry over time (left panel). In the inset, a representative dot plot analysis of flow cytometry studies is depicted. The mean fluorescence intensity (MFI) of Kb expression is indicated in the inset. Experiments were performed twice with similar results. In another sets of experiments, chloroquine or anti-HMGB1 Ab (or isotype Control Ig) were added to the coculture prior to analysis at time 120 min (right panel). (b ). C orrection o f the de fective an tigen p resen ta tion by T lr4 -/- D C in v itro . WT or T lr4 -/- DC were loaded with 1 mg/ml of OVA holoprotein or irradiated EG7 cells in the absence or presence of the indicated concentrations of chloroquine or bafilomycin A1. Six hours later, BM-DC were washed and incubated with the B3Z hybridoma for 48 h as in Fig . 1a . Similar results (means of triplicates ±SEM, n=3) were obtained in two experiments. * p<0.01. (c ). A cce le ra ted fus ion be tw een phagosom es and endo lysosom es in TLR 4 de fic ient D C . DC prelabelled with Lysotracker Red were fed with dying EG7 labeled with the green fluorescent dye CFSE. These DC contained red endosomes/lysosomes and greenish particles, the latter being considered as phagosomes containing engulfed apoptotic bodies. Fusion of apoptotic body-containing phagosomes with endolysosomes resulted in the colocalisation of Lysotracker Red and green CFSE, which caused the fused phagosomes to turn yellowish. The number of cells containing yellow phagosomes was determined out of 120 cells at different time points in WT versus T lr4 -/- BM-DC (percentages indicated on the left and representative picture on the right).

Supplemental Figure 4

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1/1 1/5 1/25

CoCo AbAnti-HMGB1siRNA CosiRNA 1siRNA 2

X-rays EG7 / DC Ratio

IL-2

(pg/

ml)

B3Z*

*

*

*

S upplem ental F igure 4 . The functional interaction betw een TLR 4 and H M G B 1 dictates cross -presentation of apopto tic cells to T ce lls . EG7 cells were transfected with a control siRNA or two independent HMGB1-specific siRNA prior to irradiation. Forty-eight hours later (when immunoblots confirm the HMGB1 depletion, see Fig . 3e ), transfected tumor cells were loaded onto WT DC and used to stimulate B3Z T cell hybridomas. Note that HMGB1 depletion did not alter cell death induction by genotoxic stress (not shown). Likewise, irradiated EG7 were cocultured with B3Z as detailed in Fig . 1 in the presence of neutralizing Ab (or isotype Control Ig). The supernatants were harvested at 24 hrs and assessed for IL-2 secretion in ELISA.

Supplemental Figure 5

TLR4TLR4

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Oxaliplatin

Anti-HMGB1

Chloroquine

Asp299Asp Asp299Gly

02040

6080

100120

140160180

-- - - +Co Ab -

++

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IFN

-γ (n

umbe

r of s

pots

)

S upplem ental F igure 5. TLR 4 Asp299G ly po lym orph ism b lunts responsiveness to LP S an d affects cross-presentation o f m elanom a antigens in v itro . Identical setting as in Fig. 6b. but using monocyte-derived DC from a different pair of normal and mutated individuals.

Supplemental Table 1

pT: pathological tumor size. HR: hormonal receptors. All data are presented as means ± standard deviation, or percentages.

Supplemental Table 1. Baseline characteristics of the individuals, tumors, and primary treatments TLR4 Asp299Asp

(n=230) TLR4 Asp299Gly

(n=50) p

Value Age – n (%) < 35 yr 24 (11.7) 6 (12.0) NS 35-49 yr 90 (39.1) 16 (32.0) NS 50-59 yr 71 (30.9) 14 (22.0) NS > 60 yr 45 (18.3) 14 (22.0) NS Mean Age (years) M± SD 50 ± 11 49 ± 11 NS Nodal status – n (%) Positive 230 (100) 50 (100) NS 1 – 3 positive nodes 140 (60) 23 (46.0) NS > 4 positive nodes 90 (40) 27 (54.0) NS Number of metastatic lymph nodes 4.5 ± 4.5 5.2 ± 5 NS Pathological tumor size – n (%) 0 – 2 cm 87 (38.3) 16 (32.0) NS 2 – 5cm 124 (54.6) 32 (64.0) NS > 5 cm 16 (7) 2 (4.0) NS Missing 3 (1.3) 0 NS Mean pathological tumor size (mm) M± SD 27 ± 1.9 25 ± 1.4 NS Histologic grade of tumor – n (%) 3 Poorly differentiated 94 (41.0) 18 (36.0) NS 2 Moderatly differentiated 106 (46.0) 25 (50.0) NS 1 Well differentiated 20 (8.6) 4 (8.0) NS Missing 13 (5.6) 3 (6.0) NS Hormone receptor status – n (%) positive 168 (73) 35 (70) NS Type of adjuvant chemotherapy – n (%) Epirubicin based regimen 230 (100) 50 (100) NS Anthracyclines + Taxanes 48 (21) 10 (20) NS Adjuvant endocrine therapy – n (%) Tamoxifen 51 (22) 14 (28) NS Aromatase inhibitor 28 (12) 4 (8) NS Missing 21 (9) 4 (8) NS Radiotherapy – n (%) 211 (92) 50 (100) NS Missing 3 (1) 0 Follow up (months) M ± SD 94 ± 64 108 ± 63 NS

Mouse strains. BALB/c (H-2d), C57BL/6 (H-2b) and nu/nu BALB/c or C57BL/6 mice were obtained from the Centre d’élevage Janvier

(Le Genest St Isle, France) and from Charles River Laboratories (L’Arbresle, France). BALB/c Tlr2-/- and Tlr4-/- mice were provided by

Grégoire Lauvau (INSERM, University of Sofia Antipolis, Valbonne, France),. CD11c-GFP/DTR mice were provided by Pierre

Aucouturier (INSERM, St. Antoine Hospital, Paris, France) and were injected intraperitonealy with 100 ng diphteria toxin (or PBS as a

vehicle control) to ablate CD11c+ cells.

Reagents and materials. Cell death was induced either with Doxorubicin (Sigma Aldrich), Oxaliplatin (Sanofi-Aventis, France), or X-rays

irradiation (RT250, Phillips). TLR4-Fc fusion protein and control protein were obtained from Alexis Biochemicals (Paris, France).

SIINFEKL peptide and TLR4 inhibitory (RKKRRQRRRGKKYSRGESIYDAFVIYSSQNEDW) and the control peptide

(RKKRRQRRRGEEYSEGESIYDAFVIYSSQN EDWV) were purchased from Eurogentec (Angers, France). Ovalbumin protein,

bafilomycin A1, chloroquine, ionomycin phorbol 12-myristate 13-acetate (PMA) were obtained from Sigma Aldrich (St Quentin Fallavier,

France). Recombinant human HMGB1 was obtained from R&D Systems (Lille, France). Monoclonal fluorochrome congugated anti-

mouse CD3, CD4, CD8, IFN-� , CD11c, I-Ab, CD40, CD80, CD86, control isotypes antibodies, Cytofix/cytoperm, brefeldin A, and

Quantikine ELISA kit for IL-2, IL-6, TNF-� and IL-12 p40 were obtained from BD Pharmingen (Le Pont de Claix, France). HMGB1

ELISA kit was obtained from SHINO-TEST CORPORATION (Tokyo, Japan). Rabbit anti-HMGB1 polyclonal antibody, isotype and goat

anti rabbit Alexa 488 polyclonal antibodies were obtained from Abcam (Paris, France). In some experiments, mice were injected with a

neutralizing anti-HMGB1 antibody provided by Huan Yang (Lexington, MA). Monoclonal unconjugated anti-mouse HSP60 and HSP90

were obtained from Stressgen (TEBU, Le Perray, France) and HSP 70 from Santa Cruz Biotechnology (Santa Cruz, USA). Mouse IgG1

antibody against fibronectin extra domain A was purchased from BD Biosciences and goat polyclonal IgG anti-� defensin 2 was

purchased from Ozyme..

Bone marrow- derived DC and T Cell hybridoma assays. Bone marrow- derived dendritic cells (DC) were propagated as already

described46 in Iscoves’s medium (Sigma Aldrich) supplemented with Penicillin (100 U/ml Gibco), Streptomycin (100 µg/ml Gibco), L-

glutamine (Gibco), 2-mercaptoethanol (50 µM, Sigma), 10% heat-inactivated and filtered, endotoxin-free FCS (Gibco), and 30% of J558

supernatant. DC were used between day 10 and day 12 when the proportion of DC within the culture was above 80% as determined by

CD11c and MHC class II labeling. The SIINFEKL-specific, H-2Kb-restricted hybridoma B3Z or ISQAVHAAHAEINEAGR-specific I-

Ab B09710 (2 x104 cells per well) were cultured with different concentration of live or 10 Gy irradiated EG7 cells or oxaliplatin-treated

EG7 (and used 24 hrs after irradiation or oxaliplatin) and 104 WT or loss-of-function DC. Supernatants were harvested 48 hours later and

IL-2 secretion was assessed by ELISA. Positive controls were SIINFEKL peptide (2µg/ml) for B3Z clone and ovalbumin protein (1mg/ml)

for B09710 clone. In some experiments, chloroquine, bafilomycin A1 (variable doses), TLR4 inhibitory peptide (10µg/ml), TLR4Fc

protein (10µg/ml) and their controls (10µg/ml) were added to the cocultures.

Priming assay. 3.106 CT26, EG7 and MCA205 cells were either untreated or treated with 20µM doxorubicin (20 µM for CT26 and 1 µM

for MCA205) or oxaliplatin (5µg/ml) for 24h. Alternatively, EG7 cells were subjected to 10Gy X-Ray irradiation . Cells were then

injected into the footpad of mice. Alternatively, DC were cocultured with EG7 cells for 2 hours, followed by the purification of CD11c+

cells with anti-CD11c mAb coupled to magnetic beads (Miltenyi Biotec, Paris, France) and injection of these purified DC into the footpad.

Five days later, gangliocytes of popliteal lymph nodes were harvested, and restimulated with SIINFEKL peptide or OVA protein.

Restimulation with MCA205 or CT26 cells was performed using 3.104 tumor cells killed by a 5 min. heating at 42°C followed by 1 cycle of

freezing/thawing in liquid nitrogen. Supernatants were harvested 72 hours later and IFN-� secretion was assessed by ELISA. In some

experiments, cells were cultured for 5 days and 1 � Ci/well of [3H] thymidine (Isotopchim) was added 18h before the end of the culture.

Alternatively, cells were stimulated with PMA and ionomycin for 1 h, followed by the addition of brefeldin A (6h at 37°C), surface

staining with Abs specific for CD3, CD4 or CD8, fixation and permeabilization (Cytofix/Cytoperm kit) and then labeled with anti-IFN-�

Abs. Immunofluorescence was analyzed with a FACSCalibur (BD Biosciences), with CellQuest software.

HMGB1 siRNAs. Irrelevant siRNA (CUUACGCUGAGUACUUCGA TT), HMGB1 siRNA 1 (GCAGCCCUAUGAGAAGAAA TT) ,

or HMGB1 siRNA 2 (GCUGAAAAGAGCAAGAAAA TT) ' synthesized by Sigma Proligo) were used to achieve HMGB1 knockdown

in vitro. The depletion of HMGB1 was assessed 48h after each transfection by immunoblot analysis as described above.

Immunoprecipitation. RAW264.7 cells were lysed in lysis buffer [in mM: 20 Tris·HCl (pH 7.5), 150 NaCl, 1 Na2EDTA, 1 EGTA, 2.5

sodium pyrophosphate, 1 -glycerophosphate, and 1 Na3VO4 with 1% Triton X-100] containing protease inhibitor cocktail (Roche,

Mannheim, Germany). Whole cell extracts were centrifuged at 14,000 rpm for 20 min to remove the debris. Immunoprecipitations were

performed by incubating whole cell extracts with indicated antibody, preincubated with recombinant protein G agarose (Invitrogen) while

rocking at 4°C overnight. Immunoprecipitates were washed three times with lysis buffer, resuspended in 50 µl of 1x Laemmli sample

buffer, and then resolved by 4–15% Tris·HCl-PAGE.

Immunoblot Analysis. Samples were lysed in lysis buffer containing 50 mM Tris HCl pH 7.5, 5M NaCl, 10% NP40, DTT, sodium

orthovanadate and antiprotease cocktail (Roche, Mannheim, Germany). The protein concentration for each sample was determined by

the Bradford assay using Bio-Rad Protein Assay kit (Bio-Rad Laboratories, Inc., Hercules, CA) according to the manufacturer’s

recommendations. After immunoblotting, immunochemical detection was revealed with an appropriate horseradish peroxidase-labeled

secondary antibody. Specific bands were visualized using an enhanced chemiluminescence detection system (Southern Biotechnologies

Associates, ECL kit from Pierce), as described in the technical manual provided by the company, with subsequent exposure to X-ray film.

Controls of equal loading of proteins were performed with anti-actin antibody for whole cells, CD47 or pancadherin antibodies for

membrane preparations and coomassie blue staining of the gel for supernatants.

Biotinylation of cell surface proteins. Cells were placed on ice and washed three times with ice-cold PBS-Ca2+-Mg2+(PBS with 0.1 mM

CaCl2 and 1 mM MgCl2). Membrane proteins were then biotinylated by a 30-min incubation at 4 °C with NHS-SS-biotin 1.25 mg/ml

(Pierce) freshly diluted into biotinylation buffer (10 mM triethanolamine, 2 mM CaCl2, 150 mM NaCl, pH 7.5) with gentle agitation. Cells

were rinsed with PBS-Ca2+-Mg2+ + glycine (100 mM) and washed in this buffer for 20 min at 4 °C to quench unreacted biotin. The cells

were then rinsed twice with PBS-Ca2+-Mg2+, scraped in cold PBS, and pelleted at 2,000 rpm at 4 °C. The pellets were solubilized for 45

min in 500µl of lysis buffer (1% Triton X-100, 150 mM NaCl, 5 mM EDTA, 50 mM Tris, pH 7.5) containing protease inhibitors. The

lysates were clarified by centrifugation at 14,000 × g for 10 min at 4 °C, and the supernatants were incubated overnight with packed

streptavidin-agarose beads to recover biotinylated proteins. The beads were then pelleted by centrifugation, and aliquots of supernatants

were taken to represent the unbound, intracellular pool of proteins. Biotinylated proteins were eluted from the beads by heating to 100 °C

for 5 min in SDS-PAGE sample buffer before loading onto a 10% SDS-PAGE gel as described above. To ensure the absence of leakage

of biotin into the cells, we systematically verified the absence of the intracellular protein actin and GAPDH in biotinylated extracts.

Phagosome-endosome/lysosome fusion assay. WT or TLR4-/- DC loaded with Lysotracker Red were incubated with CFSE labeled EG7

pretreated with 5µg/ml oxaliplatin during 24 hours. Unfused phagosomes that contained CFSE-labeled target cells were seen in green,

whereas phagosomes fused with endosomes/lysosomes were seen in yellow due to the coexistence of both fluorochromes. The number of

fused phagosomes (yellowish vesicles) was determined on 120 cells for each group of DC.

Detection of peptide/MHC class I complexes at the surface of DC, using the specific 25D1.16 Ab. H-2b DC (TLR4-/- or TLR4+/+) were

incubated at 37°C with OVA-transfected TS/A (H-2d) killed by incubation with 5µg/ml of oxaliplatin during 24h. DC-TSA ratio was 1/5.At

different time points sample were analysed for their expression of Kb and Kb/SIINKEKL on CD11c + cells by cytometry. Baseline

Kb/SIINFEKL fluorescence signal was determined using DC cultured alone. Positive control of Kb/SIINFEKL labelling was made using

DC loaded 30mn at 37°C with SIINFEKL peptide (10µg/ml). The sensitivity of the Kb/SIINFEKL mAb was assessed using dilution of

SIINFEKL peptide. We could detect signal when DC were incubated with 0.1 ng/ml. In some experiments, 50µM of chloroquine or of

anti-HMGB1 antibody were added in the culture.

Cross presentation settings in human DC

Mel96 HLA-A2 negative, Mart1 positive melanoma tumor cells were treated with oxaliplatin for 24 hours. Dying melanoma cells were

loaded onto HLA*A0201 human monocyte-derived dendritic cells (generated in three days in GM-CSF and IFNα from individuals

carrying a TLR4Asp299Asp or Gly allele) cocultured at a 1:1 ratio together with the Mart1 -specific, HLA*A0201 -restricted LT12 clone

for 24 hrs on ELISPOT wells containing anti-huIFNγ Ab. The experiments were run in triplicates with or without chloroquine (0.5 µM) or

anti-HMGB1 antibody (10 µg/ml or control Ig) during the DC/CTL coculture. IFNγ -producing cells were detected by ELISPOT.

Primers and PCR conditions. The following primers were used for the Asp299Gly polymorphism: forward 5’-

CCATTGAAGAATTCCGATTAGCATA-3’ and reverse 5’-CACTCACCAGGGAAAATGAAGAA-3’. Dual labelled ASOs (Applied

Biosystems) were designed for the wild-type Asp299 allele and the 299Gly allelic variant with sense sequences: 5’-[VIC™]-

CCTCGATGATATTATT-[MGB][NFQ]-3’ and 5’-[6-FAM]-CTCGATGGTATTATTG-[MGB][NFQ]-3’, respectively (the italicized

bases indicate the polymorphic site for each ASO). For PCR tests, 5ng of DNA in 2 µl was amplified by adding 5 µl of 2X Universal

Taqman Master Mix (Applied Biosystems), 0.25µl of 40X PCR primers, Taqman MGB probes and 2.75µl H20. PCR conditions were as

follows: initial denaturation at 95 °C for 10 min, 45 cycles of denaturation for 15 sec at 92 °C and annealing for 60 sec at 60 °C.

Fluorescence signals were measured automatically on an ABIPrism 7700 Sequence Detector (Applied Biosystems).

Clinical study design. We retrospectively constructed a patient database using data obtained from three different centers (Institut Gustave

Roussy, Centre René Huguenin and the French National E3N cohort). Approval was obtained from the local institutional review boards

“CCPPRB du Val de Marne” Eligible patients had histologically confirmed, axillary node positive- sporadic breast cancer. Patients were

selected to have been treated with primary surgery (+/- radiation therapy according to surgical procedure and local guidelines). All

patients were selected to have received an adjuvant anthracycline-based chemotherapy after surgery. The chemotherapy regimen

included anthracycline in all cases. The performance of taxanes in adjuvant setting was not an exclusion criteria. Patients with evidence

of metastasis at the time of diagnosis or with incomplete surgical resection of the primary tumor were excluded from the study. A total of

280 patients fulfilled the inclusion criteria. Analyses will therefore focus on these 280 patients. Adjuvant endocrine therapy was

recommended to all patients with hormone receptor positive tumors. The primary endpoint of the study was metastasis free survival,

defined as time from diagnosis to the occurrence of metastasis. Age at diagnosis, pathological tumor size, lymph node involvement, tumor

grade, hormone receptors, endocrine treatments, occurrence of events and follow-up were extracted from medical files and recorded in

the database (Supplemental Table 1). After generation of the patient database and collection of genomic DNA samples, genotyping and

statistical analyses were performed in a blinded fashion. All patients provided written informed content for enrollment. For the analysis of

clinical data, continuous variables were compared with Mann-Whitney U test and categorical variables by Chi-square. Kaplan Meier

survival curves were constructed using the time at diagnosis as baseline. The Log Rank test was used for analysis of survival curves.