Supplementary Figure 1. Combinatorial ligand stimulation of TRIF and MyD88
shows the greatest synergy in C57BL6-derived bone marrow macrophages. (a)
Cytokine production and (b) fold synergy in C57BL/6 bone marrow-derived
macrophages (BM-DM) in response to increasing doses of combinatorial TLR ligand
stimulation. Cells were stimulated with the indicated combinations and doses of TLR
ligands indicated in Figure 2a for 24 h and cytokine concentration in cell-free
supernatants were measured by ELISA. Data shown is representative of three
independent experiments. Student’s t-test was performed to compare double TLR ligand
stimulation (coloured lines) with the sum of single stimulations (white symbols, black
lines) *p<0.05, **p<0.01.
Supplementary Figure 2. Combinatorially stimulated cells show reduced apoptosis
and increased cytokine production. (a) BM-DM were either non-treated for 24 h, “NT
24”, treated with 10 μg/ml poly(I:C) for 24 h (I24), 25 ng/ml R848 for 24 h (R 24), 10
μg/ml poly(I:C) and 25 ng/ml R848 simultaneously for 24 h (IR 24), or 10 g/ml
poly(I:C) for 24 h followed by 25 ng/ml R848 for 24 h (I24R24). Cells were then stained
with Annexin-FITC and 7AAD (Ebioscience). Cells that stained for Annexin-V alone
were taken as early apoptotic cells, while cells that double stained for Annexin-V and
7AAD were taken as necrotic. (b) Graphical representation of data obtained in (a). (c)
BM-DM were stimulated with 10 μg/ml poly(I:C) for 8 or 16 h, 25 ng/ml R848 for 8 h,
10 μg/ml poly(I:C) and 25 ng/ml R848 simultaneously for 8 h or 10 μg/ml poly(I:C) for 8
h followed by 25 ng/ml R848 for 8h “I8R8”. Cells were stained with F4/80-FITC and
either IL-6-PE or ILp12p40-PE. Analysis was performed with CyAn™ ADP Analyzer
(Beckman Coulter) and data was analyzed using Summit v4.2 software. Data is reported
as histograms for the PE channel (either IL-6 or IL-12p40), of cells that stain positive for
F4/80-FITC.
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Supplementary Figure 3. The role of Type I Interferons in cytokine synergy. (a)
BM-DM were treated with 10 ng/ml, 100 ng/ml or 1 μg/ml of IFNAR blocking antibody
(IFNAR Ab) (Ebioscience) 30 min prior to stimulation or left untreated (white bar). Cells
were then stimulated with 10 μg/ml poly(I:C) “I”, 25 ng/ml R848 “R”, 10 μg/ml
poly(I:C) and 25 ng/ml R848, simultaneously “IR” or 10 μg/ml poly(I:C) for 8 h followed
by 25 ng/ml R848. Data shown are representative of three independent experiments.
Student’s t-test was performed and p values of less than 0.05 were deemed significant.
(b) Exogenously added IFN-β is unable to enhance IL-6 and IL-12p40 production to
levels comparable to that of poly(I:C) pretreatment. BM-DM were either pretreated with
10 μg/ml poly(I:C) for 8 h followed by R848 (I8R), or with increasing doses of
recombinant mouse IFN-β (0.5-5000 U/ml) or, not pretreated for 8 h and then stimulated
with R848 for 24 h (O8R). Cell-free supernatants were collected 24 h after the addition of
TLR ligands and cytokine production assayed by ELISA as described in the methods
section.
Supplementary Figure 4. Low production of IL-12p70 and IL-23 under synergistic
conditions. BM-DM were pretreated with one TLR agonist for 0, 4, 8, 16 or 24 h (0 h
pretreatment denotes simultaneous stimulation), followed by a second TLR agonist for 24
h, as described for main Figure 3. [IR combinatorial, dark grey] symbolizes 10 μg/ml
of poly(I:C) as the first TLR agonist, followed by 25 ng/ml of R848 as second TLR
agonist. [RI combinatorial, black] indicates the same stimulations in reverse order. The
hypothetical additive effects are computed for [IR additive, white] and [RI additive,
light grey] as described in Figure 2a. IL-10, TNF, IL-6 and IL-12p40 in cell-free
supernatants were measured by ELISA. Data are means ± s.d. of triplicate measurements
in a representative experiment. Similar results were obtained in two other independent
2
experiments.
SUPPLEMENTARY METHODS
Intracellular cytokine staining
Cells were stimulated as described in Supplementary Figure 3b. Brefeldin A was added 6
h prior to collection of the cells to prevent exocytosis of the cytokines produced. Cells
were then fixed with 4% paraformaldehyde in PBS for 10 min followed by washing with
PBS. Fc receptors on the surface of the cells were blocked with Fc block for 15 min on
ice and then the cells were permeabilized with 0.1 % saponin in 5% BSA/PBS.
Subsequently, cells were stained with F4/80-FITC and either IL-6-PE or ILp12p40-PE
for 30 min on ice in the dark. Analysis was performed with CyAn™ ADP Analyzer
(Beckman Coulter) and data was analyzed using Summit v4.2 software. Data is reported
as histograms of the PE channel (either IL-6 or IL-12p40), of cells that stain positive for
F4/80-FITC.
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