Bourquin, C., Hotz, C. et. al.: R848 tolerance 7/15/11
1 Supplemental figure legends
Fig. 1 supplemental: Dose response of cytokine induction after a single R848 injection in vivo
Mice were injected with varying doses of R848 subcutaneously (2.5 - 20 µg). Two, 4 and 8 h p.i. serum was analyzed for cytokines by ELISA. Sera of three mice were pooled for analysis.
Fig. 2 supplemental: Fractionated R848 tumor therapy is similar in efficacy to a DC vaccine combined with CpG
Mice (n = 4 to 5 per group) were inoculated with 2.5 x 105 CT26 colon carcinoma cells s.c. on day 0. On days 7, 11, 15 and 19 (arrows) mice were treated as follows: Controls were left untreated, the R848 group obtained 20 µg R848 four times per treatment cycle as indicated by the schedule below the graph. For DC + CpG 2 x 105 tumor-loaded DC per mouse were applied contralaterally followed by treatment with 200 µg CpG. Asterisks (**, p < 0.01; ***, p <
0.001) indicate significant differences to the untreated group.
Fig. 3 supplemental: Cox1/2 inhibitors do not affect tolerance induction
Bone marrow cells were stimulated for 24 h with 0.1 µg/ml R848 or left unstimulated in the presence of increasing concentrations of Cox1/2 inhibitors, washed and restimulated with 0.1 µg/ml R848. Asterisks (*, p < 0.05; **, p < 0.01; ***, p < 0.001) indicate significant differences between cells that were prestimulated with R848 (black bars) and cells that were not prestimulated (white bars). 'bdl' indicates below detection limit. Data are representative of two independent experiments.
Fig. 4 supplemental: R848 pretreatment in the ng/ml scale does not induce tolerance Primary bone marrow cells (4 x 105 per well) were stimulated for 24 h with indicated concentrations (in ng/ml) of R848, washed and restimulated for 6 h with 1µg/ml R848.
Cytokine levels were determined by ELISA. Secretion of cytokines was normalized to cells that were not prestimulated. Asterisks (*, p < 0.05; **, p < 0.01; ***, p < 0.001) indicate
2 that were not prestimulated (white bars).
Bourquin, Hotz et al.: R848 tolerance
Figure S1
IL-12p70
none 2.5μg
5 μg 10μg
20μg 0
500 1000 1500
2000 2h
4h 8h
R848 dose
pg/ml
IL-6
none 2.5μg
5 μg 10μg
20μg 0
500 1000 1500 2000
2500 2h
4h 8h
R848 dose
pg/ml
Figure S2
0 3 6 9 12 15 18 21
0 50 100
150 Untreated
R848 DC+CpG
***
*** ***
** ***
*** ***
Days after tumor induction Tumor size (mm2)
0h 4h 6h 24h
Bourquin, Hotz et al.: R848 tolerance
Figure S4
0 0.05 0.5 5 50 500
0.0 0.5 1.0 1.5 2.0 2.5
**
*** *** ***
0 0.05 0.5 5 50 500
0.0 0.5 1.0 1.5
***
*** ***
R848 (ng/ml ) R848( ng/ml)
IL-12p70 (fold change) IL-6 (fold change)
