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IJC_25451_sm_suppinfofigs1-4.doc2220KSupplementary fig.1. The sizes of the tumors at time of treatment onset and isolation of splenocytes. The mice were inoculated s.c. with 1 × 105 tumor cells/mouse. CTX was administrated i.p. at a dose of 50 mg/kg on day 8 and ATRA was given i.p. at a dose of 2.5 mg/kg continuously for 5 days starting from day 9. Supplementary fig.2. One representative photo of three individual experiments was shown. C57BL/6 mice were inoculated s.c. with 1 × 105 tumor cells/mouse and the tumors were allowed to grow to 60 mm2 before the mice received our vaccination protocol in addition to ATRA and CTX as described in Materials and Methods. Supplementary fig.3. Tumor growth and survival curve during peptide vaccination or adoptive immunotherapy in combination with ATRA and CTX. C57BL/6 mice were subcutaneously injected with 100 μg mE6Δ/mE7/TBhsp70Δ and boosted 7 days later. Splenocytes were prepared as effector cells 14 days after the last vaccination. All of the mice were subcutaneously inoculated with 1 × 105 TC-1 cells/mouse on day 0. To test the peptide vaccination method, CTX was administrated intraperitoneal on day 4 prior to immunization with a subcutaneous injection of 50 μg of either the specific peptide (HPV-16 E7 aa 49–57, RAHYNIVTF) or the control peptide (OVA aa 257–264, SIINFEKL) emulsified in incomplete Freund's adjuvant (IFA). For the adoptive immunotherapy method, a total of 4 × 106 effector cells per mouse were adoptively transferred via the tail vein on day 7 and 14. ATRA was administered intraperitoneal continuously for 14 days starting at day 7. (A) Tumor sizes (mm2) from the peptide vaccine groups. (B) Survival curve of the combined therapy using the peptide vaccine. (C) Tumor sizes (mm2) from the adoptive transfer groups. (D) Survival curve of the combined therapy using adoptive transfer. Each group included eight mice. *, P<0.05 and **, P <0.01, compared with the control group, #, P <0.05 and ##, P <0.01, compared with the group of peptide vaccination or adoptive immunotherapy group. Supplementary fig.4. The effect of tritherapy was declined beyond the optimal therapeutic time with the increased percents of Tregs and MDSCs. C57BL/6 mice were inoculated s.c. with 1 × 105 tumor cells on day 0. The tritherapy was initiated on day 7 (group 3), day 10 (group 2) and day 13 (group 3). At day 31, mice were sacrificed and tumors and spleens were harvested. (A) Diagram depicting the standard tritherapy with the different starting point. (B) The tumor growth curve for the tritherapy initiated at various time points. (C) Splenocytes and tumor-infiltrating mononuclear cells were analyzed by flow cytometry using anti-FoxP3, anti-CD4, and anti-CD25 antibodies. We Gated on CD4+ cells. The percentages of CD4+CD25+ FoxP3+Tregs in CD4+ splenocytes from mice in each group are shown. (D) Splenocytes and tumor-infiltrating mononuclear cells were analyzed by flow cytometry using anti-Gr-1, anti-CD11b antibodies. Gating on forward and side scatter, larger cells were analyzed. The percentages of Gr-1+CD11b+ cells of splenocytes from mice in each group are shown. **, P <0.01 compared with control group; #, P<0.05 and ##, P<0.01 compared with group 2.
IJC_25451_sm_suppinfotables1-2.doc39KSupplementary table.1 Comparison of the body weight from each group (n=10; body weight: mean ± SD (g)). Supplementary table.2 Comparison of the level of cytokines in serums from the indicated groups

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