A) Median survival of MMC-tumor bearing neu/tg mice after intratumoral injection of PBS, Ad.zero, and Ad.ISP vectors. Tumor size was measured every other day. The day tumors reached a volume of 400mm³ represented the endpoint in Kaplan-Meier survival studies. Median survival was calculated using GraphPad-Prizm4 software. N=5.
B) and C) studies in CB17-SCID/beige mice. B) MMC tumor cells were implanted and low-dose CY (100mg/kg) or PBS was injected i.p. six days later. Tumor volumes at day 16 (end of the observation period) were significantly lessin CY vs PBS treated groups (p<0.001);see also Suppl. Fig. 3A. C). In another experiment, tumor-bearing CB17-SCID/beige mice received 250μg of antiCD25 or control antibody 6 days after MMC cell implantation. Shown is the tumor volume in individual mice. Tumor volumes at day 16 (end of the observation period) did not significantly differ between Control Ig and antiCD25 treatment groups (p=0.07); see also Suppl. Fig. 3B. D) Treg depletion by antiCD25 in neu/tg mice. Mice were intraperitoneally injected with 250μg antiCD25 or control antibody and 4 days later, splenocytes were analyzed for CD4, CD25, and FoxP3 by flow cytometry. N=3. CD4+/CD25+ Co Ig vs. antiCD25: p=0.004; CD4+/FoxP3+ Co Ig vs antiCD25: p=0.019; CD25+/FoxP3+: p=0.009. E) Effect of antiCD25 on tumor growth. MMC tumor bearing neu/tg mice were injected with 250μg antiCD25 or control antibody 4 days before intratumoral injection of PBS or 2×10⁹ pfu of Ad.zero. The day tumors reached a volume of 400mm³ represented the endpoint in Kaplan-Meier survival studies. N=8. antiCD25+PBS vs antiCD25+Ad.zero: p=0.0061; control Ig+PBS vs. control Ig+Ad.zero: p=0.0058; antiCD25+PBS vs control Ig+PBS: p=0.9677; antiCD25+Ad.zero vs control Ig+Ad.zero: p=0.5722
F) Effect of antiCD25/Ad.αCTLA4 injection on tumor growth. MMC tumor bearing neu/tg mice were injected with 250μg antiCD25 or control antibody four days before intratumoral injection of 2×10⁹ pfu of Ad.zero or Ad.αCTLA4. The day tumors reached a volume of 400mm³ represented the endpoint in Kaplan-Meier survival studies. N=10. antiCD25+Ad.αCTLA4 vs antiCD25+Ad.zero: p=0.0066; antiCD25+Ad.αCTLA4 vs control Ig+Ad.αCTLA4: p=0.0104; antiCD25+Ad.αCTLA4 vs Mock+Ad.αCTLA4: p=0.0105.

A) Median survival of TC-1 tumor bearing C57Bl/6 mice after intratumoral injection of PBS, Ad.zero, and Ad.ISP vectors. Tumor size was measured every other day. The day tumors reached a volume of 400mm³ represented the endpoint in Kaplan-Meier survival studies.
B) Survival of TC-1 tumor bearing mice after treatment with CY (100mg/ml) 4 days before PBS or Ad injection. The day tumors reached a volume of 400mm³ represented the endpoint in Kaplan-Meier survival studies. N=8. PBS+CY vs Ad.zero+CY: p=0.076; PBS+CY vs Ad.αCTLA4+CY: p=0.028; PBS+CY vs Ad.αCD3/Ad.CD80+CY: p=0.013; Ad.zero+CY vs Ad.αCTLA4+CY: p=0.664; Ad.zero+CY vs Ad.αCD3/Ad.CD80+CY: p=0.433; Ad.αCTLA4+CY vs Ad.αCD3/Ad.CD80+CY: p=0.556.
C) and D) Effect of vaccination with Ad.αCTLA4 or Ad. αCD3/Ad.CD80 infected TC-1 cells on growth of pre-established TC-1 tumors. A total of 5×10⁴ TC-1 cells was injected subcutaneously into the right inguinal flank of C57Bl6 mice. When tumors reached a diameter of ~2mm, mice received an intraperitoneal injection of low dose CY. Four days later a total of 1×10⁶ Ad-transduced TC-1 cells were transplanted into the left inguinal flank. TC-1 cells were infected ex vivo with Ad vectors at a total MOI of 100pfu/cell. (ThisMOI efficiently inhibits in vivo growth of infected cells). Twenty-four hours after Ad infection, cells were trypsinized, washed and used for transplantation. The day tumors reached a volume of 200mm³ represented the endpoint in Kaplan-Meier survival studies. N=8
C) PBS+CY vs Ad.zero+CY: p=0.059; PBS+CY vs Ad.αCTLA4+CY: p=0.0004; Ad.zero+CY vs Ad.αCTLA4+CY: p=0.0021.
D) PBS+CY vs Ad.zero+CY: p=0.0102; PBS+CY vs Ad.αCTLA4+CY: p=0.0014; Ad.zero+CY vs Ad.αCTLA4+CY: p=0.0174.

Tissues were collected from antiCD25+Ad.αCTLA4 treated mice with MMC tumors at day 40 (see Fig. 2F) and from Ad.aCTLA4+Cy treated mice with TC1 tumors at day 30 (see Fig. 4C). Representative H&E-stained sections are shown.

MMC cells were subcutaneously transplanted into neu-tg mice. A–D) Two weeks after transplantation, tumor sections were stained for E-cadherin (A), laminin (B), FoxP3 (red) and CD4 (green) (C), and CD8 (green) (D). Cell nuclei are stained with DAPI (blue). E)β-galactosidase expression 2 days after intratumoral injection of 1×10⁹ pfu of Ad.bGal.

TC-1 cells were subcutaneously transplanted into C57Bl/6. Two weeks after transplantation, tumor sections were stained for E-cadherin (A), laminin (B), FoxP3 (red) and CD4 (green) (C), and CD8 (green) (D). Cell nuclei are stained with DAPI (blue). E)β-galactosidase expression 2 days after intratumoral injection of 1×10⁹ pfu of Ad.bGal. The scale bar represents 40μm.

A total of 1×10⁴ TC-1 cells were subcutaneously injected into the left inguinal flank of C57Bl/6 mice at day 0. At day 5, mice received an intraperitoneal injection of CY. At day 9, 1×10⁶ Ad.zero or Ad.αCTLA4 transduced TC-1 cells were injected into the right inguinal flank.
A) At day 22, tumors, tumor-draining lymph nodes, and spleen were harvested and analyzed for CD4, CD8, FoxP3, CD3 and NK1.1 by flow cytometry. White bars: Ad.zero transduced TC-1-cells, back bars: Ad.αCTLA4 transduced cells. N=4, *p<0.05
B and C) Four days before vaccination with Ad. αCTLA4 (B) or Ad.zero (C) transduced cells, mice received intraperitoneal injection of CD4, CD8 or NK-cell depleting antibodies. Injections were repeated every 3 days for the time of monitoring. Shown is the tumor volume of individual mice. Statistical analysis was performed using tumor volumes measured at the end of the observation period (day 22); see also Suppl.Fig. 5.
Ad.aCTLA4/CY:
PBS vs CD4 depletion: p=0.964
PBS vs CD8 depletion: p=0.029
PBS vs NK depletion: p=0.001
Ad.zero/CY:
PBS vs CD4 depletion: p=0.678
PBS vs CD8 depletion: p=0.191
PBS vs NK depletion: p=0.00003
Ad.aCTLA4/CY-PBS vs. Ad.zero/CY-PBS: p=0.020