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1.
Figure 6

Figure 6. Survival analysis after treatment in a spontaneous tumor model. From: ?-Galactosylceramide enhances the protective and therapeutic effects of tumor cell based vaccines for ovarian tumors.

Kaplan & Meier analysis showing the survival of the MISIIR-TAg transgenic mice treated with irradiated MOVCAR tumor cells and/or α-GalCer. Female MISIIR-TAg transgenic mice (5 per group) were injected with 1 × 106 /mouse of irradiated MOVCAR tumor cells and/or 1 μg /mouse of α-GalCer. A group of mice was left untreated as a control. (* indicates p < 0.05 as compared to the control).

Youn Seok Choi, et al. Vaccine. ;26(46):5855-5863.
2.
Figure 1

Figure 1. ELISA assay characterizing the levels of IFN-γ in the serum of vaccinated mice. From: ?-Galactosylceramide enhances the protective and therapeutic effects of tumor cell based vaccines for ovarian tumors.

Groups of C57BL/6 mice (5 per group) were vaccinated intraperitoneally with 1 × 106/mouse of irradiated MOSEC tumor cells together with 1 μg/mouse of α-GalCer or each component alone. A group of untreated mice were used as a control. Five hours after the injection, sera were collected from the naïve and vaccinated mice and assayed for levels of IFN-γ using ELISA. The bar graph shows the levels of IFN-γ in picograms /ml. The data shown is from one representative experiment of two performed.

Youn Seok Choi, et al. Vaccine. ;26(46):5855-5863.
3.

Figure 4. In vivotumor treatment experiments. From: ?-Galactosylceramide enhances the protective and therapeutic effects of tumor cell based vaccines for ovarian tumors.

C57BL/6 mice (5 per group) were challenged i.p. with 2 × 105 /mouse of MOSEC-luc tumor cells on day 0. Five days after tumor challenge, the mice were treated i.p. with 1 × 106 /mouse of irradiated MOSEC tumor cells and/or 1 μg /mouse of α-GalCer twice with a 1-week interval. A group of untreated tumor-bearing mice was used as a control. Tumor-bearing mice were imaged on days 0, 7, 14, 21 and 28 after tumor challenge using the IVIS Imaging System Series 200. Bioluminescence signals were acquired for one minute. (A) Left panel; representative bioluminescence images of MOSEC-luc tumor-bearing mice treated with the various combinations of α-GalCer and irradiated MOSEC tumor cells. Right panel; line graph depicting the kinetic expression of luciferase in MOSEC-luc tumor-bearing mice treated with the various combinations of α-GalCer and irradiated MOSEC tumor cells over time (mean±s.e.). (B) Kaplan & Meier survival analysis of MOSEC-luc tumor-bearing mice treated with irradiated MOSEC tumor cells and/or α-GalCer. (* indicates p < 0.05; ** indicates p < 0.01 as compared to the control).

Youn Seok Choi, et al. Vaccine. ;26(46):5855-5863.
4.
Figure 7

Figure 7. Characterization of tumor-specific CD8+ T cells in vaccinated MISIIR-TAg transgenic mice. From: ?-Galactosylceramide enhances the protective and therapeutic effects of tumor cell based vaccines for ovarian tumors.

Female MISIIR-TAg transgenic mice (5 per group) at the age of 6 weeks were treated with 1 × 106 /mouse of irradiated MOVCAR tumor cells and/or 1 μg /mouse of α-GalCer. A group of mice was left untreated as a control. One week after the last treatment, splenocytes were harvested from the vaccinated mice. Pooled splenocytes were cultured in vitro for 16 hrs with or without 2 × 105 live MOVCAR cells with Golgistop and then stained for IFN-γ using intracellular cytokine staining followed by flow cytometry analysis. (A) Representative flow cytometry data for each group of treated transgenic mice. The numbers in the upper right hand corner represent the number of IFN-γ-secreting, CD8+ T cells per 3×105 harvested splenocytes. (B) Bar graph showing the levels of IFN-γ-secreting, CD8+ T cells per 3×105 harvested splenocytes (mean±s.e.).

Youn Seok Choi, et al. Vaccine. ;26(46):5855-5863.
5.
Figure 2

Figure 2. In vitro cytoxicity assay with splenocytes from vaccinated mice. From: ?-Galactosylceramide enhances the protective and therapeutic effects of tumor cell based vaccines for ovarian tumors.

Groups of C57BL/6 mice (5 per group) were vaccinated intraperitoneally with 1 × 106 /mouse of irradiated MOSEC tumor cells and/or 1μg/mouse of α-GalCer twice with a one-week interval. A group of naïve mice was used as a control. Two weeks after the last vaccination, the mice were challenged intraperitoneally with 2 × 105 /mouse of MOSEC tumor cells for in vivo antigenic stimulation. One day after tumor challenge, splenocytes were harvested from the various groups of mice and plated in 96-well plates with luciferase expressing MOSEC tumor cells (MOSEC-luc) in increasing effector: target cell ratios (E:T ratios). Cell viability was demonstrated using luciferase activity and detected using the IVIS Imaging System Series 200. Bioluminescence signals were acquired for one minute. (A) Representative figures of wells showing the luciferase expression in MOSEC-luc cells incubated with splenocytes from the various groups of mice. (B) Line graph quantifying the luminescent activity of the MOSEC-luc cells incubated with splenocytes from the vaccinated mice (mean±s.e.). (* indicates p < 0.05 as compared to control).

Youn Seok Choi, et al. Vaccine. ;26(46):5855-5863.
6.

Figure 3. In vivotumor prevention experiments. From: ?-Galactosylceramide enhances the protective and therapeutic effects of tumor cell based vaccines for ovarian tumors.

C57BL/6 mice (5 per group) were vaccinated intraperitoneally with 1 × 106 /mouse of irradiated MOSEC tumor cells and/or 1 μg /mouse of α-GalCer twice with a one-week interval. A group of naïve mice was used as a control. Two weeks after the last vaccination, the mice were challenged i.p. with 2 × 105 /mouse of MOSEC-luc tumor cells (day 0). Mice were imaged on days 0, 7, 14, 21 and 28 after tumor challenge using the IVIS Imaging System Series 200. Bioluminescence signals were acquired for one minute. (A) Left panel; representative images of the various groups of mice vaccinated with irradiated MOSEC cells and/or α-GalCer prior to tumor challenge with MOSEC-luc. Right panel; line graph depicting the kinetic expression of luciferase (mean±s.e.) in the various groups of mice vaccinated with irradiated MOSEC cells and/or α-GalCer prior to tumor challenge with MOSEC-luc. (B) Kaplan & Meier survival analysis of mice vaccinated with irradiated MOSEC cells and/or α-GalCer prior to tumor challenge with MOSEC-luc. (* indicates p < 0.05; ** indicates p < 0.01; *** indicates p < 0.001 as compared to the control). The data shown is from one representative experiment of two performed.

Youn Seok Choi, et al. Vaccine. ;26(46):5855-5863.
7.
Figure 5

Figure 5. In vivoantibody depletion experiment. From: ?-Galactosylceramide enhances the protective and therapeutic effects of tumor cell based vaccines for ovarian tumors.

Groups of C57BL/6 mice (5 per group) were vaccinated i.p. with 1 × 106 /mouse of irradiated MOSEC tumor cells and 1 μg /mouse α-GalCer twice with a 1-wk interval. One week after the last vaccination, mice were depleted of immune cells expressing CD4, CD8 or NK1.1 markers using 200 μg /mouse of purified rat monoclonal antibodies GK1.5 (anti-CD4), mAb 2.43 (anti-CD8), or mAb PK136 (anti-NK1.1). The mice were injected with antibodies 3 times for the first week and then once every week. One group of vaccinated mice without depletion was used as a control. Two weeks after the last vaccination, the non-depleted and depleted mice were challenged i.p. with 2 × 105 /mouse of MOSEC-luc tumor cells. The growth of the tumor in mice was imaged on days 0, 14, 21 and 28 after tumor challenge using the IVIS Imaging System Series 200. Bioluminescence signals were acquired for one minute. (A) Representative images of MOSEC-luc tumor challenged mice with no depletion, CD4 depletion, CD8 depletion or NK1.1 depletion. (B) Line graph depicting the kinetic expression of luciferase in MOSEC-luc tumor challenged mice with or without depletion (mean±s.e.). (* indicates p < 0.05; ** indicates p < 0.01 as compared to the control).

Youn Seok Choi, et al. Vaccine. ;26(46):5855-5863.

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