Combination of JSI-124 treatment and immunotherapy. (A) MethA sarcoma was established in BALB/c mice by sc inoculation of 5×105 cells. When tumor became palpable mice were split into 4 groups (5 mice per group) with equal size of the tumors. Immunization with DC-Adp-53 and treatment with JSI-124 or DMSO (VC) was performed as described in Materials and Methods. Tumor size was monitored constantly during mice treatment (B) Mice were sacrificed 5 weeks after tumor inoculation. Lymph node cells were isolated from DC-Ad-p53 immunized mice treated with JSI-124 or VC, or from tumor-bearing non-immunized mice and used in ELISPOT assay. Cells were stimulated with MHC class I bound control or MethA-specific peptides. Number of spots was measured using CTL reader. Data are presented as average number of INF-γ producing cells per 1×106 cells per group. * - statistically significant difference between cells stimulated with control and MethA-specific peptides (p<0.05). (C) C3R tumor was established by s.c. inoculation of 5×105 cells. Mice were immunized in opposite flank with 100 μg of RAHYNIVTF peptide in IFA (Immun). In control mice were injected with IFA alone. Immunizations were performed on days 6, 11, and 14. JSI-124 (Immun + JSI-124, JSI-124) or DMSO (Control, Immun) were administered i.p. from day 18 till day 30. Each group included 5 mice. (D) Mice described above were sacrificed 34 days after tumor inoculation, lymph node cells were stimulated in quadruplicates with specific (RAHYNIVTF) or irrelevant control peptide (SIINFEKL). Both peptides bind H2Kb. The number of IFN-γ producing cells was evaluated in ELISPOT assay. The number of spots in response to control peptide was subtracted from the number of spots in response to the specific peptide and calculated per 106 cells. Each group included 3 mice. Mean ± st. error are shown. Immun. - mice immunized with RAHYNIVTF, Immun. + JSI-124 - mice immunized with the peptide and treated with JSI-124. (E). Treatment of MethA sarcoma-bearing mice was performed essentially as described in Fig. 5A. Mice were treated with DC-control or with DC-Ad-p53 vaccine as indicated. JIS-124 was administered in three cycles, 4 days each with 3 days break. Tumor size was monitored. Each group included 6 mice and Mean ± st. error are shown.