Article Figures & Data
Figures
- Fig. 1.
Characterization of C26-interleukin (IL)-27 transfectant in vitro. A, secretion of IL-12 and IL-27 proteins from C26-IL-12 and C26-IL-27, respectively, which was detected by immunoprecipitation of each culture supernatant with anti-FLAG, followed by Western blot analysis with anti-FLAG. Immunoglobulin H chain indicates the immunoglobulin heavy chain of monoclonal antibody used for the immunoprecipitation. B, enhanced proliferation of activated primary CD4+ T cells by the culture supernatant of C26-IL-12, but not of C26-IL-27 or C26-vector. Purified primary CD4+ T cells were stimulated with plate-coated anti-CD3 for 4 days, and after washing, they were additionally cultured overnight. Resultant activated primary CD4+ T cells were incubated with various concentrations of each culture supernatant (0.1, 1, and 10%) for 2 days and pulsed with [3H]thymidine for 6 h, and [3H]thymidine incorporation was measured in triplicate. Data are shown as the mean; bars, ±SD. C, enhanced proliferation of naive CD4+ T cells by the culture supernatant of C26-IL-27 but much less of C26-IL-12 or not of C26-vector. Purified naive CD4+ T cells were stimulated with plate-coated anti-CD3 in the presence of various concentrations of each culture supernatant (0.1, 1, and 10%) for 4 days and pulsed with [3H]thymidine for 20 h, and [3H]thymidine incorporation was measured in triplicate. Data are shown as the mean; bars, ±SD.
- Fig. 2.
Potent antitumor activity of interleukin (IL)-27 in vivo with enhanced IFN-γ production and augmented tumor-specific cytotoxic T lymphocyte (CTL) activity. A, inhibition of in vivo tumor growth by C26-IL-27. BALB/c mice (n = 3) were injected s.c. with each C26 transfectant (2 × 105 cells) or a mixture of C26-vector and C26-IL-27 (ratios of C26-vector, C26-IL-27 = 99:1 and 90:10; total 2 × 105 cells), and tumor volume was monitored and compared after 2 weeks. Tumor volume was calculated using the following volume equation: 0.5(ab2), where a is the long diameter, and b is the short diameter. Data are shown as the mean; bars, ±SD. Similar results were obtained in two independent experiments. B, minimal tumor growth of C26-IL-27 in vivo. BALB/c mice (n = 5) were injected s.c. with each C26 transfectant (2 × 105 cells), and tumor growth was monitored. Data are shown as the mean; bars, ±SD. Similar results were obtained in four independent experiments. C, prolonged survival time of mice inoculated with C26-IL-27. BALB/c mice (n = 7–8) were injected s.c. with each C26 transfectant (2 × 105 cells), and survival rate was monitored. Similar results were obtained in two independent experiments. D, enhanced IFN-γ production from spleen cells by IL-27. BALB/c mice (n = 3) were injected s.c. with each C26 transfectant (2 × 105 cells), and 2 weeks later spleen cells obtained from each mouse were cultured with irradiated parental C26 cells for 2 days, and the culture supernatant was analyzed for IFN-γ production by ELISA. Data are shown as the mean; bars, ±SD. Similar results were obtained in four independent experiments. E, augmented tumor-specific CTL activity of spleen cells by IL-27. Two weeks after the inoculation, spleen cells obtained from each mouse (n = 3) were restimulated with irradiated parental C26 for 5 days, and CTL activity of resultant effector cells was measured against 51Cr-labeled parental C26 or irrelevant syngeneic MethA tumor targets in a standard 51Cr release assay. Data are shown as the mean; bars, ±SD. Similar results were obtained in four independent experiments.
- Fig. 3.
Induction of tumor-specific protective immunity by interleukin (IL)-27. A, induction of tumor-specific protection by IL-27. Recovered mice (n = 5) from inoculation with C26-IL-27 or nontreated mice were challenged by parental C26 or irrelevant syngeneic MethA, and tumor growth was monitored. Data are shown as the mean; bars, ±SD. Similar results were obtained in three independent experiments. B, enhanced IFN-γ production by IL-27. Spleen cells of recovered mice (n = 3) from inoculation with C26-IL-27 or C26-IL-12 were restimulated in vitro with irradiated parental C26 for 2 days, and the culture supernatant was assayed for IFN-γ production. Data are shown as the mean; bars, ±SD. ∗P < 0.05 and ∗∗P < 0.005, respectively, compared with nontreated mice. Similar results were obtained in three independent experiments. C, augmented tumor-specific CTL activity by IL-27. Spleen cells of the recovered mice (n = 3) were restimulated in vitro with irradiated parental C26 for 5 days, and cytotoxic T lymphocyte (CTL) activity of resultant effector cells was measured against 51Cr-labeled parental C26 or irrelevant syngeneic MethA tumor targets in a standard 51Cr release assay. Data are shown as the mean; bars, ±SD. Similar results were obtained in three independent experiments.
- Fig. 4.
Involvement of CD8+ T cells, IFN-γ, and CD4+ T cells in the induction of antitumor activity by interleukin (IL)-27. BALB/c-nu/nu mice (n = 5) were injected s.c. with each C26 transfectant (2 × 105 cells), and tumor growth (A) and survival rate (B) were monitored. Data are shown as the mean ± SD. Similar results were obtained in two independent experiments. C, BALB/c mice (n = 5) were injected s.c. with each C26 transfectant (2 × 105 cells) and injected i.p. with anti-IFN-γ neutralizing monoclonal antibody (mAb), anti-CD8 or anti-CD4 depleting mAbs, or control antibody as described in “Materials and Methods,” and tumor growth was monitored. Data are shown as the mean; bars, ±SD. Similar results were obtained in three independent experiments.
- Fig. 5.
A critical role of T-bet, but not signal transducer and activator of transcription (STAT) 4, in the induction of antitumor activity by interleukin (IL)-27. Wild-type, T-bet-deficient, and STAT4-deficient mice (n = 5) were injected s.c. with C26-vector (A) or C26-IL-27 (B) transfectants (2 × 105 cells), and tumor growth was monitored. Data are shown as the mean; bars, ±SD. Similar results were obtained in two independent experiments.
Volume 64, Issue 3
