Ectopically Expressed Membrane-bound Form of IL-9 Exerts Immune-stimulatory Effect on CT26 Colon Carcinoma Cells

Abstract

IL-9 is a known T cell growth factor with pleiotropic immunological functions, especially in parasite infection and colitis. However, its role in tumor growth is controversial. In this study, we generated tumor clones expressing the membrane-bound form of IL-9 (MB-IL-9) and investigated their influences on immune system. MB-IL-9 tumor clones showed reduced tumorigenicity but shortened survival accompanied with severe body weight loss in mice. MB-IL-9 expression on tumor cells had no effect on cell proliferation or major histocompatibility complex class I expression in vitro. MB-IL-9 tumor clones were effective in amplifying CD4⁺ and CD8⁺ T cells and increasing cytotoxic activity against CT26 cells in vivo. We also observed a prominent reduction in body weights and survival period of mice injected intraperitoneally with MB-IL-9 clones compared with control groups. Ratios of IL-17 to interferon (IFN)-γ in serum level and tumor mass were higher in mice implanted with MB-IL-9 tumor clones than those observed in mice implanted with control cells. These results indicate that the ectopic expression of the MB-IL-9 on tumor cells exerts an immune-stimulatory effect with toxicity. To exploit its benefits as a tumor vaccine, a strategy to control the toxicity of MB-IL-9 tumor clones should be developed.

Keywords: Cancer vaccine; Colon carcinoma; Interleukin-9; Membrane-bound; Toxicity.

Figure 1

Generation of CT26 tumor clones expressing the membrane-bound form of IL-9. (A) The chimeric MB-IL-9 cDNA comprised the cDNA encoding the CY (from −75 to −45) and TM (from −44 to −24) of murine TNF-α and IL-9 cDNA without signal sequence (54–435). A spacer sequence (Gly-Gly-Ils) was inserted between TNF-α and IL-9. (B) MB-IL-9 transfectants were analyzed for IL-9 expression using RT-PCR. Two positive clones of the membrane-bound form (MB-1 and MB-7 clones) were chosen for further experiments. One million cells of MB-IL-9 tumor clones were cultured for 24 h and culture supernatants (C) and cells (D) were separated to quantitate MB-IL-9 protein expression by ELISA. A representative result was presented from more than 3 independent experiments. (E) The expression of IL-9 on cell surface was analyzed by flow cytometry analysis using polyclonal anti-IL-9 antibody. The statistical significance of the differences between mock-vector group vs. MB-IL-9 groups were analyzed by 1-way ANOVA. CY, cytoplasmic domain; TM, transmembrane domain; SS, signal sequence; Medium, complete RPMI medium; P, wild-type CT26; M, mock vector-transfected clone; NS, not significant. ^**p<0.01.

Figure 2

Tumor growth and survival of mice implanted with MB-IL-9 tumor clones. (A) BALB/c mice (n=5) were s.c. injected with indicated number (1, 0.5, or 0.1 million) of MB-IL-9 tumor clones (MB-1 and MB-7 clones) and tumor growth monitored. Comparable tumor volume measured at the indicated dates after injection was presented. The statistical significance of the differences between mock-vector group vs. MB-IL-9 groups were analyzed by 1-way ANOVA. (B) One of mice from each group was randomly chosen and sacrificed at indicated dates to visualize tumor mass. The weight of isolated tumor mass was also measured. (C) Survival of mice in each experiment was estimated. Mean survival (days) was indicated insets. Two independent survival experiments were repeated in case of 0.5 and 0.1 million cell injections. One of representative results was presented. (D) Body weights of mice injected with 0.5 million cells of MB-IL-9 tumor clones were simultaneously measured. A group of mice (n=5) with same age was used as control for body weight change. P, wild-type CT26; M, mock vector-transfected clone; NS; not significant. ^*p<0.05.

Figure 3

Effects of MB-IL-9 tumor clones on T lymphocytes subsets in spleens. (A) Two mice per group were implanted with MB-IL-9 tumor clones or control cells (1 million cells), and one of mice per group was sacrificed at day 15 to analyze CD4⁺ and CD8⁺ T cell subsets. (B) The other mouse of each group was also sacrificed at day 17 for the same analysis, and average proportions of T cell subsets were presented. P, wild-type CT26; M, mock vector-transfected clone. The p-values were ^*p<0.05, ^**p<0.01, ^***p<0.001. Difference alphabets mean follow as; ^aaverage±standard deviation from tumor bearing mice, ^bstatistical significance was assessed by 1-way ANOVA to evaluate differences in T lymphocytes between MB-IL-9 groups and mock controls.

Figure 4

Cytotoxic activity of spleen cells immunized with MB-IL-9 tumor clones. (A) Splenocytes were isolated from mice pre-immunized with MMC-inactivated CT26 cells and stimulated in vitro for 56 h with MMC-inactivated MB-IL-9 clones at different ratios (spleen cells: stimulators=10:1 or 50:1). The morphology of cells in the mixed cell culture was recorded under a microscope. A representative result was shown from 3 independent experiments. (B) Spleen cells activated in vivo and in vitro were assayed for cytotoxic activity against wild-type CT26 cells. The activated spleen cells were harvested and incubated for 3.5 h with CFSE-stained wild-type CT26 cells at 3 different E:T ratios (1;1, 5:1, 50:1) in triplicate, followed by their treatment with PI before flow cytometry analysis. The ratio of PI⁺ cells among CFSE⁺ population was estimated cytotoxic activity of splenocytes. A representative result at 50:1 ratio was presented from 2 independent experiments. The p-value between M and MB-1 or MB-7 was less than 0.07 or 0.08, respectively. P, wild-type CT26; M, mock vector-transfected clone.

Figure 5

Toxicity of MB-IL-9 tumor clones in vivo. BALB/c mice (n=5) were i.p. injected with one million MB-IL-9 tumor clones. Body weights (A) and survival (B) of mice were monitored daily. (C) At day 12, one of mice from each group was randomly chosen to sacrifice and the presence of lumps of tumor cells in the abdominal cavity examined. Arrows indicate the lumps of tumor cells. P, wild-type CT26; M, mock vector-transfected clone.

Figure 6

Levels of cytokines in the serum and tumor mass. At day 21 after the s.c. injection of 0.5 million MB-IL-9 clones per mouse, blood was collected from the eye artery (A) and tumor mass isolated (B) to quantitate various cytokines. To analyze cytokines in tumor mass, 0.5 g of tumor mass from each group was lysed using a lysing buffer. The IL-17/IFN-γ ratio was calculated and presented at the lowest panel (C). Cytokines were quantitated by ELISA. The statistical significance of the differences between mock-vector group vs. MB-IL-9 groups were analyzed by 1-way ANOVA. Control; tumor free mice, P; wild-type CT26, M; mock vector-transfected clone. ^*p<0.05, ^**p<0.01.