2019 Dec 31
Cell-Based IL-15:IL-15Rα Secreting Vaccine as an Effective Therapy for CT26 Colon Cancer in Mice
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Cell-Based IL-15:IL-15Rα Secreting Vaccine as an Effective Therapy for CT26 Colon Cancer in Mice
Interleukin (IL)-15 is an essential immune-modulator with high potential for use in cancer treatment. Natural IL-15 has a low biological potency because of its short half-life and difficulties in mass-production. IL-15Rα, a member of the IL-15 receptor complex, is famous for its high affinity to IL-15 and its ability to lengthen the half-life of IL-15. We have double-transfected IL-15 and its truncated receptor IL-15Rα into CT26 colon cancer cells to target them for intracellular assembly. The secreted IL-15:IL-15Rα complexes were confirmed in ELISA and Co-IP experiments. IL-15:IL15Rα secreting clones showed a higher anti-tumor effect than IL-15 secreting clones. Furthermore, we also evaluated the vaccine and therapeutic efficacy of the whole cancercell vaccine using mitomycin C (MMC)-treated IL-15:IL15Rα secreting CT26 clones. Three sets of experiments were evaluated; (1) therapeutics, (2) vaccination, and (3) longterm protection. Wild-type CT26-bearing mice treated with a single dose of MMC-inactivated secreted IL-15:IL-15Rα clones prolonged survival compared to the control group. Survival of MMC-inactivated IL-15:IL-15Rα clone-vaccinated mice (without any further adjuvant) exceeded up to 100%. This protection effect even lasted for at least three months after the immunization. Secreted IL-15:IL-15Rα clones challenging trigger anti-tumor response via CD4
+ T, CD8 + T, and natural killer (NK) cell-dependent cytotoxicity. Our result suggested that cell-based vaccine secreting IL-15:IL-15Rα, may offer the new tools for immunotherapy to treat cancer.
CT26 colon carcinoma; IL-15:IL15Rα complex; cell-based vaccine; immunotherapy; interleukin 15 receptor alpha; interleukin-15.
Conflict of interest statement
The authors have no potential conflicts of interest to disclose.
Fig. 1. Expression of exogenous IL-15 and/or IL-15Rα in CT26 colon carcinoma cells
(A) Construction of expression vectors encoding IL-15 or IL-15Rα. In the expression vectors, the natural signal peptide sequence (SS) of IL-15 or IL-15Rα were replaced with that of IL-2. TM, transmembrane domain; CY, cytoplasmic domain. (B) Quantitation of IL-15 or IL-15:IL-15Rα complex, secreted from 1 × 10
6 cells of transfected clones for 24 h incubation by ELISA. The monoclonal antibody used to detect IL-15 (DuoSet ELISA kit; R&D System) did not bind to the IL-15:IL-15Rα complex. The monoclonal antibody to the IL-15:IL-15Rα complex (Platinum ELISA; Affymetrix) did not bind to IL-15 alone. (C) Immunoprecipitation (IP) of IL-15 and IL-15Rα from the culture supernatants of the indicated transfectants. The amount of immunoprecipitated proteins were analyzed by immunoblotting (IB) with the indicated antibodies. Then, the relative intensive was shown in the bar graph. (D) Transfected cell proliferation analysis in vitro by MTT assay. The cell proliferation was correlated with the absorbance at optical density 570 nm. (E) Secreted exogenous IL-15 and IL-15:IL-15Rα complexes stimulated splenocyte proliferation in vitro. Culture supernatants from 1 × 10 6 cells of each transfected tumor clone were collected after 24 h. The splenocytes from normal BALB/c mice were re-suspended in the medium containing the indicate culture supernatants for 72 h. Then, the proliferation of splenocytes was assayed by MTT assay. Data are mean ± SEM of three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant.
Fig. 2. Tumorigenicity of CT26 tumor clones expressing IL-15 or IL-15:IL-15Rα complexes
After implantation of tumor clones (1 × 10
6 cells/mouse) expressing IL-15 or IL-15:IL-15Rα into BALB/c mice (n = 5) subcutaneously at the right flank, tumor growth and survival of mice were monitored daily basis. (A) Tumor size was measured with a Vernier calipers and the tumor volume was calculated. ** P < 0.01; *** P < 0.001. (B) Long term survival analysis. Plot of the percentage of survival rate of the groups. (C) The changes in body weight after the tumor cell implantation was shown. Body weight of same age mice (n = 2) without tumor implantation was observed as a control.
Fig. 3. IL-15:IL-15Rα complexes stimulate anti-tumor cytotoxic effect through CD4
+, CD8 +, and NK cells in vitro
(A) Splenocytes, obtained from BALB/c pre-immunized with CT26, were mixed and cultured with MMC-inactivated tumor clones expressing IL-15 or IL-15:IL-15Rα. The ratio of splenocytes to tumor clones is 50:1. After 48 h, the morphology of MMC-inactivate tumor clones in the mixed-cell cultures was recorded under a microscope. A representative result was shown from 3 independent experiments. (B) Pre-immunized splenocytes were stimulated
in vitro with MMC-inactivated wild-type cells (antigen sources) and culture supernatants of tumor clones expressing IL-15:IL-15Rα complexes (cytokine sources) for 48 h. The culture supernatant from a parent or mock-transfected clones was used as control. The stimulated splenocytes and CFSE-labeled wild-type CT26 target cells were mixed for at ratio of 20:1 or 50:1. After 6 h incubation, the cell mixtures were collected and stained with PI. The percentage of CFSE +PI + cells (dead cells) were counted by FACS. (C and D) The activated splenocytes, as described in (B) were pre-incubated with 10 μg/ml anti-CD4 or anti-CD8 antibodies for 30 min before mixing with CSFE-labeled CT26 target cells. After 6 h incubation, the percentage of CFSE +PI + cells were analyzed by FACS. (E) The activated splenocytes, as described in (B) were mixed with CFSE-labeled YAC-1 target cells (ratio 50:1). After 6 h incubation, the cells were harvested and stained with PI. CFSE +PI + cells were counted by FACS. * P < 0.05; ** P < 0.01; *** P < 0.001.
Fig. 4. Therapeutic and vaccination effects of tumor clones expressing IL-15:IL-15Rα complexes against CT26 cells
(A) The schedule of tumor cell injection for therapeutic effect. Normal mice were implanted with wild-type (WT) CT26 cells (1 × 10
5 cells/mouse) subcutaneously at the right flank, and injected with MMC-inactivated mock or IL-15:IL-15Rα tumor clones (1 × 10 6 cells/mouse) at near sites after 3 days (n = 5 for each group). (B and C) Tumor growth (B) and survival (C) of mice were monitored. (D) The schedule of tumor cell injection for vaccination. Mice were subcutaneously vaccinated with MMC-inactivated mock or tumor clones expressing IL-15:IL-15Rα (5 × 10 5 cells/mouse) at right flank. After two weeks, WT CT26 cells (5 × 10 5 cells/mouse) were subcutaneously implanted at the left flank. (E and F) Tumor growth (E) and survival (F) of mice were monitored. * P < 0.05.
Fig. 5. Vaccination with MMC-inactivated tumor clones expressing IL-15:IL-15Rα complexes, stimulates memory phenotypes in splenic cells of mice
(A) The splenocytes from sacrificed vaccinated mice described in Figures 4D–4F were counted. (B) They were stained to analyze for percentages of effector memory (CD44
+ CD62L −) and central memory (CD44 + CD62L +) of CD4 + or CD8 + T cells and NK subsets (total NK cells; CD49b +, effector NK cells; CD49b + CD11b +, NKT cells; CD3 + CD49b +). (C) The indicated numbers of CD4 +, CD8 +, and NK cells in splenocytes were graphed. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.
Fig. 6. Long-term anti-tumor memory immune response is acquired by vaccination with MMC-inactivated tumor clones expressing IL-15:IL-15Rα complexes
(A) The schedule of tumor implantation. (1st) Mice were subcutaneously primed tumor clones expressing IL-15:IL-15Rα (1 × 10
6 cells/mouse) at day 0 on the left flank. (2nd) One month after the priming, tumor-free mice were subcutaneously challenged with 1 × 10 6 cells of live wild-type (WT) CT26 cells on the right flank. (3rd) Two months after the boosting step, the tumor-free mice were re-challenged subcutaneously with 1 × 10 6 cells of live WT CT26 cells on the left flank. (B and C) After the boosting step, the average tumor growth (B) and the survival of mice (C) were monitored daily basis. (D-F) After the re-challenging step with WT CT26, the tumor growth (D) and survival of mice (E) were recorded. On day 18 after the re-challenging, mice with tumor growth were sacrificed, and spleens and tumor masses (F) were photographed. * P < 0.05; ** P < 0.01; *** P < 0.001.
Fig. 7. Challenging with live tumor clones expressing IL-15:IL-15Rα stimulated long-term memory phenotypes in splenic cells
(A) Tumor-bearing mice same as Figure 6F were sacrificed, and the total number of splenocytes were counted. (B and C) The splenocytes obtained from the indicated mice were stained with antibodies to analyze the indicated CD4
+, CD8 +, and NK cell subsets. * P < 0.05; ** P < 0.01; *** P < 0.001.
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