Preliminary study of the inhibitory effect and mechanism of B16F10-ESAT-6-gpi/IL-21 vaccine on the pulmonary metastasis in mouse models of melanoma / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the effect and mechanism of B16F10-ESAT-6-gpi/IL-21 tumor cell vaccine on pulmonary metastasis in mouse model of melanoma.</p><p><b>METHODS</b>Twelve 8-week old female C57BL/6 mice were used in this study. The mice were injected with wild-type B16F10 cells through tail vein after immunization with B16F10-ESAT-6-gpi/IL-21 tumor cell vaccine, and the pulmonary metastasis was observed. The CD4(+) and CD8(+) T cells were isolated by magnetic activated cell sorting, and then used for the detection of CFSE/7-AAD cytotoxicity by flow cytometry. Serum from the mice immunized with tumor-cell vaccine was used to detect IFN-γ expression by ELISA. The expression of TGF-β2, ZEB1, E-cadherin, and N-cadherin of tumor tissues was detected by RT-PCR and immunofluorescence, respectively.</p><p><b>RESULTS</b>The mice vaccinated with B16F10-ESAT-6-gpi/IL-21 had significantly fewer nodules in the lung and lower lung weight [(285.8 ± 19.01) mg vs. (406.3 ± 27.12) mg], with lower levels of TGF-β2, ZEB1 and N-cadherin proteins but higher level of E-cadherin protein within the tumor tissue, as compared with the control mice. Meanwhile, the immunized mice had significantly increased CD8(+) T cell killing activity [(42.62 ± 3.465)% vs. (22.29 ± 1.804)%] and IFN-γ expression level [(55.200 ± 7.173) pg/ml vs. (6.435 ± 1.339) pg/ml] over the control mice.</p><p><b>CONCLUSIONS</b>The B16F10-ESAT-6-gpi/IL-21 vaccine can inhibit the metastasis of melanoma in the lung in vaccinated melanoma-bearing mice. This inhibitory effect is associated with CD8(+) T cell immune response and a higher level of IFN-γ, which may influence on the mesenchymal-epithelial transition of tumor cells.</p>

Study on anti ovarian cancer efficacy elicited by CD34~+ umbilical cord blood hematopoietic stem cells transfected with IL-21 in nude mouse model / 中华微生物学和免疫学杂志

Objective To investigate the CD34~+ umbilical cord blood hematopoietic stem cells (CD34~+ UBSC) transfected with interleukin 21 (IL-21) against the ovarian cancer effect in tumor-bearing nude mice. Methods CD34~+ UBSC were obtained from the UBSC by a magnetically activated cell sorting technique and then transfected with recombinant plasmid plRES2-IL-21-EGFP after the CD34~+ UBSC were proliferated in vitro. The therapeutic effect was evaluated by the size of the tumor and the life span in nude mice treated with the CD34~+ UBSC-IL-21. The expression of IL-21 and its bioactivity in CD34~+ UBSC-IL-21 and in local neoplasitc tissues were respectively detected by reverse transcription-polymerase chain reaction (RT-PCR), immune fluorescence technique, ELISA, Western blot, immunohistochemistry and splenocyte proliferative activity. The NK cell cytotoxicity and the numbers of NK cells, serum level of IFN-γ and TNF-αwere simultaneouly detected by FCM and ELISA, respectively. Results CD34~+ UBSC were cultured and transfected with pIRES2-IL-21-EGFP successfully. CD34~+ UBSC-IL-21 could inhibit the tumor growth and extended nude mice life span compared with other groups (P < 0.01). The expression of IL-21 in the neo-plastic tissue, serum level of IFN-γ and TNF-α , NK cell activity and the numbers of NK cells of mice origin and of human origin in splenocytes were increased significantly in the nude mice treated with CD34~+ UBSC-IL-21 compared with other groups(P <0.01). Conclusion The CD34~+ UBSC transfected with IL-21 have competent against ovarian cancer in tumor-bearing nude mice. The findings may establish a foundation for gene therapy of the ovarian cancer by CD34~+ UBSC-IL-21 in clinic application.

Anti-tumor mechanisms of lymphopenic mice transferred with tumor-specific lymphocytes and immunized with mIL-21 tumor vaccine / 中华微生物学和免疫学杂志

Objective To explore the mechanism of anti-tumor effects of transferring tumor-specif-ic lymphocytes obtained from pre-immunized BALB/c mice with inactive rolL-21 tumor vaccine (mIL-21-Sp2/0)to syngeneic mice, associated with mIL-21 tumor vaccine immunization, in the condition of cyclo-phosphamide (Cy)-induced lymphopenia. Methods Activated lymphocytes of spleen and lymph nodes ob-tained from pre-immunlzed syngeneic mice with irradiated mIL-21-Sp2/0 cells were infused into BALB/cmice treated with Cy 2 days before, subsequently vaccinated with mlL-21 tumor vaccine, after 7 days, chal-lenged with Sp2/0 tumor cells, observed the growth of tumor of mice. T lymphocyte subsets differentiation was measured by flow cytometry (FCM) analysis. The proliferation and cytotoxie activities of activated lym-phocytes were analyzed by FCM, respectively, staining with CFSE and 7-AAD. The number of IFN-γ-secre-ting cells was evaluated by ELISPOT. Results The lymphopenic mice were transferred with activated lym-phocytes and inoculated with raiL-21 tumor vaccine might provide superior anti-tumor immunoprotection, re-tard tumor growth of the mice. The proliferating capabilities and killing rate of transferred tumor Ag-specific lymphocytes enhanced obviously, the number of IFN-γ-secreting cells was significantly higher compared with the control groups. Conclusion Under Cy-induced lymphopenia condition, tumor Ag-specific lymphocytes sensitized by raiL-21 tumor vaccine were transferred to mice and immunized with mlL-21 tumor vaccine at the same time, benefit the proliferation of transferred effective cells and immune cells itself, assist to form and sustain special anti-tumor effects.

Investigation on the anti-tumor effect and mechanisms of the glycosyl phosphatidylinositol modified routine IL-21 tumor vaccine / 中华微生物学和免疫学杂志

Objective To construct the murine IL-21 (mIL-21) tumor vaccine modified by glyco-syl phosphafidylinositol(GPI), and to evaluate its anti-tumor effect and mechanisms. Methods The IL-21-GPI gene was acquired by overlap PCR and inserted into PeDNA3.1. The recombinant plnsmid pcDNA3.1/ IL-21-GPI was transformed into cell B16F10, and the expression of mIL-21 on cell membrane was deter-mined by cell indirect immumofluorescence and flow cytometry (FCM). The bioactivity of mIL-21 was iden-tiffed according to its effects on the proliferation of mouse spleen cells. The anti-tumor effect was evaluated depending on the tumor size and the survival of tumor-beating mice after the tumor vaccine was inoculated into C57BL/6 mice. And the activity of cell-mediated immunity in immunized mice was detected at the same time. Results The recombinant plasmid pcDNA3.1/IL-21-GPI was correctly constructed, which could ex-press mIL-21 binding the membrane with good bioactivity. The vaccine had good anti-tumor effect, and the cell-mediated immunity had been improved in immunized mice. Conclusion The GPI modified mIL-21 tumor vaccine with anti-tumor activity was constructed successfully, which provided a good foundation for studying anti-tumor immunity and therapy in future.