| The recombinant MUC1-MBP fusion protein anti-tumor vaccine was prepared in our laboratory.The screening assays for vaccine adjuvants,CpG ODN1826 induce both of MUC1-specific humoral immune response and cellular immune response through combination of MUC1-MBP.Due to suppressed tumor volume was uneven,the dosage form changed to improve the effiency of the vaccine.CpG 1826,which is TLR9 agonists,can contribute to the mature and activation of DCs and Th1 immune response.MF59 is an oil-in-water emulsion and it is one of the earliest approved of adjuvants.MF59 alone can extend the residence time of antigen in lymph node macrophages regions and follicular DCs regions.Meanwhile,other report that the MF59 induced uptake and present of antigens to draining lymph nodes and accelerated the differentiation of mononuclear cells and granulocytes to DCs.In recent years,the studied showed that the combination of MF59 and CpG-ODN can induce biased Th1 immune response.Gene chip analysis showed the common target genes of MF59 and CpG 1826 include of IL-6,IL-1b,CSF-1,CXCL-10,TNFRSF-1b,IFNAR-2,etc.However,the combination of MF59 and CpG-ODN as tumor vaccine adjuvants needed to explore and reveal the unclear mechanism of action.In order to explore the application value of composite adjuvant MF59/CpG 1826 in MUC1-MBP tumor vaccine,we firstly study the combination induced mice immune activity and antitumor effect.And then to DCs as the breakthrough point,the mechanism of antitumor was deeply study.The theory basis of new type MUC1-MBP antitumor vaccine and composite adjuvant were illuminated.This research mainly includes the following aspects:1.The research on immunological activity induced by the combination of compound adjuvants MF59/CpG 1826 and MUC1-MBP.(1)The compound adjuvants MF59/CpG 1826 induced the production of MUC1-specific antibody.In this research,we chose Al(OH)3 adjuvant,MF59 or BCG combined with CpG 1826 respectively immune mice along with MUC1-MBP.At the 7 days after last immunization,the eyeball blood was collected and antibody subtypes were detected by ELISA.The results showed that the MF59/CpG 1826 induced the highest Ig G2c/Ig G1 among the groups,and represented the strongest Th1 response.(2)Effect of compound adjuvants MF59/CpG 1826 and MUC1-MBP on the secretion of cytokines in mouse spleen cells.In order to study the combination of adjuvants MF59/CpG 1826 and MUC1-MBP induced MUC1-specific Th cells in the spleen.We collected spleen lymphocytes 7 days after last immunization.The cytokines of Th1,Th2,Th17 and Treg in the supernatant were analysis by the chip.The results showed that the expression of IFN-? and IL-12p70 in MF59/CpG 1826 group were higher compared to that in CpG 1826 group,meanwhile,IL-5 and IL-6 were lower.The cytokines of Th17 and Treg subsets have no obviously changed.These results indicated that the combination of adjuvants MF59/CpG 1826 and MUC1-MBP significantly induced Th1 response superior to CpG 1826.(3)The compound adjuvants MF59/CpG 1826 induced NK and MUC1-specific CTL cytotoxicity.To further study on the immune activity of NK cells in mice,we collected spleen lymphocytes at 7 days after last immunization as effector cells,and YAC-1 as target cells.The cytotoxicity of NK was detected at the different ratio of effector and target cells.There was no statistical difference between each group of NK cytotoxicity.To study the immune activity of MUC1-specific CTL cells in mice,we collected spleen lymphocytes 4 days after last immunization.The spleen lymphocytes were restimulation by MUC1-MBP for five days as effector cells invitro.The mice B16-MUC1 melanoma cell line was used as a target cell and B16-neo as control.The cytotoxicity of CTL was detected at the different ratio of effector and target cells.The results showed that there was no statistical difference in B16-neo between each group of CTL cytotoxicity.The cytotoxicity of B16-MUC1 cells in each group increased accompained with the increase of the effective target ratio,which was most obvious in the composite adjuvant MF59/CpG 1826 group and CpG 1826/BCG group.The results above showed that each group induced MUC1 specific cytotoxicity,which suggesting that the MF59/CpG 1826 group and CpG 1826/BCG was the highest.2.Anti-tumor effect of compound adjuvant MF59/CpG 1826 combined with MUC1-MBP vaccine on melanoma.To evaluate the anti-tumor effect of the adjuvant MF59/CpG 1826 in combination with MUC1-MBP,C57 BL mice were immunized four times,B16-MUC1 was inoculated 7 days after the last immunization,and dynamic changes in tumors were observed.(1)The research on anti-tumor effect of MF59/CpG 1826 combined with MUC1-MBP on melanoma.1)The tumor from the tumor-bearing mice was weighted and recorded at the 19 days after tumor injection.The results showed that tumor growth were inhibited in all adjuvant groups.The adjuvants MF59/CpG 1826 combined with MUC1-MBP showed a stronger effect of tumor suppression.The good uniformity of tumor weight was in MF59/CpG 1826 group,superior to CpG 1826,MF59 and CpG 1826/BCG.2)The study on immunological activity induced by MF59/CpG 1826 in combination with MUC1-MBP in tumor-bearing miceTo study the mechanism of the adjuvant on tumor,we analyzed the immunological activity of tumor-bearing mice on the 19 th day after tumor inoculation.The spleen lymphocytes were restimulation by MUC1 or MUC1-MBP for five days in vitro.The culture supernatant was collected for analysize IFN-? and IL-4 secretion by ELISA.(1)The effects of different adjuvant on MUC1-specific cytokinesThe mice were immune with different adjuvant vaccine with MUC1-MBP for 4 times.The mice B16-MUC1 melanoma cell was injected at the 7 days after last immunization.At the 19 days after tumor injection,the spleen lymphocytes were collected and detected the expression of MUC1-specific IFN-? and IL-4 in the serum by ELISA.The highest level of IFN-? was observed in MF59/CpG 1826,which indicated induction of Th1 response,significantly higher than CpG 1826 group(P < 0.01),suggesting a role of Th1 response involve in anti-tumor effect of MF59/CpG 1826 in combination with MUC1-MBP.(2)The effects of different adjuvant effect on the production of MUC1-specific antibodies from mice19 days after tumor injection,the eyeball blood was collected and detected the expression of Ig G1,Ig G2 c and total Ig G antibody in the serum by ELISA.The CpG 1826,MF59/CpG 1826 group and CpG 1826/BCG group all induced MUC1-specific humoral immune responses,which was a Th1-prone immune response.CpG 1826 induced both of Th1-and Th2-responses and the Th1 response induced by CpG 1826 was significantly lower than that of MF59/CpG 1826(P < 0.01),consistent with our cytokines results.(2)Effect of compound adjuvant MF59/CpG 1826 combined with MUC1-MBP recombinant fusion protein on survival of tumor-bearing miceTo further evaluate the anti-tumor effect of adjuvant MF59/CpG 1826 combine with MUC1-MBP,we monitored the survival of mice in a prophylactic and therapeutic tumor-bearing mouse model,respectively.1)The combination adjuvants MF59/CPG 1826 and MUC1-MBP extended the survival in prophylactic model.(1)Establishment of a prophylactic tumor-bearing mouse modelFor long-term observation the preventive effect on of melanoma through the combination composite adjuvants MF59/CpG 1826 and MUC1-MBP,the preventive model was established in our research.The groups of immune mice as followed(10 in each group,half of male and female): MF59/CpG1826+MUC1-MBP,CpG 1826+MUC1-MBP,MF59+MUC1-MBP,MF59/CpG 1826 groups,NC group.The mice were immune with different adjuvant vaccine with MUC1-MBP for 4 times.The mice B16-MUC1 melanoma cell(5×106 cells /ml,each mouse injected 100 ml,5×105/mouse)was injected in the back of the neck at the 7 days after last immunization.Record the weight with vernier caliper and monitored the survival on alternate days.(2)The dynamic tumor observation of mice in the prophylactic model.In prophylactic model,the size of the tumor was observed at the 10 days after tumor injection.The tumor growth in MF59/CpG 1826 combined with MUC1-MBP group was the most slow,while tumor growth in MF59 combined with MUC1-MBP group,NC group and CpG 1826 combined with MF59 group was faster.The tumor size of each group was analyzed 31 days after tumor inoculation.Compared with NC group tumors,tumors in MF59/CpG 1826 group were significantly smaller(P < 0.01),and tumors in CpG 1826 group were also smaller(P < 0.05).(3)MF59/CpG 1826 combined with MUC1-MBP prolongs the survival of mice in prophylactic modelAt the 60 days after tumor injection,the survival of MF59/CpG 1826+MUC1-MBP,CpG 1826 + MUC1-MBP,MF59+MUC1-MBP,MF59/CpG 1826 groups,NC group was 80%,60%,30%,20%,10% respectively.At 70 days after tumor injection,the survival rate of MF59/CpG 1826 group was 80%,P < 0.01 compared with NC,P < 0.05 compared with CpG 1826 combined with MUC1-MBP group..2)The combination composite adjuvants MF59/CpG 1826 and MUC1-MBP extended the survival in therapeutic model.(1)Establishment of a therapeutic tumor-bearing mouse modelThe therapeutic anti-tumor effect of MF59/CpG 1826 combine with MUC1-MBP was further studied in a therapeutic tumor-bearing mouse model.The groups of mice as followed(10 in each group,half of male and female): MF59/CpG 1826+MUC1-MBP,CpG 1826+ MUC1-MBP,MF59+MUC1-MBP,MF59/CpG1826 groups,NC group.The mice B16-MUC1 melanoma cell(5 × 106 cells per ml,each mouse injected 100 ml,5 × 105/mouse)was injected in the back of the neck.At the 7 days after tumor injection,the mice were immune with different adjuvant vaccine with MUC1-MBP for 4 times for 4 weeks.Record the weight with vernier caliper and monitored the survival on alternate days.(2)The dynamic tumor observation of mice in the therapeutic modelIn therapeutic model,the size of the tumor was observed at the 7 days after tumor injection.The tumor growth in MF59/CpG 1826 combined with MUC1-MBP group was the slowest,followed by Cp G 1826 combined with MUC1-MBP group,while tumor growth in MF59 combined with MUC1-MBP group,NC group and CpG 1826 combined with MF59 group was faster.The tumor size of each group was analyzed 31 days after tumor inoculation.Compared with NC group tumors,tumors in MF59/CpG 1826 group were significantly smaller(P < 0.01),and tumors in CpG 1826 group were also smaller(P < 0.05).(3)The survival of mice in the therapeutic modelAt the 50 days after tumor injection,the survival of MF59/CPG 1826+MUC1-MBP,CpG 1826+MUC1-MBP,MF59+MUC1-MBP,MF59/CpG 1826 groups,NC group was 60%,40%,20%,20%,0% respectively.At the 70 days after tumor injection,the survival of MF59/CpG 1826+MUC1-MBP,Cp G 1826+MUC1-MBP,MF59+MUC1-MBP was 40%,20%,10% respectively.The mice in other two groups all died.The combination composite adjuvants MF59/CpG 1826 and MUC1-MBP inhibited the growth of tumor and extended the survival significantly.3.The effect of MF59 on DC uptake of CpG 1826 in vitro,local retention and cell localization.(1)MF59 significantly promoted DCs uptake of CpG 18261)Effect of MF59 on DCs Uptake of CpG 1826 by Flow cytometry.To investigate the effect of MF59/CpG 1826 on the uptake of BMDCs,We first labeled CpG 1826 with FITC(FITC-CpG 1826).The fluorescence intensity wasanalysied by flow cytometry at 2h,6h,12 h and 24 h after stimulated by FITC-CpG 1826 or FITC-CpG 1826/MF59.The results showed that the fluorescence intensity of BMDCs in the MF59/CpG 1826 group was higher than that in the CpG 1826 group at each time point,suggesting that MF59 combined with CpG 1826 significantly increased the uptake capacity of BMDCs.2)The effect of MF59 on DCs uptake CpG 1826 detected by fluorescence microscopy.We labeled CpG 1826 with green fluorescent FITC(FITC-CpG 1826)and MF59 with red fluorescent Dil(Dil-MF59).The uptake of CpG 1826 by BMDCs was observed by fluorescence microscopy at 6 hours after stimulation.The results showed that the fluorescence intensity of BMDC in MF59/CpG 1826 group was more strong,suggesting that MF59/CpG 1826 significantly promoted the uptake capacity of BMDC.(2)MF59 extended local retention time of CpG 1826 in the lymph nodes at the injection site.To investigate the effect of MF59 on the migration and retention of CpG 1826 in vivo,FITC-CpG 1826 was used.The mice were scanned by small animal in vivo imaging at 0h,4h,9h,and 24h after immunization.The results showed that after 9 hours of immunization with CpG 1826 alone,the fluorescence at the lymph nodes was stronger,but disappeared completely within 24 hours.While partial fluorescence was still visible at 24 hour when CpG 1826 combined with MF59,suggesting that MF59 can prolong the retention of CpG 1826 in vivo.(3)MF59 or CpG 1826 extend the local residence time of MUC1-MBP at the injection siteTo investigate the effect of CpG 1826 or MF59 on the migration and retention of MUC1-MBP in vivo,FITC-MUC1-MBP was used.MF59,CpG 1826 or MF59/CpG 1826 were combined with MUCl-MBP,respectively and inoculated in the subcutaneous inguinal region of mice,The mice were scanned by small animal in vivo imaging at 0h,4h,9h,and 24 h after immunization.The results suggest that MF59 or CpG 1826 significantly prolonged the retention of MUC1-MBP in lymphnodes,while the effect of MF59/CpG 1826 on the retention of MUC1-MBP in lymph nodes was not significantly different from that of MF59 or CpG 1826.(4)MF59/CpG 1826 are mainly co-localized DCs and T cell regions in mouse lymph node.To further find the cells where CpG 1826 and MF59 localized after immunization,we used FITC-labeled CpG 1826 and Dil-labeled MF59.MF59/CpG 1826 was inoculated subcutaneously inoculation in the groin of mice.The mice were euthanized 24 hours after the immunization.Lymph nodes were collected to prepare frozen sections.Four sections were continuously cut at the same position in the lymph nodes,and the following immune cells were stained: Violet-CD11b(Macrophages),Violet-CD11c(dendritic cell),Violet-B220(B cell),Violet-CD3(T cell),respectively.Immunofluorescence results showed that macrophages and B cells were mainly located around the lymph nodes,and DCs and T were mainly located in the central region of the lymph nodes.Red and green and yellow fluorescence were visible in the central region,suggesting that MF59/1826 mainly co-localized DCs and T region in the center of the lymph nodes.4.MF59 in combination with CpG 1826 promotes Th1 differentiation of CD4+ T Cell by inducing maturation of dendritic cells.The research above indicated that MF59 promotes the uptake of CpG 1826 by DC cells and prolongs the retention of CpG 1826 and MUC1-MBP at the injection site.MF59/CpG 1826 mainly localized DC cell regions and T cell regions in mice lymph node.DC cells are important antigen presenting cells(APC).We speculate that MF59/CpG 1826 may promote DC maturation.Subsquently,we investigate the effects of MF59/CpG 1826 on DC maturation in vitro and in vivo and whether it promoted Th1 polarization of CD4+ T cells.(1)MF59 /CpG 1826 promoted the maturation of DCs in vitro.To further investigate the effect of compound adjuvant MF59/CpG 1826 on the maturation of DCs,C57BL/6-derived mouse bone marrow cells were isolated and cultured in vitro stimulated with IL-4 and GM-CSF for 6 days.The expression of CD40,CD80,CD86,CCR7,MHC? and MHCII molecules on the surface of DCsafter stimulated with adjuvant was analyzed by flow cytometry.The results showed that the expression of CD40,CD80,CCR7 and MHCII was significantly increased(P < 0.05)in MF59/CpG 1826 group,compared with CpG 1826 group.Next,we examined the expression of IL-12p70 in the culture supernatant by ELISA.The results showed that MF59/CpG 1826 significantly promoted the expression of IL-12p70.In summary,MF59/CpG 1826 significantly induced the maturation and activation of DCs.(2)Combination of MF59 and CpG 1826 Promotes the Th1 Polarization of CD4+ T Cell co-cultured with DCsTo investigate whether BMDC treated with the combination of MF59/CpG 1826 affect the CD4+ T cell polarization,CD4+ T cells and BMDC cells were sorted.The purity of sorted CD4+ T and BMDC cells were analyzed by flow cytometry.The CD4+ T cells were cocultured with the BMDCs at a ratio of 50:1 and cultured for 48 h.The culture supernatant was collected 48 hours after stimulation.IFN-? and IL-4 level was detected by ELISA.The proliferation of co-cultured cells was examined by WST-1.The experiments above showed that MF59/CpG 1826 synergistically promoted the maturation of DCs.To investigate whether the maturation of DCs promoted by MF59/CpG 1826 would affect the Th1 polarization of CD4+ T cells,CD4+ T cells and BMDC cells were sorted.The purity of sorted CD4+ T and BMDC cells were analyzed by flow cytometry.The CD4+ T cells were cocultured with the BMDCs at a ratio of 50:1 and cultured for 48 h.The proliferation of co-cultured DCs and CD4+ T cells were detected using WST-1.Results showed the compound adjuvant MF59/CpG 1826 significantly promoted the proliferation of co-cultured cells.Supernatants were harvested and the levels of IFN-? and IL-4 were measured by ELISA.Co-culturing CD4+T cells with BMDCs stimulated with MF59/CpG 1826 significantly increased the production of IFN-? compared with NC groups.But the level of IL-4 was lower than either MF59 or CpG 1826 alone,suggesting the maturation and activation of the DCs stimulated by MF59/CpG 1826 promoted the polarization of the CD4+ T cells towards the Th1phenotype.(3)MF59/CpG 1826 combined with the MUC1-MBP vaccine significantly promoted the maturation of DCs in lymph node.To further investigate the effect of MF59/CpG 1826 on the maturation of DCs in vivo,the mice lymph nodes were harvested after four times immunization.Mouse lymph nodes were made into single cell suspension,and DC surface molecular expression was analyzed by flow cytometry.Results showed that the expression of CD80,CD86 and CCR7 was slightly increased(P<0.05)while MHC? and MHCII significantly increased(P<0.01)in mice immunized with CpG 1826,compare to NC group.The results in MF59 group are similar to those of the CpG 1826 group.In the MF59/CpG 1826 group,the expressions of CD40,CD86,CCR7,MHC? and MHCII were significantly increased(P<0.01),compared to CpG 1826 group.It is suggested that MF59/CpG 1826 combined vaccine MUC1-MBP significantly promotes the maturation of DCs in lymph node,compared with CpG 1826 combined MUC1-MBP group.5.Compound adjuvant MF59/CpG 1826 partly promotes DC maturation by down-regulating IL-6/STAT3.Compared with the CpG 1826 group,MF59/CpG 1826 significantly promoted DC maturation.Recent studies have found that CpG 1826 is also regulated by IL-6/STAT3 negative signals in addition to activation of My D88 and TRIF signaling pathways.IL-6 inhibited DC maturation through the JAK2-STAT3 signaling pathway.Therefore,IL-6 was as a key point for further mechanism study on DC maturation promoted by MF59/CpG 1826.(1)Research on MF59/CpG 1826 co-targeted genes by q PCR.Mosca detected changes in the transcriptional profiles of MF59,CpG and Al(OH)3 adjuvants by gene chip.It was found that the target genes shared by MF59 and CpG mainly include IL6,CSF1,CCL2,CCL4,CCL5,CCL12,IL2,IL-1b,IL-13,CXCL-10,TNFRSF-1b,IFNAR2,etc.Based on the chip results,six target genes were selected for q PCR detection: IL-6,CSF-1,TNFRSR-1b,IFNAR2,CCL-5,CXCL-10.We isolated BMDCs and stimulated with adjuvantfor 48 hours in vitro.q PCR results showed IL-6 secretion by CpG 1826 alone was extremely high while down-regulated significantly when CpG 1826 combined with MF59.It has been reported IL-6 inhibits DC maturation through the STAT3 signaling pathway,which need to be studied in the following experiment.(2)MF59 combine with CpG 1826 significantly down-regulated the expression of IL-6 and p STAT3 at protein level.To further explore the DC maturation mechanism by MF59/CpG 1826,IL-6 secretion was analyzed by ELISA,p STAT3 was analyzed by western blotting.The results showed that the expression of IL-6 and p STAT3 induced by MF59/CpG 1826 were significantly downregulated when compared with CpG 1826 group.These results suggest that IL-6/STAT3 signaling pathway was significantly down-regulated when MF59 combine with CpG 1826.(3)Exogenous IL-6 significantly inhibited the promotion of MF59/CpG 1826 on the maturation of DCs.To further validate the role of IL-6 in the maturation of DCs by CpG1826/MF59,exogenous IL-6 was used when DC stimulated with MF59/CpG 1826.In the IL-6 group,cells were pretreated with different concentrations of IL-6(50ng/ml or 100ng/ml)on the third day of isolation.On the fifth day,half of the medium was changed and the concentration of IL-6 was supplemented.On the sixth day of culture,IL-6(50ng/ml),IL-6(100ng/ml),IL-6(50ng/ml)+Cp G1826/MF59,IL-6(100ng/ml)+CpG1826/MF59,Cp G1826/MF59 were added to the well and culture for 48 hours.Cells were harvested of each group.The expression of DC surface markers was analyzed by flow cytometry.The expression of MHCII and CCR7 were significantly inhibited when IL-6 pretreatment.The expression of CD40?MHCII and CCR7 were significantly downregulated stimulated in IL-6(50ng/ml or 100ng/ml)combined with MF59/CpG1826 group(P<0.01),compared with MF59/CpG1826 group,suggesting maturation of DCs promoted by MF59/Cp G1826 was inhibited by IL-6.(4)IL-6 reverse the inhibition of p STAT3 by MF59/Cp G1826To investigate the effect of IL-6 on the inhibition of p STAT3 expression in DCcells induced by MF59/CpG1826,DC were pretreated with exogenous IL-6 and the expression of p STAT3 was analyzed by western blotting.Results showed that IL-6 promotes the expression of p STAT3.The expression of p STAT3 was upregulated when MF59/CpG1826 was combined with IL-6(50ng/ml or100ng/ml),compared with MF59/CpG1826(P<0.01).Conclusion: In summary,our results demonstrate that MF59/CpG1826 combined MUC1-MBP has a good anti-tumor effect on the mouse melanoma in prophylactic and therapeutic model through MUC1-specific cellular and humoral immune responses.MF59 promoted the uptake of CpG1826 by DC and prolonged the retention of CpG1826 and MUC1-MBP in lymph nodes.MF59 and CpG1826 co-localized in DC and T cell region in lymph nodes.Further,MF59/CpG 1826 synergistically promoted the maturation of DC cells in vitro and in vivo and indirectly promoted Th1 differentation of CD4+ T cell co-cultured with DC.Meanwhile,MF59/CpG 1826 significantly promoted DC maturation partly by down-regulating the expression of IL-6/STAT3 signaling pathway-associated proteins.This study clarified the mechanism of MF59/CpG 1826,revealed its adjuvant role in the MUC1-MBP vaccine,and demonstrated the application value of MF59/CpG 1826 as a vaccine complex adjuvant,laying foundation for the development of a new MUC1-MBP vaccine. |
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