Study On The Mechanism Of Antitumor Effect Mediated By CDC1 Activation Induced By MUC1-MBP Combined With CpG 2006

Mucin-1(MUC1)is a tumor-associated antigen.Maltose binding protein(MBP)is a TLR2/TLR4 ligand with immune-enhancing property.To enhance the immunogenicity and antigen presenting cell(APC)uptake,our group generated a recombinant MUC1-MBP fusion protein(MUC1-MBP,M-M)by the fusion of MUC1 and MBP by genetic engineering.Subsequently,different adjuvants were screened to develop an antitumor vaccine which can combined with it and improve its activity.CpG2006 exhibited superior antitumor activity,therefore M-M was combined with CpG2006 to prepare a vaccine named recombinant M-M fusion protein vaccine.Effective cellular immunity,particularly the response of tumor-specific CD8+ T cells,is critical in antitumor immunity.In cancer vaccine development,vaccine-antigen-specific CTL responses correlate strongly with tumor suppression and host survival in mouse models and clinical trials [1,2].Our previous studies have demonstrated that M-M combined with CpG 2006 vaccine has a significant antitumor effect in B16-MUC1 tumor-bearing mice,inducing MUC1-specific CTL killing activity and Th1 activation [3,4].However,the mechanism by which this vaccine,as an exogenous antigen,successfully induces MUC1-specific CTL killing activity is not understood fully.Recent studies have shown that cDC1s express the chemokine receptor XCR1 and C-type lectin receptor DNGR-1/CLEC9 A.They specialize in cross-presentation of exogenous antigens to CD8+ T cells via MHC I.Would it be possible that M-M in combination with CpG 2006 induces CTL killing via cDC1 cross-presentation?Therefore,this study aims to investigate the role of cDC1s in CTL killing induced by the combination of M-M and CpG 2006,using DC subpopulation as a target.Furthermore,the mechanism of IFN-I signaling as a starting point to regulate cDC1activation-induced CTL killing will be further explored.M-M combined with CpG 2006 vaccine induced CTL cytotoxicity and inhibited B16-MUC1 tumor growth in miceB16-MUC1 melanoma cells expressing human MUC1 were used to establish a prophylactic mouse model to evaluate the antitumor effects and induced immune responses of different vaccine doses in mouse models.C57BL/6 mice were subcutaneously immunized five times weekly with different vaccine doses(25,50,and100 μg).B16-MUC1 tumors were inoculated subcutaneously 7 days after the final immunization,and tumor growth was monitored.14 days post-inoculation,the mice were euthanized,and the tumors were dissected.The tumor suppression rate was calculated,MUC1-specific antibodies were detected in the serum,IFN-γ and IL-4 were detected in the supernatants of splenocyte cultures,and the proportions of Th1 and Tc1 in the spleens and d LNs were determined.Splenocytes were stimulated with MUC1-MBP for 5 days to use as effector cells,B16-MUC1 cells were used as target cells,and CTL killing curves were monitored by RTCA to evaluate the MUC1-specific killing rate.1.M-M combined with CpG 2006 vaccine inhibits B16-MUC1 tumor growth in miceThe groups vaccinated with doses of 25 μg,50 μg,and 100 μg of the vaccine demonstrated significant tumor inhibition.The rate of tumor inhibition increased in a dose-dependent manner,with the 100 μg group showing a tumor inhibition rate as high as 82.9%.2.M-M in combination with the CpG 2006 vaccine induces MUC1-specific humoral immune response and enhances Th1 activation in B16-MUC1 tumorbearing miceDetection of MUC1-specific antibodies in serum by ELISA showed that antiMUC1-specific Ig G,Ig G1 and Ig G2 c antibodies were induced by different doses of MM + CpG 2006,and the Ig G2c/Ig G1 ratio was increased in the 100 μg group;Cytokines in culture supernatants of splenic lymphocytes stimulated with specific antigen(M-M)were determined by double-antibody sandwich ELISA,and the results showed that 50μg and 100 μg of the vaccine increased the secretion of IFN-γ and decreased the secretion of IL-4;Flow cytometry results showed that Th1 subpopulation(CD3+CD8-IFN-γ+)was significantly increased in the d LNs of the 50 μg and 100 μg vaccine groups P < 0.05).These results suggest that the M-M combined CpG 2006 not only induced a MUC1-specific humoral immune response,but also induced enhanced Th1 activity.3.M-M combined with CpG 2006 vaccine induced antitumor CTL immune responses in B16-MUC1 tumor-bearing miceFlow cytometry analysis showed that the 50 μg and 100 μg vaccine groups upregulated the proportion of Tc1 subpopulation(CD3+CD8+IFN-γ+)in the spleens and d LNs.RTCA results showed that MUC1-specific CTL killing was induced in the 25μg,50 μg and 100 μg vaccine groups,and that the killing activity was increased incrementally with vaccine dose.The results suggest that M-M combined with CpG2006 vaccine can effectively induce MUC1-specific CTL immune response in B16-MUC1 tumor-bearing mice.The effect of M-M combined with CpG 2006 vaccine on DC subpopulationsIn this section,we study the impact of M-M combined with the CpG 2006 vaccine on the immune function of the DC subpopulations,both in vivo and in vitro.Additionally,we analyze the effect of cDC1s on the vaccine-induced MUC1-specific CTL killing activity.1.In vivo studies of the effects of vaccine on DC subpopulationsThe vaccine and its components were applied to immunize mice to study the effect of the vaccine on DC subpopulations in mice and the role of DC subpopulations in the induction of CTL killing activity.C57BL/6 mice received five subcutaneous immunizations of M-M(100 μg),CpG 2006(100 μg),and M-M + CpG 2006(100 μg each),administered once a week.7 days following the last immunization,we evaluated the proportion of each DC subpopulation and maturation marker(MHC Ⅰ,CD40,CD80,CCR7)in the spleens and d LNs.Flow cytometry sorting of CD8+ T,p DC,and cDC1 cells from the spleen was performed for 3 days of culture in the presence of M-M,B16-MUC1 cells were used as target cells,and CTL killing curves were monitored by RTCA to calculate the MUC1-specific killing rate.1.1 M-M combined with CpG 2006 vaccine enhances the ratios of p DC1 s and cDC1s,and the maturation of p DC1 s,cDC1s,and cDC2s in mice.This enhancement is dependent on CpG 2006.Flow cytometry analysis showed that both the M-M + CpG 2006 group and the CpG 2006 group up-regulated the ratios and maturation of p DCs and cDC1s,and were able to up-regulate cDC2 maturation without any significant effect on cDC2 ratio.These effects were observed in both spleen and d LN,and there was no statistically significant difference between the two groups.Furthermore,M-M did not show any significant effect on the ratio of DC subpopulation and maturation.1.2 cDC1s are the dendritic cells that play a pivotal role in eliminating MUC1-specific CTLs induced by our vaccine,and the IFN-Ⅰ signaling pathway in cDC1s regulates CTL immune response induced by the vaccine.Co-culture of DC subpopulations with CD8+T cells revealed that the cDC1subpopulation alone mainly triggered the MUC1-specific CTL killing activity.Addition of p DCs slightly increased the CTL killing activity(CTL+cDC1 vs.CTL+cDC1+p DC for 24.66% vs.29.12%).These findings suggest that cDC1s is the DC subpopulation that plays a significant role in inducing MUC1-specific killing.IFNAR1 antibody was applied to block IFNAR1 on cells in the DC subpopulationCD8+T co-culture system,and CTL killing activity was again assessed by RTCA method.The results showed that the MUC1-specific CTL killing activity was reduced by 61.9% after IFNAR1 antibody was blocked on all cells;the reduction in CTL killing activity was most obvious in the cDC1 blocking group(55.63%).The results suggest that IFN-Ⅰ signaling plays an essential role in the induction of CTL killing activity,and it regulates the vaccine-induced CTL killing activity mainly by action on cDC1s.2.The effect of M-M combined with CpG 2006 vaccine on DC subpopulations in vitroIn vitro,FL-DCs were successfully differentiated from C57BL/6 mouse bone marrow cells cultured with Flt3 L and low-dose GM-CSF.The vaccine components(MM,CpG 2006,M-M + CpG 2006)were used to stimulate FL-DCs,in which the proportion of each DC subpopulation and changes in maturation markers(MHC Ⅰ,CD40,CD80,CCR7)were analyzed.Cytokine expression including IL-12p40,IL-6,IFNα,and IFNβ,as well as the secretion of IL-12p70 and IL-6 was detected.Gene expression changes in the two DC subpopulations were analyzed by flow-sorting cDC1s and cDC2s and transcriptome sequencing.2.1 M-M combined with CpG 2006 vaccine up-regulates the ratios of p DC1 s,cDC1s and the maturation of p DC1 s,cDC1s and cDC2s in FL-DCs,and this modulation is dependent on CpG 2006The flow cytometry analysis revealed that the M-M + CpG 2006 vaccine substantially increased the proportions of p DCs(2.54-fold)and cDC1s(4.13-fold)in FL-DCs.The proportion of cDC2s was only slightly up-regulated(1.26-fold).Meanwhile,M-M + CpG 2006 vaccine was able to up-regulate the expression of MHCⅠ,CD40,CD80,and CCR7 on p DCs,cDC1s,and cDCs.And this regulatory effect was also dependent on CpG 2006,which was generally consistent with the results of in vivo experiments.2.2 M-M combined with CpG 2006 vaccine up-regulates cytokine production and secretion in FL-DCs.IL-12p40,IL-6,IFNα,and IFNβ m RNA expression in FL-DCs was assessed by q RT-PCR after vaccine stimulation,and IL-12p70 and IL-6 levels in culture supernatants were measured by ELISA.The results showed that M-M + CpG 2006 could up-regulate IL-12 and IL-6 m RNA expression and secretion in FL-DC,as well as promote the expression of IFNα and IFNβ m RNA,and this effect was mainly attributed to the adjuvant CpG 2006.2.3 M-M combined with CpG 2006 mainly affects TLR and cytokine pathway in cDC1sThe transcriptome sequencing results of the effect of M-M combined with CpG2006 on DCs were analyzed.The differential gene analysis revealed that CpG 2006 and the combination group had the highest number of differential genes for both cDC1s and cDC2s.These findings support that the effects of M-M + CpG 2006 on cDC1s and cDC2s were mainly due to CpG 2006.Moreover,differential genes which were significantly up-regulated were mostly found in cDC1s,while the differential genes which are significantly down-regulated were primarily located in cDC2s.KEGG analysis demonstrated that cDC1s showed enrichment of TLR pathway and cytokinerelated pathways in M-M + CpG 2006 group and CpG 2006 group,whereas cDC2s did not show enrichment of TLR pathway,suggesting that M-M + CpG 2006 mainly affected TLR pathway in cDC1s.M-M combined with CpG 2006 regulates cDC1 immune function through the IFNI signaling pathway.The above studies have shown that the up-regulation of cDC1subpopulation and maturation through the M-M + CpG 2006 vaccine is dependent on the adjuvant CpG2006.Additionally,these studies found that the IFN-I signaling pathway in cDC1s is involved in regulating MUC1-specific CTL killing activity.As the adjuvant CpG 2006 is a TLR9 agonist,we hypothesized that IFN-I may influence cDC1 immune function by modulating the TLR9 pathway on cDC1s.We therefore validated this hypothesis.The impact of vaccine components on TLR9 expression and TLR pathway in cDC1s was initially assessed.Following that,we studied the alterations in TLR9 expression and TLR pathway in cDC1s upon anti-IFNAR1 and IFNα challenge.3.1 M-M combined with CpG 2006 regulates the IFN-Ⅰ-STAT1 signaling pathway in cDC1s.FL-DCs were treated with M-M and CpG 2006 in the presence of either antiIFNAR1 or IFNα.cDC1 cells were isolated using FACS and the m RNA expression of STAT1 and STAT3 was analyzed via q RT-PCR.The study revealed that the M-M +CpG 2006 vaccine significantly regulated the IFN-Ⅰ-STAT1 pathway in cDC1s,whereas it had no significant effect on the IFN-Ⅰ-STAT3 pathway.Anti-IFNAR1/IFNαsignificantly down/up-regulated the IFN-Ⅰ-STAT1 pathway,but had no significant effect on the IFN-Ⅰ-STAT3 pathway,indicating that M-M + CpG 2006 can regulate the IFN-Ⅰ-STAT1 signaling pathway in cDC1s.3.2 M-M combined with CpG 2006 vaccine promotes TLR9 expression in cDC1s and activates TLR9 downstream signaling pathwayM-M and CpG 2006 were used to stimulate FL-DCs.Afterwards,cDC1s were isolated.The m RNA expression of TLR9 in cDC1s was analyzed using q RT-PCR assay,while the protein level of TLR9 was detected through Western Blotting assay.The study determined that the M-M + CpG 2006 vaccine markedly increased TLR9 expression in cDC1s,primarily due to the effect of CpG 2006.cDC1s were isolated from FL-DCs that were stimulated using M-M,CpG 2006,and M-M + CpG 2006.QRT-PCR assay was performed to analyze the m RNA expression of TLR9 downstream signaling molecules,including My D88,TRAF6,and IRF7.Additionally,Western blotting assay was conducted to detect the protein levels of TLR9 downstream signaling molecules,including My D88,TRAF6,and IRF7,as well as p-IκB,IκB,p-Erk,Erk,p-p38,and p38.The results showed that M-M + CpG2006 vaccine significantly up-regulated TLR9 downstream signaling molecules in cDC1s,and this up-regulation was also mainly dependent on the activity of CpG 2006.3.3 The promotion of TLR9 expression in cDC1 by M-M combined with the CpG2006 vaccine depends on the IFN-I-STAT1 signaling pathway.FL-DCs were stimulated with M-M + CpG 2006 in the presence of anti-IFNAR1 or IFNα.cDC1s were subsequently isolated and TLR9 m RNA expression was assessed using q RT-PCR.TLR9 protein levels were determined by Western blotting.The results showed that TLR9 m RNA expression and protein levels were significantly up-regulated in the COM(M-M + CpG 2006)group compared with the NC group.This up-regulation was abolished after M-M + CpG 2006 combined with anti-IFNAR1,whereas antiIFNAR1 alone had no effect on TLR9 m RNA expression and protein levels.IFNα alone can up-regulate the expression and protein levels of TLR9.When the vaccine was combined with IFNα,there was no significant change in TLR9 m RNA,but the TLR9 protein level increased further in comparison to the vaccine group.The findings indicate that the M-M + CpG 2006 vaccine substantially up-regulated TLR9 in cDC1s,and this outcome was attained through the IFN-I signaling pathway.3.4 Activation of TLR9 downstream pathway in cDC1s induced by M-M combined with CpG 2006 vaccine is regulated by IFN-Ⅰ-STAT1 signalling pathwayFL-DCs were stimulated with M-M + CpG 2006 in the presence of anti-IFNAR1 or IFNα,and cDC1s were sorted by flow cytometry.The m RNA expression of TLR9 downstream pathway molecules My D88,TRAF6 and IRF7 was analysed by q RT-PCR assay,and the molecular protein levels of TLR9 downstream pathway were detected by Western blotting.The results showed that the activation of My D88-TRAF6-NF-κB,My D88-TRAF6-MAPK and My D88-TRAF6-IRF7 downstream of TLR9 in cDC1s induced by M-M combined with CpG 2006 vaccine was significantly down-regulated after blockade by anti-IFNAR1 and up-regulated after combined with IFNα.It was demonstrated that the up-regulation of TLR9 downstream pathway by M-M combined with CpG 2006 vaccine was regulated by IFN-Ⅰ signalling pathway.3.5 The proportion and maturation of the cDC1s in FL-DCs increased by the MM with CpG 2006 vaccine is regulated by the IFN-Ⅰ-STAT1 signalling pathwayFL-DCs were stimulated with M-M + CpG 2006 in the presence of anti-IFNAR1 or IFNα,and the proportion of cDC1s and the expression of surface maturation markers(MHC Ⅰ,CD40,CD80,and CCR7)on cDC1s in FL-DCs were detected by flow cytometry.It was demonstrated that the IFN-Ⅰ-STAT1 pathway was involved in the regulation of increased proportion and maturation of cDC1s induced by M-M + CpG2006 vaccine.3.6 IL-12 and IL-6 production and secretion in FL-DCs promoted by M-M combined with CpG 2006 vaccine is regulated by IFN-Ⅰ signalling pathwayFL-DCs were stimulated with M-M + CpG 2006 in the presence of anti-IFNAR1 or IFNα.q RT-PCR was performed to detect the expression of IL-12p40 and IL-6m RNA,and ELISA was performed to detect the secretion levels of IL-12p70 and IL-6in culture supernatants.The results showed that anti-IFNAR1 antibody blockade significantly reduced the m RNA expression and cytokine secretion of IL-12 and IL-6induced by M-M + CpG 2006,whereas IFNα was able to further increase the m RNA expression and cytokine secretion of IL-12 and IL-6 induced by M-M + CpG 2006.It is suggested that IFN-Ⅰ-STAT1 pathway is involved in IL-12 and IL-6 m RNA expression and secretion in FL-DCs induced by M-M + CpG 2006.CONCLUSION: In this study,the mechanism of M-M combined with CpG 2006 in inducing antigen-specific CTL response was explored in depth for the first time from the perspective of DC subpopulation,which elucidated the positive regulatory role of the IFN-Ⅰ signalling pathway in vaccine-induced MUC1-specific CTL killing activity,and revealed the possible mechanism by which cDC1s cross-present exogenous antigens,facilitated by DC subset cross-talk to activate CTLs.This study lays the experimental foundation for the drug development of recombinant M-M fusion protein vaccine,and provides a new idea for the combination immunotherapy of recombinant M-M fusion protein vaccine with cytokines,especially IFN-Ⅰ.