Study On The Effect And Mechanism Of Combined Immunization With Two Listeria-vectored Cervical Cancer Vaccines On Tumor Prevention And Treatment

Background and ObjectiveCervical cancer is caused by persistent infection with high-risk human papilloma virus(HPV).Globally,the burden of cervical cancer is increasing continuously,and it has become a female health problem that cannot be ignored.After HPV infection,the virus can be cleared automatically in most people within 2years.However,in a minority of people with risk factors,such as poor lifestyle habits and immune deficiencies,the virus would persist in the body for a long time.Persistent infection with high-risk HPV-16 and HPV-18 types accounts for approximately 70%of cervical cancer cases.The HPV prophylactic vaccines induce effective antibody protection against viral infection.A 6-year follow-up cohort study has reported the rate of protection against infection of HPV-16 and HPV-18 in women who vaccinate the bivalent HPV vaccine is 95.3%.The HPV prophylactic vaccines also significantly reduce the incidences of cervical intraepithelial neoplasia(CIN)and genital warts,but the vaccines cannot provide immune protection for women who has been infected with HPV.On the other hand,the incidence of cervical cancer is still increasing in low-and middle-income countries and regions,where the vaccination coverage and the cervical cancer screening rate are low and health care measures are inadequate.Compared with traditional treatments,immunotherapy provides a new method for tumor treatment.Immunotherapy improves the anti-tumor immune responses actively to cure disease or reduce the disease burden.Tumor vaccine is one approach of the immunotherapies,which utilizes a kind of vector to deliver tumor-associated antigens(TAA),so that to activate the body’s immune system and induce cellular immune responses to produce cytotoxicity T lymphocytes(CTL),resulting in the lysis of tumor cells and the clearance of tumor finally.At the same time,the immune system can also establish the immune memory to eliminate residual tumor cells or prevent tumor recurrence.Listeria monocytogenes(LM)is one of the ideal vectors for presenting antigens in tumor vaccine due to its ability of intracellular proliferation and intercellular transmission within antigen presenting cells(APCs),and then to induce strong CTL responses.LM-vectored tumor vaccines have been extensively used in cervical cancer,liver cancer,breast cancer and other diseases.The data from clinical trials show that LM-vectored tumor vaccines can significantly improve the anti-tumor immune responses in tumor patients,demonstrating their good clinical therapeutic values.Our preliminary study also found that LMΔE6E7and LIΔE6E7,the two cervical cancer therapeutic vaccines using attenuated LM and attenuated Listeria ivanovii(LI)as vectors respectively,could induce the specific T cell response against tumor antigens in mice in vivo.The heterologous cross combined immunotherapy of LMΔE6E7 and LIΔE6E7 showed a good therapeutic effect in the tumor bearing mice.In addition,the combined immunotherapy could not only activate an anti-tumor immune response,but also reduce the proportion of regulatory T cells(Treg)infiltration at the tumor site.This result was consistent with the study of Wallecha A et al that LM-LLO-vectored tumor vaccines reduced the infiltration of Treg at tumor sites in mice with breast cancer and prostate cancer.It is suggested that the Listeria-vectored tumor vaccines have two-sided regulatory ability on the immune responses during immunotherapy.Immunosuppressive status is prevalent in cancer patients,which is a big problem of hindering tumor treatment.How to break immunosuppression is a problem that oncology clinicians and researchers are committed to solve.Currently,researches on tumor vaccines focus more on the therapeutic effect,and there are few reports on the immune regulatory mechanism of tumor vaccines in vivo.Therefore,this study explores the regulatory effect on immune responses of cervical cancer vaccines LMΔE6E7 and LIΔE6E7during the treatment process,to clarify the regulatory mechanisms of tumor vaccines.This study has important scientific exploration values and could supplement the basic research data related to tumor vaccine immunotherapy.At the same time,these data have important guiding roles for optimizing immunotherapy protocols of tumor vaccine,to improve therapeutic effects.In addition,researchers have found that the mice cured by tumor vaccine were able to resist the reattack of tumor cells,and the increased level of memory T cells(Tm)was detected,suggesting that vaccination of tumor vaccine may prevent tumor recurrence through T-cell immune memory.Tm cells are sensitive to antigens and could generate fast and strong immune responses.Therefore,by vaccination of tumor vaccine to induce T-cell immune memory against tumor antigens,and utilizing Tm cells to generate robust anti-tumor immune response is a reasonable and feasible approach for tumor prevention,which also has scientific exploration values.This study would investigate whether the combined immunization with cervical cancer vaccines,LMΔE6E7 and LIΔE6E7,has the ability of tumor prevention protection for healthy organisms,which can provide direct data to support the exploration of new tumor prevention methods for population who are with HPV infection and cervical cancer high risk and further expand the clinical application value of tumor vaccine.Therefore,in this study,two Listeria-vectored cervical cancer vaccines,LMΔE6E7 and LIΔE6E7,which are successfully constructed by our lab,are used as the research objects.On the one hand,mice are preimmunized with LMΔE6E7 and LIΔE6E7,after T-cell immune memory is established in the body,tumor cells are subcutaneously inoculated.We investigate the tumor prevention effect and mechanism of LMΔE6E7 and LIΔE6E7 by observing the tumor growth and assessing the anti-tumor immune responses and tumor tissues.On the other hand,using the subcutaneous tumor-bearing mouse model,we observe the therapeutic effect of combined immunotherapy with LMΔE6E7 and LIΔE6E7 and evaluate the modulatory effect on immune responses,and further investigate the immune regulatory mechanism through the detection for biological indicators of biological samples and cellular experiments.MethodsPart 1 Study on the preventive effect and mechanism of preimmunization with vaccines against tumor in miceTo investigate whether the preimmunization with LMΔE6E7 and LIΔE6E7 has the ability to induce the body to establish T-cell immune memory against tumor antigens and the effect on tumor prevention,this part of the study was performed on female C57BL/6J mice.Mice were divided into PBS group(PBS),vaccine vectors group(LMΔ&LIΔ)and cervical cancer vaccines group(E6E7).After preimmunization,all mice were housed normally for 40 days.After stable T-cell immune memory was established in the body,TC-1 cells were subcutaneously inoculated and then the survival situation and tumor volumes of mice were monitored and recorded,so as to explore the effect of preimmunized with LMΔE6E7and LIΔE6E7 on preventing tumor.Before and after tumor cells inoculation,flow cytometry was used to detect the levels of central Tm cells and effective Tm cells in the spleen and lymph nodes of preimmunized mice,as well as the proportions of immune cells in the spleen,including CD8~+T cells,macrophages(M1,M2),myeloid derived suppressor cells(MDSCs),natural killer cells(NK)and Treg,to explore the role of immune cells in the prevention of tumor formation.Besides,the expressions of CD31,Ki67 and CD3 in the tumor tissues were assessed using Immunohistochemical staining,and the expressions of immune molecules were detected by RT-q PCR to investigate the mechanism of tumor prevention of the preimmunization with LMΔE6E7 and LIΔE6E7.Part 2 Study on immunotherapeutic effect and mechanism of combined immunotherapy with vaccines on tumorIn order to investigate the therapeutic effect of combined immunization with LMΔE6E7 and LIΔE6E7 on tumor and the regulatory mechanism,this part of study,we established a cervical cancer subcutaneous tumor-bearing mouse model by subcutaneous inoculation of TC-1 cells in the abdomen of female C57BL/6J mouse,and performed immunotherapy on the tumor-bearing mice.Mice were divided into the PBS group(PBS),vaccine vectors group(LMΔ&LIΔ)and cervical cancer vaccines group(E6E7).The survival situation and tumor volumes of the tumor-bearing mice were monitored and recorded to evaluate the tumor treatment effect of combined immunization with LMΔE6E7 and LIΔE6E7.During the process of treatment,the proportions of immune cells in the spleen and tumor infiltrating lymphocytes(TILs)of mice were detected respectively,before the inoculation of tumor cells,before the immunotherapy,one week after completing the immunotherapy and at the endpoint,including CD8~+T cells,macrophages(M1,M2),MDSCs,NK and Treg,to clarify the effect of combined immunotherapy on the regulation of immune status in the systemic and tumor microenvironment(TME)of tumor-bearing mice.Besides,the cytokine profiles in mouse serum were determined and the cytokines that may play an important role in the immunotherapy process were analyzed.By using bioinformatics analysis,the relevant immune regulatory pathways were predicted.And the interaction between the change of immune status and regulatory pathways was summarized and analyzed.Part 3 Study on the molecular mechanism of JAK-STAT signaling pathway in the regulation of suppressive immune response during combined immunotherapyThe results in Part 2 showed that during the immunotherapy,in addition to a significant increasement of anti-tumor immune response,the level of tumor-induced suppressive immune response was also significantly suppressed in vivo,especially the proportions of MDSCs in the spleen and tumor sites of the tumor-bearing mice.The results of enrichment analysis of serum cytokine profiles suggested that the janus kinase(JAK)-signal transducer and activator of transcription(STAT)signaling pathway might play an important role in the regulation of immune response during the treatment process.Therefore,in this part,to clarify the molecular regulation mechanism of inhibiting suppressive immune response by combined immunotherapy,we assessed the expressions of genes and proteins in tumor-bearing mice that related to the regulation on the differentiation and development of MDSCs and immunosuppressive function of MDSCs induced by JAK-STAT signaling pathway.To explore the mechanism of JAK-STAT signaling pathway in reducing the level and immunosuppressive function of MDSCs during combined immunotherapy.ELISA was used to detect the levels of cytokines in the spleen tissues of tumor-bearing mice after one week of immunotherapy so that to investigate the regulatory effect of combined immunotherapy on factors of induction and recruitment of MDSCs.Western Blot was used to detect the activation levels of the key proteins STAT1 and STAT3 in spleen tissues,regulating the differentiation and expansion of MDSCs.The cellular experiments were performed to compare the proportions of MDSCs differentiated from bone marrow cells induced by grinding supernatant of spleen tissues from tumor-bearing mice in different treatment groups,so that to explore the effect of combined immunotherapy on regulating MDSCs differentiation through JAK-STAT signaling pathway.Western Blot and RT-q PCR were performed to detect the phosphorylation levels of proteins in JAK-STAT signaling pathway and the expressions of downstream regulatory genes and effector molecules in tumor tissues.Though the above experiments,we tried to comprehensively investigate how the LMΔE6E7 and LIΔE6E7 combination immunotherapy to regulate MDSCs level in tumor-bearing mice in vivo through the JAK-STAT signaling pathway,and summarized the regulatory mechanism diagram.ResultsPart 1 Study on the preventive effect and mechanism of preimmunization with vaccines against tumor in micePreimmunization with LMΔE6E7 and LIΔE6E7 prevented the development of tumor.After inoculating the tumor cells,the tumors in the PBS group and LMΔ&LIΔgroup mice grew rapidly and led to the death of mice.However,the mice preimmunized with LMΔE6E7 and LIΔE6E7 were able to resist the formation of tumors with the 60%tumor prevention rate.In the E6E7 group,even the mice that developed tumors,the tumor growth rate was significantly inhibited and survival time was significantly prolonged.The results of the immune status of mice showed that preimmunization with LMΔE6E7 and LIΔE6E7 did not affect the Tm proportions in mice before tumor cells inoculation.Although the proportions of Tm in all groups mice increased after tumor cells inoculation,a significant CTL response against tumor antigens in spleen cells was detected only in the E6E7 group mice(P<0.001).This indicated that the tumor-specific T cells induced by preimmunization with LMΔE6E7 and LIΔE6E7were transformed into the corresponding Tm which kept in vivo,and these Tm were able to generate a rapid and specific secondary immune response against tumor cells.40%of the mice that preimmunized with LMΔE6E7 and LIΔE6E7 developed tumors,but the tumor volumes grew slowly.Immunohistochemical analysis of tumor tissues showed that the cell proliferation level in tumor tissues in E6E7 group was significantly decreased compared with that of the PBS group(P<0.05),and the proportions of T cells infiltration in tumor tissues in the E6E7 and LMΔ&LIΔgroups showed an increasing trend(P>0.05),the angiogenesis of these two groups showed a decreasing trend(P>0.05)compared with the PBS group.Further detection results revealed that the m RNA expression levels of L-selelctin and Ccr7 were significantly increased(P<0.05)and Vegf was decreased(P>0.05)in the tumor tissues of the E6E7 group mice compared with the PBS group or the LMΔ&LIΔgroup.It is suggested that the preimmunization with LMΔE6E7 and LIΔE6E7 may inhibit tumor progression by increasing the infiltration of T cells at the tumor site and exerting a specific immune response to inhibit angiogenesis and cells proliferation.Part 2 Study on immunotherapeutic effect and mechanism of combined immunotherapy with vaccines on tumorThe combined immunotherapy with LMΔE6E7 and LIΔE6E7 had good therapeutic effect on tumor.The tumor volumes of E6E7 group mice were significantly smaller compared with the PBS group and LMΔ&LIΔgroup after one week of completing the immunotherapy,and the cure rate of tumor was 40%.The combined immunotherapy with LMΔE6E7 and LIΔE6E7 could also significantly prolong the survival time of tumor-bearing mice(P<0.001).The results of the immune status of tumor-bearing mice showed that the proportions of NK and CD8~+T cells in the spleen increased in the early stage of tumor formation.In the absence of any treatment and as the tumor progressed,the proportions of immune cells that had anti-tumor ability,such as CD8~+T cells(P<0.001),NK(P<0.001)and M1-type macrophages(P<0.05)were significantly lower;the proportions of immune cells that related to suppressive immune response,including MDSCs(P<0.05)and Treg were higher in the spleen of tumor-bearing mice compared with the early stage of tumor formation.At last,the anti-tumor immune response was weakened,the suppressive immune response was increased and the immune status was out of balance.However,at one week after immunotherapy,the proportions of CD8~+T cells(P<0.05)and M1-type macrophages(P<0.05),the M1/M2 ratio(P<0.05)were significantly higher in the spleen of the tumor-bearing mice that treated with the combined immunotherapy compared with the PBS group or the LMΔ&LIΔgroup mice,and simultaneously,the proportion of MDSCs was significantly lower(P<0.05)and the proportion of Treg also showed a decreasing trend.In the TILs,the proportions of CD8~+T cells(P<0.001)and M1-type macrophages(P<0.05),the M1/M2 ratio(P<0.001)were significantly higher in the E6E7 group mice compared with those of the PBS group or the LMΔ&LIΔgroup,while the proportions of Treg(P<0.001)and MDSCs(P<0.05)were significantly lower.These results suggested that the combined immunotherapy with LMΔE6E7 and LIΔE6E7 could not only improve the anti-tumor immune response in the tumor-bearing mice,but also reduce the level of Treg and MDSCs in vivo,especially in TME,to break the tumor-induced immunosuppression.The results of immune response in mice at the endpoint revealed that the proportions of CD8~+T(P<0.001)and NK(P<0.05)in the spleen of vaccines-cured mice were significantly higher than those of tumor-bearing mice in the PBS and LMΔ&LIΔgroups,and the proportions of CD8~+T cells(P<0.05)in the spleen and NK(P<0.05)in the TILs were also significantly higher in uncured mice of the E6E7 group than those of the PBS group or LMΔ&LIΔgroup mice.These results suggested that the anti-tumor immune response induced by combined immunotherapy may be responsible for inhibiting tumor progression and prolonging survival time in tumor-bearing mice.The determination and comparative analysis of cytokine profiles in mouse serum revealed that LMΔE6E7 and LIΔE6E7 combination immunotherapy regulated the levels of interleukin-1β(IL-1β),IL-2,IL-3,IL-5,IL-9,IL-10,IL-13,IL-17A,keratinocyte-derived chemokine(KC),granulocyte-colony stimulating factor(G-CSF),granulocyte-macrophage colony stimulating factor(GM-CSF),tumor necrosis factor-α(TNF-α),monocyte chemoattractant protein-1(MCP-1),macrophage inflammatory protein-1β(MIP-1β),interferon-γ(IFN-γ),and regulated on activation in normal T-cell expressed and secreted factor(RANTES),suggesting that these cytokines may play a role in immunotherapy.The further analysis of these proteins for pathway prediction and enrichment showed that the JAK-STAT signaling pathway was involved in multiple pathways in the regulatory network of these cytokine interactions,which also indicated that the JAK-STAT signaling pathway might play an important immunomodulatory role in the process of combined immunotherapy with LMΔE6E7 and LIΔE6E7.Part 3 Study on the molecular mechanism of JAK-STAT signaling pathway in the regulation of suppressive immune response during combined immunotherapyOne week after completing the immunotherapy,the levels of IL-1β,IL-5,IL-6,MCP-1 and CXCL12 were significantly lower(P<0.05),and the levels of IL-4,G-CSF and GM-CSF were also decreased(P>0.05)in the spleen of the E6E7 group mice compared with those of the PBS group or the LMΔ&LIΔgroup mice.Compared with the PBS group or LMΔ&LIΔgroup,the phosphorylation level of STAT1 protein in the spleen of the E6E7 group showed a decreasing trend(P>0.05)and the phosphorylation level of STAT3 protein was significantly decreased(P<0.01).The results of cell experiments also confirmed that the proportion of MDSCs induced by grinding supernatant of spleen tissues of the E6E7 group mice was similar to the level of MDSCs induced by the healthy mice(P>0.05),while the proportion of MDSCs induced by grinding supernatant of spleen tissues of the PBS group mice was significantly higher compared with the healthy and E6E7 group mice(P<0.001),suggesting that the LMΔE6E7 and LIΔE6E7 combination immunotherapy reduced the proportion of MDSCs in tumor-bearing mice,via decreasing the expression levels of induction factors of MDSCs,mainly inhibiting the phosphorylation level of STAT3 protein to reduce the generation and amplification ability of MDSCs in vivo,as well as decreasing the recruitment factors of MDSCs and inhibiting their enrichment to tumor tissues.In TME,the LMΔE6E7 and LIΔE6E7 combination immunotherapy reduced the m RNA expression levels of gene Arg-1(arginase-1)(P<0.001),S100A8/A9(the recombinant S100 calcium binding protein A8/A9)(P<0.05),immunosuppressive molecules secreted by MDSCs,and the cell proliferation gene c Myc(P<0.05),and simultaneously upregulated the m RNA expression level of Irf8(interferon transcriptional regulatory factor 8)(P<0.001),via inhibiting the phosphorylation levels of JAK1-STAT1 and JAK2-STAT3 signaling pathways.Importantly,the protein expression levels of indoleamine2,3-dioxygenase(IDO),arginase-1(ARG-1)and inducible nitric oxide synthase(i NOS)in tumor tissues of E6E7group were reduced compared with those in the PBS group or LMΔ&LIΔgroup(P>0.05).The above results indicated that the combined immunotherapy attenuated the immunosuppressive function of MDSCs by inhibiting the phosphorylation levels of JAK1-STAT1 and JAK2-STAT3 pathways in the tumor tissues.ConclusionThe combined immunization with two Listeria-vectored cervical cancer vaccines,LMΔE6E7 and LIΔE6E7,has both preventive and therapeutic effects on tumor.On the one hand,preimmunization with LMΔE6E7 and LIΔE6E7 is able to establish memory T-cell immunity against tumor antigens,with a 60%tumor prevention rate and significantly delays tumor progression.Tm response could be activated rapidly by tumor cells.The effect on inhibiting tumor formation and delaying tumor progression is achieved by increasing T cells infiltration at tumor sites and exerting specific anti-tumor immune response,reducing angiogenesis and cell proliferation.On the other hand,in addition to reactivate the anti-tumor immune response in vivo,the combination immunotherapy could also break the immunosuppressive status induced by tumor progression mainly via reducing the level of MDSCs.Breaking immunosuppressive status facilitates the immunotherapy.The tumor cure rate is 40%and the survival time of tumor-bearing mice is significant prolonged.The combined immunotherapy with LMΔE6E7 and LIΔE6E7 reduces the expression levels of MDSCs inductive factors in tumor bearing mice,which mainly inhibiting the phosphorylation level of STAT3 protein,the regulator of MDSCs differentiation,and thus decreases the formation level of MDSCs in vivo.The combined immunotherapy also reduces the expression levels of recruitment factors in tumor bearing mice,further inhibiting the enrichment of MDSCs into tumor sites.Meanwhile,the LMΔE6E7 and LIΔE6E7 combination immunotherapy reduces the expression of immunosuppressive molecules to attenuate the immunosuppressive function of MDSCs by inhibiting the phosphorylation levels of JAK1-STAT1 and JAK2-STAT3 pathways in tumor tissues.Innovation points1.This study is the first to elucidate that combined immunotherapy with LMΔE6E7 and LIΔE6E7 could break the immunosuppressive status by reducing the formation level and immunosuppressive function of MDSCs in tumor bearing mice via the JAK-STAT signaling pathway.The related studies have not been reported.2.In this study,we clarified that the combined immunotherapy with LMΔE6E7and LIΔE6E7 had the two-sided regulatory ability on the immune statue in the tumor bearing mice by reactivating the anti-tumor immune response and inhibiting the suppressive immune response level.3.The results of this study confirmed that the preimmunization with LMΔE6E7and LIΔE6E7 could induce the establishment of stable memory T-cell immunity against tumor antigens so that to prevent tumor.4.The results of this study showed that the combined immunotherapy with cervical cancer vaccines LMΔE6E7 and LIΔE6E7 have the effect on both tumor prevention and treatment.