Enhancement Of Antitumor Immunity Of Hyperthermia Treated Tumor Vaccine Co-using With CpG-ODN In Mouse Melanoma

PartⅠEffect of different thermal dose of hyperthermia on HSP70 expression in mouse melanomaPurpose:It is well reported that hyperthermia can influence the immune phenotype of tumor cells.More recently,some researchers reported that the mechanism of immunogenic augmentation against cancer after heat treatment is associated with HSP70 expression which can activate the host.However,the influence on tumor cells varies on hyperthermic temperatures.In addition,the response of tumor cells and their susceptibility to immune effector cells is strongly dependent on the model system,on the magnitude and duration of the thermal stress and on the time of recovery after heat exposure.To find out the hyperthermic temperature which can enhance the immunogenicity of malignant melanoma cell line B16 best,the expression and transcriptional level of HSP70 were detected in this experiment.Methords:B16 cells in the logarithmic growth phase were heated at 42℃,45℃and 50℃for 60min,15min and 5min respectively by directly immersion of the cell plates into a temperature controlled water bath. Once heating was over the medium was changed and the cells were placed back into the 37℃incubator for recovery culture.After recovery culture for 2h,12h,24h,48h respectively,the cells were harvest and the expressional and gene transcriptional level of HSP70 were detected by western blot and RT-PCR respectively.Results:The western blot results show that the hyperthermia induced significant up-regulation of expression of HSP70 at all temperatures(p＜0.05).However,the peak value and its time point of each group are different.The HSP70 expression level of 42℃group reached its peak at 2h after heat exposure;while the time point of 45℃and 50℃hyperthermia reaching their peak value is 24h after heat exposure.Among the three hyperthermic temperature,the peak value of 45℃is the highest(p＜0.01).The RT-PCR examination shows the similar results with the western blot examination.Conclusion:45℃hyperthermia induced the highest expressional and transcriptional level of HSP70 at 24h's recovery time after heat exposure which is likely contributing to the enhancement of immunogenicity of tumor cells.Thus,hyperthermia at 45℃for 15min could be the better hyperthermic thermal dose to prepare the HSP70-rich tumor cell lysates vaccine when compared to 42℃60min or 50℃5min. PartⅡThe experimental study on the antitumor immune responses induced by heated tumor cell lysates combination with CpG-ODN in mouse melanomaPurpose:Tumor vaccines represent a promising new strategy for immunotherapy against cancer,which is an important method to prevent the recurrence and metastasis of tumor.Recently,much attention is given to that heat and heat shock proteins can activate multiple anti-tumor immunological effects through serveral pathway so as to improve the prognosis and prolong the survival time.Moreover,many studies reported that hyperthermia affects not only the immunological reactions of leucocytes but also the immunogenicity of certain tumor cells.Therefore, hyperthermia may be a valuable way to prepare tumor cell vaccine.The heat induced HSPs can be uesd as a multivalent,autologous vaccine preparation with undefined tumor-associated antigens,removing the need to identify the epitopes of tumor associated antigens.Thus,HSP-peptide complexes offer the possibility of highly immunogenic and multivalent cancer vaccines.However,because of neoplastic load and immunological tilt of tumor host,HSP70 upregulation in tumor cells or HSP70-rich cell lysates increases tumor immunogenicity and protects animal models from challenge by wild-type tumor to some extent though,it is not effective enough to obtain an stable and significant inproved outcome that feasible for clinical use.Therefore,to optimize the HSP-related vaccine,we want to find out a kind of adjuvant which can intensify the remedial effect of tumor vaccine. Oligodeoxynucleotide(ODN) containing unmethylated cytosine-phosphate -guanosine(CpG) motifs was originally isolated from components of bacterial DNA.CpG-ODN immuno-therapy has been studied as a strategy for tumor prevention as well as for treatment of immune disorders.Recent studies have demonstrated that CpG-ODN induce strong Th1-like innate and acquired immune responses.In particular,NK and dendritic cells are induced to produce pro-inflammatory cytokines such as IFN-γand IL-2,and mediate cellular cytotoxicity.Even though CpG-ODN has already been used as an adjuvant for tumor therapy,we showed for the first time that the protective and immunotherapeutic signifficance of the combination of CpG-ODN and tumor cell lysates induced by hyperthermia at 45℃for 10min.Methods:48 C57BL/6 mice were divided into 4 groups at random: PBS control group,heated tumor cell lysate treatment group(HCL), CpG-ODN treatment group or heated tumor cell lysate and CpG-ODN co-used treatment group(HCL+CpG).On day0,6,12,18 and day24, PBS(0.2mL/mouse),HCL(2×10~7/0.2ml/mouse),CpG-ODN2395 (30μg/0.2ml/mouse),HCL+CpG({HCL(2×10~7)+CpG-ODN(30μg)} /0.2ml/mouse) was respectively injected into the four groups of mice every 5 days by subcutaneous(SC) at bilateral axillary space and inguinal region,five times totally.To investigate the influence on the body's immune function mediated by vaccination,on day 30,3 mice from each group were randomly picked out and killed,splenocytes were collected to detect the specific proliferation activity,specific cytotoxic activity and Th1 cytokine(IFN-γ,IL-2) secretion activity of splenocytes.The peripheral blood was also collected to detect the CD4~+ T cells,CD8~+ T cells and CD4~+CD25~+ regular cells' population by flow cytometer. Meanwhile,on day 30,the rest mice of each group were challenged with 1×10~5 viable B16 cells,thereafter the tumor growth and survival period were observed untill the experiment was over at day70(40 days after tumor challenge).Results:All the three arms of HCL,CpG ODN or combination of HCL and CpG ODN can suppress the growth of tumor(p＜0.05) and prolong the survival period when compared with PBS group.Especially, the HCL+CpG vaccination therapy obtained the best protective immunological effect including 35%of mice in this group were able to survive without any tumor developed in the injection site.On the other hand,our immunological results show that the co-use of HCL and CpG ODN can siginficantly improve the immune system's function.The ratio of CD4~+/CD8~+ T lymphocyte subsets and the quantity of Th1 cytokines(IFN-γ,IL-2) in the culture supernatant of mouse splenocytes sitimulated by inactivated B16 cells were siginificantly increased contrasting with group of PBS,HCL or CpG ODN(p＜0.05).Consisting with Th1 cytokine secretion's up-regulation,there is obvious enhancement of CTL cell's specific cytoxicity and splenocytes' proliferation activity when compared to other three groups(p＜0.05). While for the CpG ODN or HCL vaccination group,there was obvious immune response induced as well though,it were much less effective than the co-use group.Especilly for the HCL vaccination,which was unable to mediated a up-regulation of IL-2 secretion and specific cytoxicity and proliferation(p＞0.05).Finally,concerning to the change of CD4~+CD25~+T regular cells' population in the peripheral blood,there was no siginificant change observed(p＞0.05). Conclusion:These results implyed co-using of heated tumor cell lysates and CpG-ODN can activate the anti-tumor immunological response,especially cell immunity which contribute to the improved outcome of protecting mice from tumor challenge or suppressing the tumor growth at least.The underlying mechanism is likely that they can induce the redistribution of T cell subsets and the release of Th1 cytokines which can form an activated immunologic micro-environment and mediate an enhanced cytotoxic and proliferational activity of the splenocytes.The applying of CpG-ODN or heated tumor cell lysates can also induce protective anti-tumor immue reponse and supress the tumor growth to some extent,but they are much less effective than the co-use treatment.PartⅢThe observation of efficacy and immunological effects in mouse melanoma treated by regional hyperthermia combination with CpG-ODNObjective:Hyperthermia is one of the oldest documented tumor treatment modalities.However,it is unsufficient to eliminate the tumor drastically by hyperthermia only,recurence become the main problem. More recently,much attention is given to that heat and heat shock proteins can activate multiple anti-tumor immunological effects through serveral pathway so as to improve the prognosis and prolong the survival time.Because of the inflammatory/immune response triggered by regional hyperthermia,this therapy has been shown to be particularly suitable for combination with a variety of immunotherapy based treatments.CpG-ODN is such a immunologic active material that have been demonstrated that it can induce strong Th1-like innate and acquired immune responses.In particular,NK and dendritic cells are induced to produce pro-inflammatory cytokines such as IFN-γand IL-2,and mediate cellular cytotoxicity.Thus,CpG-ODN may be an optimal adjuvant for hyperthermia.Even though CpG-ODN has already been used as an adjuvant for tumor therapy,we showed for the first time that the immunotherapeutic signifficance of the combination of CpG-ODN and regional hyperthermia.Methods:40 tumor-bearing C57BL/6 mice were divided into 4 groups at random:①Control group(without any treatment);②CpG-ODN treatment group(CpG-ODN2395 30μg/0.2ml/mouse,inject subcutaneous into the area around tumor nodule at 4 points);③Regional hyperthermia (directly applying regional hyperthermia to the tumor nodule with a heating machine maken by Tsinghua University and the Guangdong University of Technology at the temperature of 50℃-52℃for 10 min);④CpG-ODN combination treatment group(RH+CpG).The treatments were carried out every 3-4 days respectively,totally three times. Thereafter,the tumor growth curve and mice survival were observed untill the experiment was over at day40(40 days after tumor implantation).To investigate the underlying mechanism,two weeks after the last treatment,3 mice from each group were randomly picked out and killed,splenocytes were collected to detect the Th1 cytokine(IFN-γ,IL-2) secretion activity by splenocytes.The peripheral blood was also collected to detect the CD4~+/CD8~+ ratio by flow cytometer.Results:The growth of tumor of three groups(RH,CpG-ODN or combination of RH and CpG-ODN was suppressed to some extent and the survival time was prolonged when compared with control group.. Especially,the RH+CpG combinational therapy obtained the best therapeutic effect including 30%of mice in this group showed tumor regression.On the other hand,our immunological results show that the combination of RH and CpG-ODN can siginficantly improve the immune system's function.The ratio of CD4~+/CD8~+ T lymphocyte subsets and the quantity of Th1 cytokines(IFN-γ,IL-2) in the culture supernatant of mouse splenocytes sitimulated by inactivated B16 cells were siginificantly increased contrasting with group of PBS,RH or CpG-ODN(p＜0.05).While for the RH group,tumor growth was inhibited significantly or even dissapeared at first.However,shortly after the treatments were over the tumor restarted to grow,there was only one mouse showed tumor regression at last.Moreover,though immune responses were induced by RH as well,they were much weaker than the CpG-ODN group or combination group.CpG-ODN treatment group were able to induce more active secretion of Th1 cytokines(p＜0.05),however, the tumor growth was not well inhibited as we expect.Conclusion:Regional hyperthermia combinated with CpG-ODN2395 inhibits the tumor growth more effectively than single use of regional hyperthermia.The significantly increased CD4~+/CD8~+ ratio in peripheral blood and Th1 cytokine(IL-2,IFN-γ) secretion by splenocytes were likely contributing to the improved theraputic outcome after combinational treatment.The single use of regional hyperthermia showed a well inhibition of tumor growth during the treatment,but the tumor recurrent quickly once the treatment was stopped.The insufficient immune activation especially the cell immunity may be one of the possible reasons.The single applying of CpG-ODN is not so effective for the treatment of developed tumor,which may due to the neoplastic load and immunological tilt of tumor host.These findings have significant therapeutic implications in clinical anti-tumor therapy suggesting that regional hyperthermia combinated with a immune modulator such as CpG-ODN could be a promising strategy.