| Heat shock proteins have been used as adjuvant for tumor vaccine due to theirabilities of activating innate immune immunity cross-presenting antigen to promoteCTL, promoting DC mature and Th1type immune response. But, the mechanism ofheat shock protein as adjuvant needed further research：(1) Heat shock protein tumorvaccine hasn’t display the broad anti-tumor effect in clinical studies, indicating thatadjuvant effect of heat shock protein needed be enhanced.(2) The adjuvant activity ofheat shock protein is associated with its dosage. For example, HSP65at low doseexerted more potent adjuvant effect.(3) In autoimmune disease model, HSP65couldinduce Th17immunity, which is benefit for tumor immunotherapy, indicating that themechanism of HSP65as adjuvant for tumor vaccine is associated with induction ofTh17type immunity.Heat shock protein tumor vaccine needed the assistant of adjuvant as it didn’tdisplay broad anti-tumor effect in clinical studies. In a clinical study for melanoma,GM-CSF and IFN-α were used to enhance the anti-tumor effect of gp96tumor vaccine[Dirk J. Reitsma, et al,2012]. Synthetic oligodeoxynucleotides containing CpG motifs(CpG ODNs), a TLR9agonist, are divided into A, B and C types based on theirfunctions. All types of CpG ODNs have been explored as adjuvants for tumor vaccinesas they could induce Th1type immunity. Of them, B-type CpG ODN has been mostlyevaluated as adjuvants for many cancer vaccines in clinical study. Therefore, B-typeCpG ODN might be used to enhance the anti-tumor effect of heat shock protein tumorvaccines.In addition, the adjuvant effect of heat shock protein is associated with its dosage.For example, autologous tumor derived gp96-peptide complex at lower dose (10μg/mouse) was sufficient to cause tumor rejection in mice withmethamphetamine-induced fibrosacroma cells, whereas the complex at a higher dose(100μg/mouse) was unable to elicit protective immunity. The dose-related differencesmight be caused by Treg induction properties of HSPs at high dosage, as demonstratedthat when used as an adjuvant of HBV peptide, gp96at a lower dose (10μg/mouse) effectively enhanced generation HBV peptide specific CTL and at a higher dose (100μg/mouse) induced significantly increased number of Tregs. HSP65at lower dosedisplayed more potent adjuvant activity when used as adjuvant for lung caner lysates,and the mechanism needed be further investigated.Recently, some heat shock proteins have been found to enhance anti-tumor effectvia induction of Th17type immunity. For example, HSP70could induce Th17typeimmunity to inhibit prostate cancer growth. HSP65could aggravate uveitis withenhancement of IL-17. Th17cells could discrupt the blood-brain barrier (BBB) andmigrate to brain and enhance infiltration of other immune cells, which are benefit forglioma immunotherapy. Glioma is the most common malignant brain tumor with poorprognosis. The failure of the conventional therapies such as surgery and radiotherapyand chemotherapy required for the novel approach such as immunotherapy. However,immunotherapy for glioma was less effective than that in other types of tumors as theBBB limited the infiltration of effector immune cells to glioma. Th17cells coulddisrupt tight junction proteins in the CNS endothelial cells via production of cytokinessuch as IL-17and IL-22and enhance subsequent infiltration of other lymphocytes. AsHSP65at low dose could induce Th17-type immunity and glioma tissue lysates (GTL)contain almost tumor antigens, HSP65combined with GTL might be a good strategyfor glioma immunotherapy.In this study, we chose HSP65-HER2fusion protein and HSP65-GTL to test theadjuvant effect of HSP65. Part1, we test the mechanism of HSP65as molecularadjuvant. In addition, we chose BW006to enhance the anti-tumor effect ofHSP65-HER2and tested the dosage effect of HSP65-HER2. Part2, we choseHSP65-GTL to investigate whether HSP65could assist the GTL to induce of Th17type immunity to exert anti-glioma effect1. Preparation of HSP65, HSP65-HER2, GTL, HSP65-GTLHSP65and HSP65-HER2protein were expressed in E. coli and purified. Theconcentrations of HSP65-HER2and HSP65were0.76mg/ml and2.3mg/ml,respectively. The LPS levels of HSP65and HSP65-HER2protein were less than100EU/mg, according with the requirements of the cell and animal experiments. The resultof SDS-PAGE showed that the purities of HSP65-HER2and HSP65were up to95%.To prepare glioma tissue lysates (GTL), mice were inoculated with GL261glioma cellsintraperitoneally, when the tumor growed to the appropriate size, solid tumors wereisolated for preparing the tumor lyastes by repeated freezing and thawing for5times. The concentration of proteins in GTL was4.3mg/ml using Brandford method.HSP65-GTL was prepared by mixing GTL with HSP65(20μg HSP65+1mg GTL/ml)for subsequent experiments in vivo or in vitro.2. B-type CpG ODN (BW006) enhanced the anti-tumor effect of HSP65-HER2To enhance anti-tumor effect of HSP65-HER2, a B-type CpG ODN (BW006) wascombined with. In the prophylactic setting, mice were immunized subcutaneously withHSP65-HER2/BW006at days-19,-12and-5and then injected intraperitoneally withHER2+B16melanoma cells at day0. In therapeutic setting, the mice were injectedintraperitoneally with HER2+B16melanoma cells at day0and immunizedsubcutaneously HSP65-HER2/BW006, HSP65-HER2, BW006and PBS at days2,9,and16. In the prophylactic setting, HSP65-HER2alone could significantly prolongsurvival of mice bearing HER2+B16melanoma, indicating that HSP65as a molecularadjuvant could assist HER2antigen to induce HER2specific immunity. In thetherapeutic setting, HSP65-HER2alone couldn’t prolong the survival of mice bearingHER2+B16melanoma, but combined with BW006could prolong the survival of micebearing HER2+B16melanoma, indicating BW006plus HSP65could be used asadjuvant for therapeutic tumor vaccines.To investigate the mechanism how BW006enhanced the anti-tumor effect ofHSP65-HER2, we tested the effect of BW006on Th1type immunity and CTL inducedby HSP65-HER2. The serum was prepared from tail bleeds of immunized mice at7days after the last immunization. ELISA assay was carried out to determineHSP65-HER2specific IgG2c and IgG1antibodies in the polled sera. The resultsshowed that BW006plus HSP65-HER2could enhance the ratio of HSP65-HER2specific IgG2c/IgG1in comparison to HSP65-HER2. For CTL analysis, mice wereinjected with HSP65-HER2(2μg or10μg/mouse), HSP65-HER2(10μg/mouse) plusBW006, BW006or PBS for three times in a7-day interval. On day21after the firstimmunization, the mice were killed for isolating spleen cells. The spleen cells werecultured with HSP65-HER2for4days, and then co-cultured with the HER2+B16melanoma cells at effector/target ratio of12.5:1,25:1,50:1, for another4h. Afterdiscarding the suspended spleen cells, the remaining adherent cells were stained bytrypan blue and counted. The splenocytes from the mice immunized withHSP65-HER2alone or plus BW006significantly killed HER2+B16melanoma cells,but there was no statistics significance between HSP65-HER2andHSP65-HER2/BW006groups. 3. HSP65-HER2at lower dose induced more effective anti-tumor immunityIt was reported that the aduvant effect of heat shock protein was related with itsdosage. To test the dosage effect of HSP65-HER2on its anti-tumor effect,HSP65-HER2(100μg or10μg/mouse)/BW006were used in a pilot study.HSP65-HER2at10μg induced more effective antitumor activity than HSP65-HER2at100μg (p=0.044). Based on the above experiments, we set HSP65-HER2alone group(10μg/mouse or2μg/mouse) to test whether HSP65-HER2at lower dose could displaymore effective anti-tumor activity. The results showed that HSP65-HER2at2μg wasmore effective in prolonging the survival of mice bearing HER2+B16melanoma,compared with HSP65-HER2at10μg (p=0.246).4. HSP65at lower dose induced more potent Th17immunityHSP65-HER2at lower dose displayed more potent anti-tumor effect and HSP65accelerated the autoimmune disease with enhancement of IL-17A [Marengo EB, et al,2009], indicating that the adjuvant effect of HSP65on HER2was associated withinduction of Th17type immunity. To test the hypothesis, we first assayed the timedynamics of HSP65on inducing Th17type immunity. Mice were injected with HSP65(2μg/mouse) or0.85%NaCl at axillary lymph node area on the both sides at day0andkilled at days1,2,3and4, lymphocytes for isolating the spleen and axillary lymphnode to assay the mRNA expressions of IL-17A and RORγt using real–time PCR. Theresults showed that HSP65could up-regulate mRNA expressions of IL-17A andRORγt in spleen cells on day3after immunizaion.To further investigate the dosage effect of HSP65on Th17type immune response,mice were injected with HSP65(100μg,10μg,2μg/mouse) or0.85%NaCl at axillarylymph node area on the both sides and killed at day3for isolating the spleen cells toassay the mRNA expression of IL-17A using real–time PCR. The results showed thatHSP65at lower dose was more effective in enhancing the mRNA expressions ofIL-17A in spleen cells.5. Therapeutic effect of HSP65-GTL on the mice bearing GL261gliomaHSP65could induce Th17type immunity and Th17cells could discrupt BBB,which is benefit for glioma immunotherapy. To test whether HSP65could assist theGTL to induce Th17type immunity to cure the mice bearing intracranial GL261glioma, we first tested therapeutic effect of HSP65-GTL in vivo. Mice were inoculatedwith GL261cells (2×104/mouse) intracranially on day0and then immunized withHSP65-GTL, HSP65, GTL or0.85%NaCl at axillary lymph node area on the both sides on days4and11. The results showed that HSP65-GTL trended to reduce thebody weight loss and significantly prolonged the survival of mice with intracranialGL261glioma, compared with NaCl, GTL and HSP65alone (p=0.029, p=0.018andp=0.006, respectively).Next, we assessed whether HSP65-GTL could induce specific CTL response in themice with intracranial GL261tumor. Mice were inoculated with GL261cells(2×104/mouse) intracranially on day0and then immunized with HSP65-GTL, HSP65,GTL,0.85%NaCl at axillary lymph node area on the both sides on days4and11. Themice were killed on day18and their spleens were isolated for CTL assay. The spleencells were cultured with GTL (100μg/ml) for4days as effector cells, and thenco-cultured with GL261cells at effector/target ratio of25:1,50:1,100:1for another8h.The cytotoxicity was determined using MTT assasy. The results showed that thesplenocytes from the mice immunized with HSP65-GTL significantly killed GL261tumor cell, compared with NaCl, GTL and HSP65(p=0.000, p=0.006and p=0.022,respectively).6. Induction of Th17type immunity by HSP65-GTL in mice bearing gliomaTo test whether the anti-tumor effect of HSP65-GTL was associated withinduction of Th17type immunity, mice were inoculated with GL261cellsintracranially on day0and then injected with HSP65-GTL, HSP65, GTL,0.85%NaClat axillary lymph node area on the both sides on day4. The mice were killed at day7for isolating the lymph node cells and spleen cells to detect the mRNA expressions ofIL-17A and RORγt using the real-time PCR and and analyze the productions of Th17cell and IL-21using flow cytometry. The results showed that HSP65-GTLenhancedmRNA expressions of IL-17A and RORγt in lymph node cells, HSP65and GTLenhanced the mRNA expressions of IL-17A and RORγt in spleen cells. Consistantly,the results of flow cytometry also showed that HSP65-GTL could significantlyenhance the ratio of Th17cells in lymph node, compared with NaCl, GTL and HSP65(p=0.01, p=0.00and p=0.00, respectively). HSP65sightly enhanced the ratio of Th17in spleen cells. GTL and HSP65-GTL could enhance the expression of IL-21in thedraining lymph node cells in comparion with NaCl (p=0.019, p=0.033, respectively)and HSP65slightly enhanced the expression of IL-21in the spleen cells.7. Activation of lymph node cells and spleen cells by HSP65–GTLTo inveatigate the machenism of anti-glioma immunity induced by HSP65-GTL,we tested the activation status of lymphocytes including NK, CD8+T cells in the axillary lymph node and spleen of vaccinated mice with intracranial GL261tumor.Mice were inoculated with GL261cells intracranially at day0and then immunizedwith HSP65-GTL, HSP65, GTL or0.85%NaCl and killed at day7for isolating lymphnode cells and spleen cells. The lymph node cells and spleen cells were directly stainedwith PE-labeled anti-CD69mAb followed by flow cytometry analysis. The resultsshowed that the GTL significantly up-regulated the expression of CD69in lymph nodecells, compared with NaCl (P=0.005), HSP65(P=0.009), HSP65-GTL (P=0.051).HSP65significantly up-regulated the expression of CD69in spleen cells, comparedwith NaCl (P=0.00), GTL (P=0.00), HSP65-GTL (P=0.00). Interestingly, HSP65-GTLcould up-regulate the expressions of CD69in both lymph node cells and spleen cells,compared with NaCl (P=0.042, P=0.047, respectively).As NK cells and CD8+T cells are important for tumor immunity, we further testedactivation status of NK cells and CD8+T cells. The lymph node and spleen cells werestaining with PE-labeled anti-CD69mAb and FITC labeled anti-NK1.1mAb and thespleen cells were staining with PE-labeled anti-CD69mAb and FITC labeled anti-CD8mAb. The results showed that GTL and HSP65could activate NK cells in lymph nodeand spleen, respectively. HSP65, HSP65-GTL could activate the CD8+T cells inspleen.8. HSP65-GTL enhanced Glioma infiltrating Th17cells and inflammary cellsAs HSP65-GTL could induce Th17type immunity, while Th17cells coulddiscrupt BBB and promote infiltration of immune cells to brain, we tested whetherHSP65-GTL could enhance glioma-inflitrating Th17cells and inflammatory cells.Mice were inoculated with2×104GL261cells at day0and immunized at days4,11.Mice were sacrificed at day18to isolate mouse glioma for detecting brain-infiltratingTh17cells and HE staining. The results showed that HSP65, HSP65-GTL enhanced theratio of Th17cells in tumor infiltrating cells and increased infiltration of inflammatorycells. |
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