Anti-glioma Effect Of Intracranial Vaccination Using Glioma Lysate Formulated With Flagellin In Mice

Gliomas are the most common primary malignant brain tumors.Patients with glioma generally died within 15 months from diagnosis because of their strong resistance to conventional therapies including surgery,chemotherapy and irradiation.Thus,more promising therapies,such as immunotherapies using adoptive T cells,immune checkpoint inhibitors and tumor vaccines are demanding urgently.The EGFRvIII peptide vaccine(CDX-110)was the most advanced in clinical trials.Although achieved the significant clinical benefit with progression-free survival(PFS)and overall survival(OS)in patients with glioblastoma(GBM)in the phase II clinical trial,the CDX-110 failed to reproduce the clinical benefit in the phase III clinical trial.The DC based vaccines were shown to improve median survival(MS)and 5-year survival of GBM patients in phase II clinical trials.Composing of whole set of potentially existed T cell epitopes for tumor specific T cells,glioma lysate(GL)prepared from autologous glioma specimens was formulated in various tumor vaccines for the treatment of glioma,exemplified by GL loaded autologous DC,GL prepared from transforming growth factor-?2(TGF-?2)anti-sense vector modified glioma cells,and GL plus granulocyte macrophage-colony stimulating factor(GM-CSF).Although showed certain clinical benefits,no GL based vaccines have been undertaken in phase III clinical trials for the treatment of glioma.The situation called for studies to develop more efficient adjuvant formulations capable of assisting the GL based vaccines to induce stronger anti-tumor immunity against gliomas.In recent years,toll-like receptor(TLR)agonists have been used as adjuvants for developing efficient tumor vaccines.Flagellin,a protein TLR5 agonist,has been also tested as a potential adjuvant for tumor vaccines.Formulated with human papillomavirus(HPV)E6/E7 peptides,flagellin could increase CD4~+T and CD8~+T cells in the draining genital lymph nodes and prolong the survival of genital tumor-bearing mice.In this study,we immunized the mice with flagellin formulated GL prepared from the in vivo developed glioma subcutaneously(s.c.)or intracranially(i.c.),and found that the intracranial vaccination activated peripheral CD8~+T cells,CD4~+T cells and natural killer(NK)cells,recruited T cells to brains,and prolonged the survival of the mice with in situ glioma.1 Administration of GL plus flagellin could prolong the survival of tumor-bearing miceTo prove whether flagellin(Fla),a TLR5 agonist,could serve as an adjuvant to assist tumor cell lysate(GL)to induce anti-glioma immunity prophylactically,we immunized C57BL/6 mice(n=6)with GL plus Fla(GL+Fla),Fla,GL,or Saline subcutaneously(s.c.)in the right forelimb on day 0 and 7.On day 14,the mice were intracranially(i.c.)inoculated with 2×10~4GL261 cells into caudate nucleus.Around10 days after the inoculation,GL261-bearing mice received Fla or Saline started to exhibit clear symptoms including weight loss,irregular breathing,hunched back,decreased activity/prostration,paresis/paralysis and convulsions.Comparatively,these symptoms developed a week later in the mice immunized with GL plus Fla.One of the six mice received Fla died on day 18 and the rest died within 24 days.The median survival(MS)of the mice received Fla,Saline or GL was 21 days,22 days or 24 days,respectively.In contrast,the MS of the mice received GL plus Fla was 28 days which were significantly longer than those of the mice receive Fla,Saline or GL(p<0.005,p<0.005 and p<0.05,respectively).Upon the results,we also tried therapeutic applications with the GL plus Fla in the glioma-bearing mice.However,no extended survival was observed.2 Intracranial vaccination of GL plus flagellin exhibited anti-glioma effectIn recent years,functional lymphatic vessels have been found existed in the central nervous system(CNS)and linked to deep cervical lymph node,suggesting that i.c.vaccination with tumor vaccines could induce the tumor specific immune responses in the peripheral lymphoid organs and the immune cells in the peripheral lymphoid organs could be consequently activated and then moved into brain to attack tumor cells.To demonstrate whether intracranial(i.c.)vaccination with GL plus Fla could induce anti-glioma immunity therapeutically,we injected the mice(n=6)with2×10~4GL261 cells i.c.on day 0,then immunized them with Fla plus GL in caudate nucleus or in axilla twice on day 0 and day 7.The glioma-bearing mice received Saline i.c.developed glioma related symptoms around day 10 after the immunization,and started to die on day 20.The rest mice died within 25 days.The mice immunized with GL plus Fla s.c.began to exhibit the glioma-related symptoms around day 12and none of the mice survived beyond 27 days.Comparatively,the mice immunized with GL plus Fla i.c.exhibited the symptoms on day 20.One of the mice in the group died on day 26 and the last one died on day 41.The MS of the mice received GL plus Fla i.c.was 31 days,while the MS of the mice received GL plus Fla s.c.was 25 days.The mice treated with GL plus Fla i.c.survived significantly longer than the mice treated with GL plus Fla s.c.or Saline treated mice(p<0.05 and p<0.005).Furthermore,we tested whether Fla used alone could prolong the survival of GL261-bearing mice.To exclude the interference of lipopolysaccharides(LPS),we prepared Fla with or without LPS removal.The LPS in the Fla with LPS removal(LPS-low-Fla)was less than 50EU/mg and in the Fla without LPS removal(LPS-high-Fla)was higher than 100EU/mg.The mice(n=8)were injected with2×10~4 GL261 cells i.c.on day 0,and inoculated with LPS-low-Fla,GL,GL plus LPS-low-Fla,GL plus LPS-high-Fla or Saline i.c.twice on day 0 and 7.The results indicated that Fla or GL alone failed to show any therapeutic advantage compared to Saline(p>0.05 and p>0.05).GL plus LPS-low-Fla significantly prolonged the survival compared with GL,LPS-low-Fla or Saline,respectively(p<0.005,p<0.05and p<0.005,respectively).In addition,no significant difference in survival was observed between the mice received GL plus LPS-low-Fla and the mice received GL plus LPS-high-Fla.3 Administration of flagellin plus GL could activate glioma-specific CTLCD69 was commonly served as the biomarker of activated T cells.To prove the ability of Fla to activate CD8~+T cells,the mice(n=3)were immunized with GL plus Fla,Fla,GL,or Saline s.c.in the right forelimb on day 0 and 7.On day 14,the mice were i.c.inoculated with 2×10~4 GL261 cells in caudate nucleus(Fig.3A).On day 28,the mice were sacrificed for isolating splenocytes and draining lymph node cells.The cells were stained with CD8 and CD69 fluorescence-labeled antibodies and analyzed by flow cytometry.The results indicated that GL plus Fla significantly increased the number of CD8~+CD69~+T cells from draining lymph nodes,compared with GL and Saline(p=0.0312 and p=0.02),and that GL plus Fla significantly increased CD8~+T cells in spleens,compared with GL and Saline(p=0.0148 and p=0.014)and the number of CD8~+CD69~+T cells,compared with GL and Saline(p=0.017 and p=0.0085).Next,we investigated whether the activated CD8~+T cells could kill GL261 cells.The mice(n=3)were immunized with GL plus Fla,Fla,GL,or Saline s.c.on day 0and 7,inoculated i.c.with GL261 cells on day 14 and sacrificed on day 28.The splenocytes were isolated and co-cultured with GL261 cells for 8 hours at effector/target ratios of 100:1,50:1 and 25:1,respectively,and their cytotoxicity was assayed.The results showed that GL plus Fla significantly induced the generation of cytotoxic T lymphocyte(CTL)against GL261 tumor cells,compared with GL and Saline(p=0.0212 and p=0.0077).4 Administration of flagellin could increase the ratio of CD8+T cell and NK cellSince that NK cells and CD8~+T cells were the cancer cell killers and CD4~+T cells could activate or help both of them to launch innate and adaptive immune responses against the cancer cells,we tested whether the recombinant Fla could mobilize and activate the NK cells,CD8~+T cells and CD4~+T cells,which were crucial to assist the tumor antigens to induce tumor specific immune response.The C57BL/6 mice(n=3)were injected with Fla or Saline s.c.on day 0,and sacrificed on day 4 and 8 for isolating splenocytes and draining lymph node cells.The cells were stained with CD8,NK1.1 and CD4 fluorescence-labeled antibodies and analyzed by flow cytometry.The results showed that Fla significantly enhanced the proliferation of NK cells in draining lymph nodes of the mice on day 4(p=0.019)and also in spleens of the mice on day 8(p=0.01),respectively,compared with Saline.Fla induced the proliferation of CD8~+T cell in both the spleens and the draining lymph nodes on day 4,compared with Saline(p=0.008 and p=0.005,respectively),and tended to induce the proliferation of CD8~+T cells on day 8.Furthermore,Fla enhanced the proliferation of CD4~+T cells in draining lymph nodes on day 8,compared with Saline(p=0.02).These results indicated that Fla could assist tumor antigens to induce more vigorous immune responses mediated by the NK cells and T cells.5 Flagellin recruited T cells to brainBased on the above results that i.c.vaccination with GL plus Fla prolonged the survival of GL261-bearing mice significantly compared with the s.c.vaccination,we tested whether Fla formulated in the GL vaccine could recruit the peripheral T cells into brains to attack the glioma cells.The mice(n=3)were inoculated with Fla plus GL,Fla,GL or Saline i.c.on day 0.On day 3,the mice were sacrificed and the brain tissues were embedded into paraffin,cut into sections,and then stained with hematoxylin and eosin(HE).We observed massive cell infiltration in brains of mice received GL plus Fla or Fla and much less filtration of the cells from the mice received GL or Saline.To identify whether the infiltrating cells contained numerous T cells,we inoculated the mice(n=3)with Fla plus GL,Fla,GL and Saline i.c.on day0 as previously described in Fig 5A.On day 3,the cells of the brain tissues were isolated by a Percoll gradient centrifugation,stained with mAbs(PE-labeled anti-CD8mAb and FITC-labeled anti-CD4 mAb)for 30 minutes and analyzed by flow cytometry.GL plus Fla,Fla or GL could significantly increase CD8~+T cells in brains,compared with Saline,respectively(p=0.005,p=0.003 and p=0.0385,respectively).GL plus Fla or Fla tended to increase CD4~+T cells in brain,compared with GL or Saline.To verify whether the Fla or Fla formulated GL could also recruit the immune cells in brains of glioma bearing mice,we injected C57BL/6 mice(n=3)with 2×10~4GL261 cells in 5?l of RPMI-1640 i.c.in right caudate nucleus of brains and then immunized them with Fla plus GL,Fla,GL or Saline through the same route.On day 7 after the tumor cell inoculation,the mice were immunized again.All the mice received Saline died within 18 days post-tumor cell injection.On day 18,all of the survived mice received Fla plus GL,Fla or GL were sacrificed for collecting brain tissues.The brain tissues were histologically examined.The massively infiltrated inflammatory cells were observed in the brain tissues of GL261 tumor-bearing mice received GL plus Fla or Fla.In contrast,much less infiltrated inflammatory cells were observed in the brain tissues of the tumor bearing mice received GL or Saline.6 Intracranial administration of flagellin influenced peripheral immune organs and cellsTo detect the effects of i.c.injection with Fla on proliferation of lymphocytes in spleens,we inoculated the mice(n=3)with Fla plus GL,Fla,GL or Saline i.c.on day0,sacrificed them on day 5 and isolated the spleens.The results showed that the weights of spleens were higher in the mice inoculated with Fla or GL plus Fla than in the mice received GL or Saline.Since that intracranially injected proteins could be recovered in peripheral blood,we examined whether the i.c.inoculated Fla could somehow get into the peripheral blood to activate the NK cells and T cells.The mice(n=3)were inoculated with Fla or Saline i.c.on day 0 and sacrificed on day 4,7 and11.Peripheral blood mononuclear cells(PBMCs)were isolated from the mice and analyzed by flow cytometry using mAbs(FITC-labeled anti-NK1.1 mAb,PE-labeled anti-CD8 mAb and FITC-labeled anti-CD4 mAb).The results showed that the i.c.applied Fla significantly increased the number of NK cells on day 4(p=0.0018),CD8~+T cells on day 7 and day 11(p=0.007 and p=0.019)(Fig 6G and H),and CD4~+T cells on day 7(p=0.016)in the PBMCs,respectively.In summary,prophylacticly subcutaneous administration of GL-Fla could prolong the survival of GL261-bearing mice and induce the activation of glioma-specific CTL.Therapeutic intracranial administration of GL-Fla could prolong the survival of GL261-bearing mice;Fla could recruit T cell into brains.The datas indicated that GL derived from glioma tissues of patients formulated with Fla could delay or prevent the recurrence of glioma,which provided a new strategy of glioma immunotherapy.