| The success of using immune checkpoint inhibitors to treat cancers implies that blocking immunosuppressive cytokines could be another approach to enhance immune responses to tumor antigens and also microbial antigens,and therefore to develop novel adjuvants for vaccines.Accumulated evidences suggest that transforming growth factor beta 2(TGF-?2),among the immunosuppressive cytokines,could be targeted in developing this type of adjuvants.TGF-?2,similar to its other two isoforms(TGF-?1 and TGF-?3),is synthesized as a precursor composed of a signal peptide,a latency-associated peptide(LAP),and a C-terminal fragment.The precursor is processed to yield a biologically active mature TGF-?2 by removing its signal peptide and LAP.The TGF-?2 is either secreted as a soluble TGF-?2(so-TGF-?2),or anchored to the cell surface as a surface-bound TGF-?2(s TGF-?2).All TGF-?isoforms trigger signaling in cells via binding to a TGF-?receptor complex composed of two type I TGF-?receptors(TGF-?RI)and two type II TGF-?receptors(TGF-?RII).Preferentially,TGF-?2 needs a co-receptor TGF-?type III receptor(T?RIII)to trigger optimal signaling.The T?RIII is expressed in dendritic cells(DCs),macrophages,T cells and B cells,thereby making these cells responsive to TGF-?2.As reported,TGF-?2 reduces the expression of MHC I molecules,MHC II molecules,CD80,CD86 and CD40 molecules on DCs,B cells and macrophages.Also,TGF-?2 inhibits activated DCs to produce soluble IL-23 and membrane-bound IL-23.The IL-23 is able to facilitate the production of high-level Ig A in virus-infected mice.TGF-?2 induces the generation of regulatory T cells(Tregs),and the generation depends on the interaction of TGF-?2 with T?RIII.Furthermore,TGF-?2 inhibits B cells to proliferate and differentiate into antibody producing cells.Oligodeoxynucleotide(ODN)-based TGF-?2 inhibitors have been evaluated as adjuvants for tumor vaccines.In mice,short hairpin RNA(sh RNA)targeting TGF-?2m RNA could enhance anti-melanoma efficacy of MART1(melanoma antigen recognized by T cells).In clinical trials,antisense oligonucleotides(ASOs)targeting TGF-?2 m RNA were tested as adjuvants for whole tumor cell vaccines for the treatment of glioblastoma,melanoma,non-small cell lung cancer,prostate cancer,colon cancer,gastric cancer and leiomyosarcoma.Noticeably,in mammalian cells,micro RNAs targeting 3'untranslated region(3'UTR)of specific m RNAs could reduce gene translation and initiate the degradation of the targeted m RNA.Together,these data support the feasibility of developing ODN-based TGF-?2 inhibitors as adjuvants for the microbial vaccines.In this study,we designed series of TGF-?2 interfering oligonucleotides(TIOs)with the sequences complementary to the conserve regions identical between human and murine3'UTR of TGF-?2 m RNA,and demonstrated that two TIOs,TIO3 and TIO1,could enhance the microbial vaccines to induce vigorous and persistent antibody responses by interfering with TGF-?2 expression in immune cells.And the TIO3-adjuvanted influenza virus vaccine could induce effective protection against the virus challenge.To understand the underlying mechanism,we demonstrated that TIO3 or TIO1 could induce the degradation of TGF-?2 m RNA in the antigen-activated immune cells,reduce the expression of surface-bound TGF-?2 on memory T cells and memory B cells,inhibit the generation of regulatory T cells,enhance the production of IL-4 from CD4~+T cells,promote the expression of MHC class II molecules,CD40,CD80,CD86 on B cells,and promote the expression of MHC class II molecules,CD40,CD80,CD86 on dendritic cells.Together,the data suggested that TIO3 or TIO1 could be developed as a novel adjuvant to the microbial vaccines by inhibiting the expression of an immunosuppressive cytokine.1.Design of oligonucleotides complementary to TGF-?2 m RNA 3'-UTRTo develop a novel type of adjuvants functioning by interfering with TGF-?2,an immunosuppressive cytokine,therefore reducing the immune response to vaccines,we tried to design ODNs targeting TGF-?2 m RNA 3'UTR.In consideration of doing pre-clinical experiments in mice and conducting clinical trials for human use,we designed a series of ODNs targeting the TGF-?2 m RNA 3'UTR in these regions identical between human and mouse.After looking into the primary and secondary structures of TGF-?2m RNA,we found three regions lying in the internal loops of TGF-?2 m RNA 3'UTR.The internal loops were identified to be favorable for the invasion of complementary antisense oligonucleotides(ASOs).With the aids of Nucleotide Basic Local Alignment Search Tool(Nucleotide BLAST)and the US National Center for Biotechnology Information(NCBI),three regions located in the TGF-?2 m RNA 3'UTR were chosen,and designated as R1 and R2 and R3,respectively.The R1,R2 and R3 were further analyzed against Mfold web server for their secondary structures and therefore selected as the targeting sequences for designing three TGF-?2 inhibitory oligonucleotides,TIO1,TIO2,and TIO3,respectively.2.Interfering effects of TIOs on TGF-?2 expression in cultured cellsTo investigate the interfering effects of TIOs on TGF-?2 expression,we chose RAW264.7 cells(mouse macrophage-like cell line),U937 cells(human monocyte cell line)and CAL-1(human plasmacytoid dendritic cells)cells to evaluate the effect of TIOs.Under the stimulation of IL-4 or LPS,we first explored the optimal dose of IL-4 and LPS.The results showed that IL-4,at 25ng/m L,or LPS,at 2?g/m L,could induce a large amount of TGF-?2 production in RAW264.7 cells.And TIOs,at 10?g/m L,could inhibit the expression of TGF-?2 m RNA and protein.The inhibitory effect of TIOs is specific,because it only inhibited the m RNA expression of TGF-?2 neither TGF-?1 nor TGF-?3 in the cells.TIO3 was more efficient than TIO1 or TIO2.And TIO3 up-regulated the expression of Arg1 and down-regulated the expression of TNF-?.To demonstrate the inhibitory effects of TIOs on TGF-?2 expression in human cell lines,we selected human monocytes and human plasmacytoid dendritic cells.The results showed that TIO1,TIO2 or TIO3 could also reduce TGF-?2 expression in U937 or CAL-1 cells.3.Cell and tissue distribution of TIO3To observe whether TIO3 could enter into TGF-?2 expressing CD4~+T cells,B cells,dendritic cells and neutrophils,the lymph node(LN)cells isolated from na?ve mice were incubated with Alexa-488-TIO3 or Cy3-c ODN for 24 h,and then stained with PE-labeled m Abs or FITC-labeled m Abs against CD4,CD19,CD11c and Ly6G,followed by counterstaining with DAPI,a blue-fluorescent DNA dye for staining the nucleus.Under confocal microscope,we observed that both of the TIO3 and c ODN could enter CD4~+T cells,B cells,dendritic cells and neutrophils.Next,we observed whether the TIO3,like Cp G ODNs,could also enter endosomes,and found that the fluorescent signals of Alexa-488-TIO3 or Alexa-488-c ODN in the LN cells were merged with the fluorescent signals of Lyso Tracker?Red DND-99,an endosome probe,indicating that the TIO3 could enter endosomes.Upon the in vitro observations,to further observe the entry of TIOs into T/B cells in vivo,we intramuscularly immunized the mice in the right thigh with Cp-ISA35-Alexa-488-TIO3 or Cp-ISA35-Alexa-488-c ODN once.0.5 h later,the cells isolated from LNs,spleens,lungs,livers,kidneys,hearts,peripheral blood and the LN draining the injection site of the sacrificed mice were analyzed in FACS.We found that the highest percentage(49.5%)of TIO3 entered cells(Alexa-488~+cells)were detected in the LN draining the injection site;18.93%,18.36%,7.21%,0.78%,0.49%,0.12%of the Alexa-488-TIO3~+cells were detected in ipsilateral inguinal,ipsilateral popliteal,ipsilateral axillary,contralateral inguinal,contralateral popliteal,and contralateral axillary LNs;3.97%,0.42%,2.01%,7.17%,0.42%,1.8%of the Alexa-488-TIO3~+cells were found in the spleen,lung,liver,kidney,heart and peripheral blood,respectively.Furthermore,we demonstrated that the TIO3 entered 20.4%,8.2%of CD4~+cells in the LN cells and CD4~+cells in the spleen cells,and 9.8%,2.4%of CD19~+cells in the LN cells and CD19~+cells in the spleen cells,respectively,in the mice at 3h post the immunization with Cp-ISA35-Alexa-488-TIO3.4.Adjuvanticity of TIOs for microbial vaccines in miceUpon their in vitro activities,we further tested whether the TIOs could be used as adjuvants for microbial vaccines to enhance the humoral immune response in mice.To start with,we explored the optimal dose of the TIOs using TIO3.On day 0 and day 14,the mice were intramuscularly immunized in the right thigh with Cp at 5?g/shot formulated with ISA35 emulsion plus TIO3 at dosages of 2.5,5,10,20 or 40?g/shot,respectively.On day 21,the mice were bled for measuring the specific Ig G antibodies in ELISA with the inactivated PCV2b as coating antigens.The results showed that TIO3 at 5,10 or 20?g/shot significantly enhanced the production of specific antibodies after the primary and secondary immunization,and the TIO3 at 10?g/shot facilitated the Cp vaccines to induce the highest level of the Ig G antibodies.Thus 10?g/shot was chosen as the dosage of TIOs(TIO1,TIO2 or TIO3)to test their adjuvanticity using various viral vaccines including Cp-ISA35(Cp at 5?g/shot in ISA35 emulsion),i IAV(inactivated influenza A virus at 0.5?g/shot in ISA35 emulsion),inactivated FMDV-ISA35(inactivated foot and mouth disease virus at 0.5?g/shot in ISA35 emulsion),inactivated IBV-ISA35(inactivated influenza B/Victoria/2/1987 lineage virus at 0.5?g/shot in ISA35 emulsion)or HBV[Hepatitis B virus surface antigen(HBs Ag)at 5?g/shot in aluminum adjuvant].These vaccines were used individually with or without the TIO1,TIO2 or TIO3,respectively.To confirm the sequence-specificity of TIOs on influencing the antibody responses to the vaccines,we used two ODNs(19bp-c ODN and 24bp-c ODN),negative control ODNs,with each of the vaccines,respectively.The 19bp-c ODN,except the sequenced order is disrupted,contains the same type and the number of nucleotides as the TIO3 does,and was inert in the in-vitro experiments.The 24bp-c ODN was demonstrated as an inert ODN in our previous study for evaluating the sequence dependence of CTLA-4 interfering ODN when used as a vaccine adjuvant.The mice were intramuscularly immunized in the right thigh with these vaccines with or without the TIO(TIO1,TIO2 or TIO3)or the c ODN(19bp-c ODN or 24bp-c ODN)on day 0 and day 14,respectively.On day 14,28,42,112 and 133,the mice were bled for detecting the antigen-specific Ig G antibodies in their sera.Noticeably,when analyzing the data obtained from these experiments,we found that 19bp-c ODN showed some degree of inhibitory activity,and thus the data derived from 24bp-c ODN was chosen and summarized to present.Resultantly,compared with c ODN,the TIO3 and TIO1,not TIO2,significantly enhanced the production of specific antibodies induced by Cp-ISA35,i IAV,FMDV-ISA35,IBV-ISA35 or HBV vaccines on day 14,28,42 and 112,respectively.The enhanced antibody response reached its peak on day 42 and lasted to day 133.TIO3 was more efficient than TIO1.To demonstrate whether the TIO3-adjuvanted viral vaccines could induce effective protection against virus,BALB/c mice were injected with i IAV[inactivated Influenza A virus(IAV)in ISA35 emulsion],i IAV-TIO3(i IAV plus TIO3),i IAV-19bp-c ODN(i IAV plus 19bp-c ODN),i IAV-24bp-c ODN(i IAV plus 24bp-c ODN)or PBS on day 0 and day 14,respectively.On day 56,the mice were intranasally inoculated with 50?l of the IAV-infected allantoic fluid(AF)at 10 LD50 or 50?l of AF.Afterwards,the mice were observed daily over the following 2 weeks for recording body weight,clinical manifestation and survival.Similar to what we noticed in the microbial vaccine immunization experiments,the 19bp-c ODN,not the 24bp-c ODN,tended to be inhibitory,and hence we chose the results derived from the 24bp-c ODN to evaluate the sequence specificity of TIO3.The results showed that the body weight of the mice received i IAV-TIO3 declined slightly,whereas the body weight of the mice received PBS underwent a dramatic and continuous loss.And the body weights of the mice received i IAV-TIO3 gradually recovered towards normal levels from day 9 post infection(p.i.).However,the body weights of the mice received PBS or i IAV-c ODN did not recover until the endpoint of the experiment.The clinical manifestations of the mice were evaluated with clinical score(CS),which is defined based on the signs of lethargy,piloerection,tremor,periorbital exudates,respiratory distress and diarrhea.The mice with a CS>3 or?3 were defined as undergoing severe infection,moderate infection,respectively.Resultantly,on day 3 p.i.,the mice began to display signs of inactivity,inappetence,hunching and ruffled fur.By day 5 p.i.,the mice received PBS developed laboured respiration,respiratory distress,periorbital exudates and tremors(with a CS>3),the mice received i IAV or i IAV-c ODN developed lethargy,piloerection(with a CS?3),while the mice received i IAV-TIO3 did not show obvious clinical signs(with a CS?2).As to survival,the mice received i IAV-TIO3 were all survived,while the mice received PBS began to die on day 5 p.i.and all died within 14 days p.i..The mice received i IAV or i IAV-c ODN began to die on day 9 p.i.and 3 of the 8 mice were alive on day 14 p.i..The results indicated that i IAV-TIO3 immunization prolonged the survival of the IAV-infected mice.In a parallel experiment,on day 5 p.i.,the mice were sacrificed and assayed/observed for their IAV load and pathological changes in the lungs of the mice.The data demonstrated that i IAV-TIO3 immunization significantly reduced the IAV titer,IAV haemagglutination titerin the lung homogenates,and the IAV RNA load in the lung tissue of the mice,compared to i IAV or i IAV-c ODN immunization.Histopathologically,the mice received PBS displayed severe consolidated lung parenchyma lesion manifested by viral inclusion body appearance in alveolar cells and injury alveoli with interstitial inflammation,profuse hemorrhage,edema,dense fibrin accumulations,hyaline membrane formation,thickened alveolar walls and massive inflammatory cell infiltration,while these pathological changes were hardly observed in the mice received i IAV-TIO3,and mild or moderate in the mice received i IAV or i IAV-c ODN.Compatibly,the lung injury score in the mice received i IAV-TIO3 was considerably lower than those in the mice treated with i IAV,i IAV-c ODN or PBS,respectively.To explore whether the TIO3 could facilitate the vaccine to activate T cells at the site of IAV infection,the mice were immunized as described above.On day 56,the mice were challenged with the IAV.8 h later,their lungs were isolated,homogenized,and detected for CD69 expression on CD4~+T cells and CD8~+T cells.The results demonstrated that i IAV-TIO3 immunization apparently elevated the ratios of CD4~+CD69~+T cells and CD19~+CD69~+B cells,indicating that TIO3 could facilitate the vaccine to activate T cells at the site of IAV infection.5.The possible mechanisms for TIO1 and TIO3 used as adjuvants for microbial vaccinesTo explore the possible mechanism about how the TIO1 and TIO3 to enhance the antibody responses to microbial vaccines,we chose the HBV vaccine with or without TIO1or TIO3 to immune mice,and then isolated immune cells from the mice to detect the effects of TIOs on TGF-?2 expression,T cell activation,Treg generation,cytokine production and expression of co-stimulatory molecules and MHC II molecules in these cells.The HBV vaccine,a commercialized vaccine consisting of the HBs Ag in the form of virus-like particles(VLPs)which resemble the overall structure of virus particles,was demonstrated to induce higher-level expression of TGF-?2 in the inguinal LN cells isolated from the immunized mice,when the cells exposed to the HBs Ag(10?g/ml)in vitro.To further confirm the sequence specificity,the c ODN(24bp-c ODN)used in microbial vaccine immunization was included in the following in vivo recall experiments.To detect the interfering effect of TIOs on TGF-?2 m RNA expression,we immunized the BALB/c mice with HBV,HBV-TIO1,HBV-TIO3 or HBV-c ODN vaccines on day 0 and day 14,and boosted on day 63 post the second immunization.3 h later,the mice were sacrificed and their LN cells and spleen cells were isolated for detecting the expression of TGF-?2 m RNA.The results showed that the TGF-?2 m RNA was significantly increased in the LN cells and spleen cells from the mice received HBV vaccine,and the increase was almost completely abolished in the LN cells and spleen cells from the mice received HBV-TIO3vaccine,partially abolished in the LN cells and spleen cells from the mice received HBV-TIO1 vaccine,and un-interfered in the LN cells and spleen cells from the mice received HBV-c ODN vaccine.Upon the observation that enhanced antibody response to TIO formulated microbial vaccines could last up to the day 133 after the first immunization,on the day 133 after the first immunization,we boosted the mice which had been immunized twice with HBV,HBV-TIO1,HBV-TIO3 or HBV-c ODN to detect the interfering effect of TIOs on TGF-?2protein expression on T cells and B cells.6 h later,the mice were sacrificed and their LN cells and spleen cells were isolated for detecting s TGF-?2 on CD4~+T cells and CD19~+B cells.Flowcytometrical analysis showed that in the LN cells and spleen cells,HBV induced over 50%increased s TGF-?2 expressing CD4~+T cells and CD19~+B cells at 6h after the boost immunization,respectively.In both of the LN cells and spleen cells,TIO3formulated HBV or TIO1 formulated HBV caused 40%or 30%reduction of s TGF-?2expressing CD4~+T cells,respectively,and TIO3 formulated HBV or TIO1 formulated HBV caused 35%or 30%reduction of s TGF-?2 expressing CD19~+B cells,respectively.To detect the effects of TIOs on activation of CD4~+T cells and CD19~+B cells,we firstly immunized the mice with HBV vaccines on day 0 and day 14,and on day 14 after the second immunization,we isolated the LN cells from the mice and incubated them with HBs Ag(10?g/ml)alone or plus TIO1,TIO3 or c ODN,at 10?g/ml.48 h later,the cells were analyzed using FACS to detect the expression of CD69 on CD4~+T cells and CD19~+B cells.Resultantly,in the in-vitro antigen recalled response,TIO3 or TIO1 significantly increased the ratios of CD4~+CD69~+T cells and CD19~+CD69~+B cells,and up-regulated the expression of CD69 on CD4~+T cells and CD19~+B cells in the LN cells,respectively.Alternatively,we immunized the mice with HBV,HBV-TIO1,HBV-TIO3 or HBV-c ODN vaccines on day 0 and day 14.48 h after the second immunization,we isolated the LN cells and immediately detected the expression of CD69 on CD4~+T cells and CD19~+B cells.The results showed that TIO3 or TIO1 could apparently increase the ratios of CD4~+CD69~+T cells and CD19~+CD69~+B cells,and markedly increase the mean fluorescent intensity(MFI)of CD69 on CD4~+T cells and CD19~+B cells in the in-vivo antigen recalled response.Considering that s TGF-?2 induces the generation of Tregs and the Tregs can release TGF-?2 to inhibit the production of IFN-?,a typical Th1 cytokine,we detected whether TIO3 and TIO1 could impact the production of IFN-?and the generation of Tregs in the immune responses to microbial vaccines.To do this,we immunized the mice with HBV,HBV-TIO1,HBV-TIO3 or HBV-c ODN vaccines on day 0 and day 14.On day 7 after the second immunization,we boosted the mice.48 h or 72 h later after the boosting,we isolated the LN cells and immediately detected the expression of intracellular IFN-?in CD4~+T cells and CD8~+T cells,and the expression of CD25 and Foxp3 in CD4~+T cells.The results demonstrated that TIO3 or TIO1 significantly increased the ratios of IFN-?producing CD4~+T cells and IFN-?producing CD8~+T cells in the LN cells at 72 h after the boosting,and TIO3 or TIO1 significantly decreased the ratio of the CD25~+Foxp3~+cells in CD4~+T cells at 48 h after the boosting.Given that activated B cells or DCs can produce TGF-?2;TGF-?2 down-regulates the expression of co-stimulatory molecules and MHC II on DCs and B cells,and TGF-?2inhibits the production of IL-4,a Th2 typically cytokine,we immunized the BALB/c mice with HBV,HBV-TIO1,HBV-TIO3 or HBV-c ODN vaccines on day 0 and day 14,and boosted the mice on day 133 after the first immunization.48 h later,we isolated the LN cells and immediately detected the expression of intracellular IL-4 in CD4~+T cells,the expression of CD40,CD80,CD86 and MHC II molecules on CD19~+B cells and CD40,CD80,CD86 and MHC II molecules on CD11c~+DCs.Resultantly,TIO3 or TIO1obviously increased the ratios of CD4~+T cells and CD19~+B cells,IL-4 expressing CD4~+T cells,CD40,CD80,CD86 and MHC II expressing CD19~+B cells,and the CD80,CD86,CD40 and MHC II expressing CD11c~+DCs,respectively.Also,TIO3 or TIO1 significantly increased the MFI for CD40,CD80,CD86 and MHC II in CD19~+B cells.6.Adjuvanticity of oligodeoxynucleotides targeting TGF-?2 m RNA 3'UTR for glioma vaccinesTo investigate whether TIOs could be used as an adjuvant for glioma vaccines,we immunized C57BL/6 mice(n=6)with TCL plus TIOs or c ODN,TCL,or Nacl in the neck on day 0 and 10.On day 14,the mice were intracranially inoculated with 2×10~4GL261cells into caudate nucleus,the results showed that prophylacticly intramuscular administration of TCL+TIO3,TCL+TIO2 or TCL+TIO1 could significantly prolong the survival of the GL261-bearing mice.To further explore the possible mechanism of the glioma vaccine with TIOs as adjuvants to enhance the anti-glioma effect,we immunized mouse neck muscles with TCL,TCL+TIO3,TCL+c ODN on day 0 and day 10,and isolated them after 8 hours.Lymph nodes were tested for TGF-?2 expression and immune cell activation.The results show that TIO3 can inhibit the expression of s TGF-?2 on the surface of CD4~+T cells and CD8~+T cells,induce glioma-specific CD4~+T cells and CD8~+T cells,and promote the mass production of IFN-?by CD4~+T cells and CD8~+T cells.To demonstrate whether TIOs could serve as an adjuvant to assist tumor cell lysate(TCL)to induce anti-glioma immunity,we intracranially inoculated the C57BL/6 mice with 2×10~4GL261 cells into caudate nucleus on day 0,and then therapeutically immunized the mice(n=6)with TCL+TIOs,TCL,or Nacl in the neck on day 4 and 14.Resultantly,therapeutic intramuscularly administration of TCL+TIO2,but not TCL+TIO1 or TCL+TIO3 markedly prolong the survival of GL261-bearing mice in situ glioma model.And TCL+TIO2 could induce specific anti-glioma immunity.The survived mice have the immune memory specifically for glioma.Histopathologically,TCL+TIO2 could significantly promote inflammatory cell infiltration in the brain of the GL261-bearing mice.7.TIO3 as a therapeutic drug for gastric cancer and glioma.To explore whether TIO3 could be a therapeutic drug for gatrical cancer and glioma,we firstly establish a model of gastric cancer heterotopic transplantation,5×10~5MFC cells(gastric cancer cells)were inoculated subcutaneously in the back in the mice,and then TIO3 was intraperitoneally administrated on day 1,3,5 and day 7,respectively.The results showed that therapeutically intraperitoneal injection with TIO3 could significantly prolong the survival of MFC-bearing mice,indicating that TIO3 could be used as an effective therapeutic drug for gastric cancer.To demonstrate that TIO3 does have anti-gastric cancer effects,we increased the density of MFC cells.On day 0,2×10~6MFC cells were injected subcutaneously into the back of mice,and then TIO3(10?g/shot)was injected intraperitoneally on day 1,3,5 and day 7,respectively.The results also confirmed that TIO3 could significantly reduce tumor volume and prolong the survival time of mice bearing gastric cancer.This result suggested that TIO3 could be used as a drug candidate for the treatment of gastric cancer.To explore the possible mechanism of TIO3 against gastric cancer,we tested TIO3 on the expression of TGF-?2 in lymph nodes in the drainage area of gastric cancer and MFC cells which were transplanted on the back.The results showed that TIO3 could down-regulate the m RNA expression of TGF-?2 in lymph nodes and protein expression of s TGF-?2 on the surface of CD4~+T cells and CD8~+T cells.TIO3 could significantly down-regulate the expression of TGF-?2 in MFC cells,and promote the expression of MHC I,IL-13,IL-15,IL-17,and IFN-?.Similarly,to investigate whether intraperitoneal injection with TIO3 alone was effective for the treatment of glioma,we firstly establish the intracranial glioma model,the GL261 cells were intracranially inoculated with 2×10~4GL261 cells into caudate nucleus on day 0,and then TIO3 was intraperitoneally administrated on day 1,3,5 and day 7,respectively.Resultantly,therapeutically intraperitoneal injection with TIO3 could significantly prolong the survival of GL261-bearing mice,indicating that TIO3 could be used as an effective therapeutic drug for Glioma.To explore the mechanism of the anti-glioma effect of TIO3,we tested the effect of TIO3 on TGF-?2,MHC I,IL-17A and IFN-?expression in tumor cells.The results showed that TIO3 could inhibit expression of TGF-?2 in GL261 cells and U-251 cells,promote the expression of MHC I,IL-17A and IFN-?,and finally enhanced the anti-tumor efficacy of tumor vaccines.In conclusion,in this study,we designed an antisense oligonucleotide drug(TIO3)targeting TGF-?2 and proved that TIO3 could enter T cells,B cells,DC cells,neutrophils and endosomes;inhibit the expression of TGF-?2;improve the activation of CD4~+T cells and CD19~+B cells;promote the production of cytokines IL-4 and IFN-?;enhance subunit vaccines,inactivated virus vaccines(A/B influenza Virus vaccine,Foot-and-Mouth disease vaccine)and Hepatitis B vaccine.Thus,TIO3,as a nucleic acid inhibitor of TGF-?2,can enhance the antibody response induced by microbial vaccines,and has the potential to develop into a novel microbial vaccine adjuvant.In addition,we found that TIO3combined with glioma cell lysate can significantly prolong the survival period of glioma model mice inoculated in situ,and induce glioma-specific CD4~+T cells and CD8~+T cells.The above results suggest that TIO3,by inhibiting the immunosuppressive cytokine TGF-?2,has the potential to be used as an adjuvant for prophylatic glioma lysate vaccine adjuvant.Prophylacticly intramuscular immunization of the tumor vaccine TIO3-TCL by mixing TGF-?2 interfering oligonucleotides combined with glioma tissue lysate could significantly prolong the survival of the mice intracranially inoculation with GL261 cells.And TIO3-TCL induced potent CD4~+T and cytotoxic T lymphocyte.Moreover,we found that TIO3 has the monotheraputic effect for glioma and gastric cancer.The postoperative tumor tissue of patients with glioma is prepared into glioma lysate,when combined with TIO3 to immunize the neck muscles,which may delay or prevent the recurrence of glioma,providing a new way of thinking for treatment patients received glioma surgery,suggesting that TIO3 could be used as a drug candidate for the treatment of glioma and gastric cancer. |
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