A LAG3 Interfering Oligonucleotide Acts As An Adjuvant To Enhance The Antibody Responses Induced By Recombinant Protein Vaccines And Inactivated Influenza Virus Vaccines

Lymphocyte activation gene-3(LAG3)is an inhibitory receptor(IR)of T cells.T cells express virous of IRs,such as CTLA-4 and PD-1,and inhibitory pathways are crucial for maintaining the self-tolerance,modulating the amplitude and duration of immune responses,avoiding tissue damage and the occurrence of autoimmune diseases.However,the sustained expression of IRs will inhibit the function of T cells and induce T cell exhaustion.Blocking IRs can reverse the anergy of T cells and enhance the immune response of anti-virus and anti-tumor.Therefore,the IRs are also regarded as immune checkpoints.Currently,Blocking CTLA-4 or PD-1/PD-L1 has been used in the clinical treatment of a variety of tumors,suggesting the feasibility of blocking T cell inhibitory signals for the treatment of diseases.However,many patients haven’t benefited from these checkpoint inhibitors and more researches about the various of immune checkpoint inhibitors are still needed.LAG3 is also an immune checkpoint,when binds to MHC class II molecule,LAG3 can deliver inhibitory signals to inhibit the first signal of T cell activation.Blocking LAG3 can enhance the activity of T cells,thereby,enhancing the immune response.Currently,the nucleic acid antagonist of LAG3 has not been reported.In this research,we designed ASOs targeting LAG3,and evaluated their effects on maintaining persistent T cell responses,and the possibility of acting as adjuvants to enhancing immunogenicity of recombinant antigen vaccines and inactivated influenza virus vaccines.When investigated whether the ASO targeting LAG3 could also be used as a tumor vaccine adjuvant to enhance the antitumor activity,we observed a lethal duodenal necrosis in the mice injected with Lewis lung cancer lysates unexpectedly,so we also explored the possible reasons of this occurrence.1.Design and screen of LAG3 interfering antisense oligonucleotidesTo develop ASOs targeting LAG3,we looked into the Homo sapiens LAG3mRNA and Mus musculus LAG3 mRNA in the NCBI Nucleotide Database,and identical the conserved regions that could be used to design ASOs with the help of BLAST web.After detecting the specificity of the conserved regions,rest fifteen regions were screened as target regions.Then,fifteen ASOs completely complementary to them were designed,and with the help of“sfold”and“mfold”web servers the ASOs were further analyzed with three assessment parameters,secondary local structures,binding free energy,and negative motifs.The result showed that two LAG3 interfering antisense oligonucleotides(LIOs),LIO-1 and LIO-2,were chose for further research.2.LIO entry of immune cellsTo observe the entry of LIOs into immune cells,especially T cells,we labeled LIO-1or LIO-2 with Cy3,and detected the uptake of LIO-1 and LIO-2 by total T cells,CD4~+T cells,CD8~+T cells,B cells and DC in the lymph node or spleen cells isolated from na?ve ICR mice by flow cytometry.Also,the location of LIO in T cells were detected by immunofluorescence assay.The result showed that the mainly immune cells in the lymph node cells and splenocytes could uptake LIO-1 and LIO-2,and the internalized Cy3-labled LIO-1 and LIO-2 were visualized in the CD4~+T and CD8~+T cells.The result indicated that the LIO-1 and LIO-2 could entry into T cells.3.The effect of LIO on inhibiting LAG3 expression of T cellsTo test whether the LIO-1 and LIO-2 could inhibit LAG3 expression,we detected the expression of LAG3 mRNA in splenocytes by RT-PCR and the expression of LAG3on CD4~+T cells or CD8~+T cells in the splenocytes and drainage inguinal lymph node(DILN)cells,as well as LAG3 expression on Jurkat cells by flow cytometry.The result showed that:1)LIO-1,not LIO-2,inhibited the expression of LAG3 mRNA in the splenocytes;2)LIO-1,not LIO-2,could decrease the expression of LAG3 on CD4~+T cells and CD8~+T cells in the splenocytes and DILN cells;3)LIO-1,not LIO-2,caused reduced expression of LAG3 on the Jurkat cells.This result indicated that LIO-1inhibited the LAG3 expression of T cells,while LIO-2 not.4.Effect of LIO on the activity of antigen-specific T cell in a recall responseTo detect the effect of LIO on the antigen-specific T cell response in a recall response,the splenocytes isolated from the mice immunized with LIO plus the recombinant CP antigen vaccines were re-stimulated by CP in vitro,and the proliferation of CD4~+and CD8~+T cells were analyzed by the T cell proliferation assay.Also,the splenocytes stimulated by CP were tested their mRNA expression of IFN-γ,IL-2,IL-4 and IL-6 by quantitative real-time PCR analysis.The result showed that:1)LIO-1 increased the ratio of the proliferation of antigen-specific CD4~+T cells,but not the ratio of antigen-specific CD8~+T cell proliferation;2)LIO-1 increased the mRNA expression of IFN-γ,IL-2 and IL-6 in the splenocytes.This result indicated that LIO-1could facilitate the recombinant antigen vaccines to induce the proliferation of CD4~+T cells and the production of IFN-γ,IL-2 and IL-6 when re-exposed to antigen in the recall response.5.Effect of LIO on maintaining activation of Ag-specific T cellsTo further detect the effect of LIO on maintaining T cell activation in vivo,we tested the expression of the T cell activation marker,CD69 on CD4~+and CD8~+T cells in the DILN and spleen from the mice immunized with LIO plus the recombinant CP antigen vaccines on 48 h after the second immunization by flow cytometry.Also,the DILN cells and splenocytes were detected the mRNA expression of IFN-γ,IL-2,IL-4and IL-6 by quantitative real-time PCR analysis.The result showed that:1)LIO-1increased the ratio of CD4~+CD69~+T cells in the DILN cells induced by the recombinant antigen vaccines.2)LIO-1 increased the IFN-γand IL-2 mRNA expression in the DILN cells.This result indicated that LIO-1 could maintain the Ag-specific CD4~+T cell activation and the production of IFN-γand IL-2.6.Effect of LIO on facilitating the antibody response induced by the recombinant antigen vaccine and inactivated influenza virus vaccineThe effect of LIO-1 on enhancing CD4~+T cell response significantly inspired us that LIO-1 may facilitate the antibody response induced by the recombinant antigen vaccine and inactivated influenza virus vaccine.To verify this,we immunized the mice with LIO-1 plus the recombinant CP antigen vaccine or inactivated FM1 influenza virus vaccine,then detected the antibody response by indirect ELISA.The result showed that:1)LIO-1 facilitated obviously increased the antibody production induced by the recombinant CP antigen vaccine.2)LIO-1 facilitated an increase of the antibody production induced by the inactivated FM1 influenza virus vaccine.This result indicated that LIO-1 could act as an adjuvant to enhance the antibody response induced by recombinant protein antigen vaccines or inactivated virus vaccines.7.Correlation of CD8~+T cells or IFN-γwith duodenal necrosis in miceIn a further investigation of whether LIO-1 could also act as an adjuvant in cancer vaccines,we unexpectedly observed that injection of the Lewis lung cancer tumor lysate vaccines(LCLV)induced the wide-spread duodenum necrosis which succumbed the mice.To find the underlying reasons,we injected na?ve mice with the LCLV,and detected the ratio of CD4~+T or CD8~+T cells in the peripheral white blood cells(PWBC),splenocytes and DILN cells,as well as the IFN-γmRNA expression in the PWBC and splenocytes of the moribund mice.The mice injected with saline were used as control.The result showed that:1)The ratio of CD8~+T cells in PWBC from the mice injected with LCLV was 5-fold increased,reaching to 50%approximately.2)The IFN-γmRNA expression in the PWBC was rocked to a level of 12-fold more than that of the normal.The result indicated that the dramatically increased CD8~+T cell and IFN-γproduction in the PWBC could contribute to the fatal duodenal necrosis of the mice.Together,we designed a LAG3 interfering antisense oligonucleotide,LIO-1,and demonstrated that LIO-1 could enter into T cells;induce the degradation of LAG3mRNA;inhibit the LAG3 expression on CD4~+T cells;maintain the prolonged proliferation of antigen-specific CD4~+T cells;promote the activation of antigen-specific CD4~+T cells;increase the production of IFN-γ,IL-2 and IL-6;enhance the antibody responses induced by recombinant antigen vaccines or inactivated influenza virus vaccines.Thus,the LIO-1 as a nucleic acid LAG3 antagonist,could facilitate vigorous antibody responses induced by vaccines,and has a potential to be used as a novel adjuvant.Unexpectedly,we found the possible correlation between the dramatically increased CD8~+T cells/IFN-γproduction in the peripheral blood and duodenum necrosis in the mice.The findings,if being further confirmed,could provide guides in clinical practice for evaluating the risk of developing the duodenal necrosis by numerating the CD8~+T cells and detecting the level of IFN-γin the peripheral blood,and for intervening the abnormally increased CD8~+T cells and IFN-γproduction to prevent the occurrence of the duodenal necrosis.