The Study On Mechanism Of IL-7 Promoting The T Cell Immune Memory And Thymic T Lymphocytes Development In Aged Mice

Tuberculosis is a chronic infectious disease caused by Mycobacterium Tuberculosis.In recent years,with the global aging population,how to prevent tuberculosis infection among the elderly has become a difficult problem.BCG（Bacillus Calmette-Guerin）,which is suitable for children to prevent the sever tuberculosis,cannot provide effective immune protection in the elderly population due to the atrophy and degeneration of thymus and the decrease of immune function in the elderly.Therefore,the development of a vaccine for the elderly population is of great significance for the prevention and control of tuberculosis.Our laboratory has long been engaged in the research of tuberculosis subunit vaccines.The pre-clinical study results of the tuberculosis fusion protein Mtb10.4-Hsp X（MH）and ESAT6-Rv2626c（LT29）combined with the adjuvant DDA and Poly I:C（DP）showed strong cellular/humoral immune response,and the protective effect was better than BCG.How to apply these tuberculosis fusion proteins to the elderly population is one of the directions we are exploring.We hope to improve the immune response of subunit vaccine in the elderly by improving the generation of central memory T cells(T_CM).Interleukin 7（IL-7）has a function of promoting the proliferation of thymocytes,naive T cells and memory T cells and maintain the survival of memory T cells.We speculate that IL-7 could promote a long-term immune response to tuberculosis subunit vaccines in the elderly.Therefore,we used aged mice to explore the long-term immune memory induced by IL-7 assisted tuberculosis subunit vaccine in the elderly and analyze its mechanism.Cytokines and transcription factors play important roles in the generation and maintenance of central memory T cells(T_CM).Transcription factors Id3,Bcl6,and Bach2 promote the formation of memory T cells,and Blimp1 induces the terminal differentiation of effector T cells.To further investigate the potential regulatory mechanism of IL-7 on immune memory,we explore the regulation mechanism of IL-7 on transcription factors related to immune memory differentiation.Ageing is closely linked to thymic atrophy which leads to reduction of the number of thymocytes and the output of na?ve T cells.The reduction of na?ve T cells reduces the number of activated T cells and vaccine efficacy after immunization.DNA methylation modification varies,i.e.the methylation level of specific loci could increase with age.The methylation in promoter region was mostly associated with gene expression.In the first part,the study was that IL-7regulated the immune response induced by tuberculosis subunit vaccine in the peripheral immune system of aged mice.In order to further study the effect of IL-7 on the thymocytes of central immune system of aged mice,we use the aged mice,further explore the effect of IL-7 on the different stages during the thymocytes development,and investigate the mechanism from the level of epigenetic modifications.Part One:IL-7 promotes tuberculosis subunit vaccine to generate the long-term immune memory in aged miceObjective:To study the effect of IL-7 as adjuvant in enhancing the long-term immune memory induced by tuberculosis subunit vaccine in aged mice and its mechanism.Methods:（1）Preparation for the tuberculosis subunit vaccine（MH+LT29）/DP.（2）r AAV-IL-7was constructed and packaged by Adeno-Associated Virus packaging system.（3）Immunization schedules:（1）Young mice（6-8 weeks）and aged mice（40-42 weeks）were immunized with（MH+LT29）/DP at 0,2 and 4 weeks.（2）The old mice were immunized with r AAV-IL-7+（MH+LT29）/DP at 0,2 and 4 weeks.The experimental mice were all C57BL/6 strain mice.（4）The immunodetection time was at 25 weeks after the last immunization,which mainly reflects the immune response mediated by T_CM.The immunodetection was including:（1）The production of IFN-γ,TNF-α,and IL-2 in splenic lymphocytes after stimulation were detected by ELISA.（2）IFN-γsecretion from the CD4+and CD8+T cells following antigen repeated stimulation was performed by intracellular cytokine staining to reflect the quality of T_CM like cells.（3）The proliferation of memory CD4+T cells after stimulation by specific antigen was detected by Ed U.（4）The production of antigen-specific Ig G was detected by ELISA.The effect of IL-7on the immune memory of aged mice was evaluated by the above method.（5）Serum IL-6 levels of young mice（6-8 weeks）and aged mice after immunization were detected by ELISA,which reflect the inflammation with ageing.（6）The changes of serum cytokine profiles in young mice,aged mice,r AAV-IL-7 treated of aged mice,and IL-7 deficient（-/-）mice were detected by the Multi-Analyte Flow Assay Kit,which could analyze the mechanism of IL-7 on the level of cytokines in aged mice.Results:（1）The tuberculosis subunit vaccine induced long-lived memory T cells in young mice（6-8 weeks）,but failed to induce long-term immune memory T cells in aged mice（40-42weeks）.It showed that the secretion of TNF-αand IL-2 from spleen lymphocytes were significantly decreased after the stimulation in aged mice（P<0.05）,the productions of IFN-γfrom memory CD4+and CD8+T cells were decreased following repeated-stimulation of antigens（P<0.01）,the proliferation of CD4+memory T cell was significantly reduced（P<0.05）and serum antigen-specific Ig G levels were decreased.（2）r AAV-IL-7 promotes the generation of long-term memory T cells in aged mice.It showed that the ability to secrete TNF-αand IFN-γof spleen lymphocytes was significantly increased（P<0.05）,the production of IFN-γfrom CD4+and CD8+memory T cells was significantly increased after repeatedly stimulation with antigens（P<0.01）,the proliferation of CD4+memory T cells was significantly enhanced（P<0.05）,and the level of antigen-specific Ig G was increased.The results suggest that IL-7 significantly promote the generation of long-term memory T cells and enhance the long-term immune response in the aged mice after immunization.（3）Serum cytokine profiles found that（1）the levels of IL-12p40,IL-12p70,IL-17A,IL-22and GM-CSF in aged mice were significantly decreased,while the level of IL-6 was significantly increased;the levels of IL-1α,IL-1β,IL-3,IL-12p70 and GM-CSF were significantly increased in aged mice after r AAV-IL-7 treatment（P<0.05）.（2）After IL-7 knockout,levels of IL-12p70,IL-22,and GM-CSF were significantly decreased,while IL-1αand IFN-γwere significantly increased（P<0.05）.These results suggest that IL-7 can restore the function of immune cells in aged mice and promote the secretion of IL-12p70 and GM-CSF.Conclusion:The long-term immune response induced by tuberculosis subunit vaccine in aged mice was weakened.IL-7 could enhance the generation of long-lived memory T cells and increase the production of antibodies in aged mice.IL-7 upregulates serum cytokines IL-12p70 and GM-CS and promotes the generation of T cell immune memory in aged mice.Part Two:IL-7,Id3 and Bcl6 promote tuberculosis subunit vaccine to generate the long-term immune memory in miceObjective:To explore the formation of long-lived memory T cells induced by IL-7 and transcription factor-assisted tuberculosis subunit vaccine.Methods:（1）Preparation for the tuberculosis subunit vaccine（MH+LT70）/DP.The LT70(ESAT6-Ag85B-MPT64_{（190-198）}-Mtb8.4-Rv2626c)and MH（Mtb10.4-Hsp X）were constructed by our lab.（2）r AAV-IL-7 was constructed and packaged by Adeno-Associated Virus packaging system.Lentivirus packaging system was used to construct and package r LV-Id3,r LV-Bcl6,r LV-Bach2 and r LV-Blimp1.（3）Immunization schedules:C57BL/6 mice were immunized by（MH+LT70）/DP plus r AAV-IL-7,and（MH+LT70）/DP plus r LV-Id3,r LV-Bcl6,r LV-Bach2 and r LV-Blimp1,respectively,at 0,2 and 4 weeks.（4）The immunodetection time was at 25 weeks after the last immunization,which mainly reflects the immune response mediated by T_CM.The immunodetection was including:（1）T_CM（CD62L+CD44+）phenotype detection.（2）The proliferation of memory CD4+and CD8+T cells was detected by Ed U.（3）IFN-γproductions from the CD4+and CD8+T cells following antigen repeated stimulation were performed by intracellular cytokine staining to reflect the quality of T_CM like cells.（4）The number of TB10.4specific CD8+memory T cells was evaluated by TB10.4 pentamer.（5）The production of antigen-specific Ig G,Ig G1 and Ig G2c was detected by ELISA.The effects of IL-7 and transcription factor on long-term memory T cells were evaluated by the above method.Based on the above,the regulatory effect of IL-7 on transcription factors was explored,and revealed the mechanism of IL-7 promoting the generation of immune memory.（5）The expression of Id3,Bcl6,Bach2 and Blimp1 in CD4+and CD8+T cells were analyzed by RT-PCR at 3,5 and 7 days after immunization with r AAV-IL-7+（MH+LT70）/DP.Results:（1）r AAV-IL-7 promoted the generation of T_CM induced by tuberculosis subunit vaccine and enhanced the immune response mediated by T_CM.It showed that the CD4+T_CM were significantly increased（P<0.01）,the proliferation of CD4+and CD8+memory T cells was significantly increased（P<0.05）,the levels of IFN-γsecretion following repeated-stimulation of antigens were obviously higher（P<0.05）,and the frequency of TB10.4 specific CD8+memory T cells was increased（P<0.05）.Besides,r AAV-IL-7 promoted the production of antigen-specific Ig G.（2）r AAV-IL-7 significantly increased the expression of Id3 and Bcl6 in CD8+T cells and Id3,Bcl6 and Bach2 in CD4+T cells（P<0.05）at 3 days after the immunization.（3）Overexpression of Id3 and Bcl6 promotes the formation of long-term immune memory induced by tuberculosis subunit vaccines.The results indicate that the CD8+T_CM were significantly increased（P<0.01）,the proliferation of CD8+and CD4+T cells was enhanced（P<0.05）,the TB10.4-specific CD8+memory T cells were increased（P<0.05）,the productions of IFN-γfrom CD4+and CD8+T cells were significantly increased after antigen restimulation（P<0.01）,and the antigen-specific antibodies were increased.（4）Overexpression of Bach2 has no significant effect on long-term immune memory induced by tuberculosis subunit vaccine.Conclusion:IL-7 promotes the generation of T_CM-like cells and enhances the long-term immune responses induced by tuberculosis subunit vaccines.IL-7 regulates immune memory by promoting the expression Id3 and Bcl6.Id3 and Bcl6 promote the formation of long-term immune memory induced by tuberculosis subunit vaccines.Part Three:Study on IL-7 of DNA methylation regulation in thymic DN3 cells of aged miceObjective:r AAV-IL-7 was applied in aged mice to investigate the effect of IL-7 on thymus double negative（DN）cells,the regulation of IL-7 on DNA methylation levels in the promoter regions of proliferation-related genes Bcl2 and c-Myc,and its mechanism were further analyzed.Methods:Using online software Meth Primer（http://www.urogene.org/cgi-/bin/Meth Primer Meth Primer.cgi）predict the Cp G islands in Bcl2 and c-Myc promoter,and designing primers for methylation PCRs.Then,C57BL/6 aged mice were treated with r AAV-IL-7,and the thymocytes subsets of double negative（DN）cells（DN1,DN2,DN3 and DN4）,double positive（DP）and single positive（SP）CD4/CD8 cells were detected by flow cytometry（FCM）at 7 days and 15 days.Genomic DNA and RNA were extracted from thymic DN3 cells after sorting by flow cytometry.Bisulfite sequencing PCR detect the DNA methylation levels in the promoter regions of Bcl2 and c-Myc genes in DN3 cells of young mice（6-8 weeks）and r AAV-IL-7-treated aged mice（50-52weeks）.Finally,the expression of Bcl2 and c-Myc and the correlation between their DNA methylation levels and expressions in DN3 cells were analyzed.Result:（1）Ageing increases DNA methylation levels in the promoter regions of Bcl2 and c-Myc in DN3 cells of aged mice.（2）r AAV-IL-7 decreases the DNA methylation levels in the promoter regions of Bcl2 and c-Myc in DN3 cells of aged mice.（3）r AAV-IL-7 increases the expression of Bcl2 and c-Myc genes in thymic DN3 cells,and their expressions negatively correlated with its methylation level.（4）r AAV-IL-7 increases the numbers of total thymocytes,DN cells and DN2-DN3-DN4 cells on the day 15 of in aged mice.Conclusion:Aging increases the DNA methylation levels in the promoter regions of Bcl2 and c-Myc in thymic DN3 cells.r AAV-IL-7 promotes the demethylation in Bcl2 and c-Myc promoter regions,and increases the thymic DN3 cells in aged mice.