The Study On The Cross Talk Between B And T Lymphocytes In Type1Diabetes With Different IA-2Epitope Conformation Peptides

BackgroundType1diabetes mellitus is one of the chronic diseases in Children endocrinediseases. Almost70000children less than15years old were diagnosed as T1DMper year all over the world. T1DM is an organ specific autoimmune diseasemediated by T lymphocytes. Some autoantibodies can be detected in the serum ofT1DM patients, such as insulin autoantibody（IAA）, Glutamic acid decarboxylaseantibody（GADA）, Insulinoma associated protein-2antibody（IA-2A） and Zinctransporter8antibody（ZnT8A） which was found in recent years. B lymphocytescan present antigens to T lymphocytes to induce immune response, But how T andB lymphocytes can communicate are not well known. In our research we explorethe mechanism between T and B lymphocyte cross talk and aim to revealpathogenesis of T1DM.Objective:Insulinoma associated protein-2（IA-2） is an important autoantigen of T1DMand we have found amino acid843-855of IA-2are important B lymphocyteepitopes. But we didn’t understand clearly how the conformation and the sequenceof amino acid can influence the crosstalk between T lymphocyte and Blymphocyte. In our study we designed and synthesized two peptides of IA-2_843-855with different conformations（the parental β-fold structure and the helical peptideanalogue） to investigate the interaction between T lymphocyte and IA-2_843-855 peptide and compare their roles in the crosstalk between T lymphocyte and Blymphocyte to confirm in this process whether the conformation or the sequenceof amino acid plays the important part. In order to tell the immunologypathogenesis, prevention and treatment of T1DM.Methods:1.1、Subjects:Patients who were diagnosed as T1DM （n=12） within1year and with hightiter IA-2autoantibodies were recruited from the second hospital of Jilinuniversity （Nov.2009to Nov.2010）; Patients who were diagnosed as T2DM（n=12） within1year were collected from the same hospital （Jan.2010toMay.2010）; Healthy volunteers （n=12） were recruited as normal controls.The mean age of T1DM group was10.5years, the mean duration of T1DMwas5.0（range0–11） months,4male and8female. The mean age of T2DM groupwas35.1years, the mean duration of T1DM was5.8（range2–12） months,7maleand5female. Type1and type2diabetes were diagnosed by1999WHOdiagnostic criteria. The12healthy control subjects（mean age22.1years）had nofamily history of diabetes and other autoimmune diseases, fasting and2hpostprandial blood glucose were normal. Informed consent was obtained from allparticipants.2. The proliferation of lymphocytes in PBMC:PBMCs were isolated from heparinized whole blood by Ficoll-Hypaquedensity centrifugation, and3×105PBMCs per well were cultured in96-well and1×106PBMCs per well in24-well trays in RPMI1640（10%fetal calf serum）medium for72hours （5%CO2,37℃）.The cells were incubated with two types ofIA-2antigens（10μg/ml）and nonspecific mitogen PHA （5μg/ml） was added as positive control.10μl cell count solutions （CCK-8kit） were added to each well for6h untill harvest. The absorbance of each well was read by microplate reader.Cellular proliferation was expressed as the stimulation index （SI=mean counts perminute incorporated in the presence of antigen divided by the mean counts perminute incorporated in antigen absence）.3. The cytokines stimulate by T cells：The suspensions collected fromculture medium after centrifuged from24-well were used to test the levels ofcytokines IL-2and IL-4.Results：The proliferation of lymphocytes: The SI of PBMC stimulated by PHAwere1.47±0.17、1.49±0.22、1.58±0.27respectively in T1DM,T2DM and healthygroup. Stimulated by the parental β-fold structure IA-2_843-855 peptide, SI inT1DM,T2DM and healthy group were1.33±0.05、1.13±0.06、1.11±0.06respectively. Stimulated by theα-helical IA-2843-855peptide, SI in the three groupswere1.27±0.06；1.19±0.05、1.09±0.05respectively. The results showed that SI inparental and analogous IA-2_843-855 peptides in T1DM groups are significantlyhigher than that in T2DM and healthy group（P<0.05）,but there is no significantdifference of SI were found between the two peptides （P>0.05）.The analyses of cytokine:IL-4（ng/L） secreted by T cells of T1DM,T2DM and healthy group were24.82±2.13、28.14±2.92、16.51±1.79respectively without any antigen. Stimulatedby PHA of the three groups were23.51±1.65、27.42±2.86、17.92±1.15respectively.Stimulated by the parental β-fold structure IA-2843-855peptide were13.61±1.17、34.36±2.28、23.13±1.96respectively and by α-helical IA-2843-855peptide were16.42±2.23、30.62±2.74、21.22±3.11. The results shows that IL-4secretion inparental and analogous IA-2843-855peptides of T1DM groups are significantlylower than that in T2DM and healthy group（P<0.05）,but there is no significant difference of secretion were found between the two peptides （P>0.05）.IL-2（ng/L） secreted by T cells of T1DM,T2DM and healthy group were15.99±1.16、13.15±2.43、14.05±1.72respectively without any antigen. Whilestimulated by PHA were14.31±1.11、12.13±2.23、16.19±1.05respectively.Stimulated by the parental β-fold structure IA-2843-855peptide were17.93±1.41、15.00±1.18、14.11±1.64respectively and by α-helical IA-2843-855peptide were19.24±2.14、13.77±0.74、14.48±1.92. The results shows that IL-2secretion inparental and analogous IA-2843-855peptides of T1DM groups are significantlyhigher than that in T2DM and healthy group（P<0.05）,but there is no significantdifference of secretion were found between the two peptides （P>0.05）.The ratio of IL-2/IL-4:This ratio in T1DM,T2DM and healthy group were0.64±0.05、0.47±0.11、0.85±0.13respectively without any antigen. Stimulated byPHA of these three groups were0.61±0.10；0.44±0.09、0.90±0.12；Stimulated bythe parental β-fold structure IA-2_843-855 peptide were1.32±0.21、0.47±0.12、0.76±0.08respectively and by α-helical IA-2843-855peptide were1.17±0.07、0.45±0.14、0.68±0.05. The results shows that IL-2/IL-4in parental and analogousIA-2843-855peptides of T1DM groups are significantly higher than that in T2DMand healthy group（P<0.01）,but there is no significant difference were foundbetween two peptides （P>0.05）.Conclusions:From these results we can conclude that the two types of IA-2_843-855 peptideswe designed and synthesized can be specific binding sites of B lymphocyte and bepresented to T cells to influence the immune responses，but different conformationhas no different effects on the crosstalk of T and B lymphocyte. And the two typesof IA-2843-855peptides can make T cells of T1DM patients to secreted more Th1cytokines thanTh2cytokines to break the balance of Th1/Th2which lead to disorder of autoimmune.