| Objective Type 1 diabetes is an chronic autoimmune disease mediated by T cells. The autoimmune process of the disease include a long pre-clinical period of diabetes (i.e.,insulitis)and overt diabetes period.Recent genetic data and preliminary trial results suggest T cells as a target for preventive strategies.The role of T cells in the pathogenesis of type 1 diabetes has become hot points in the study of type 1 diabetes. Sufficient histological analysis and transfer study of T cells suggests that both CD4+ T cells and CDS+ T cells are essential in the development of type 1 diabetes,the relative contribution of these two T cell subsets in triggering insulitis remains poorly defined.Therefore,we put that if CD8 + T lymphocytes launched insulitis? If so, how CD8 + T cells trigerred the diseases?Method Non-obese diabetic(NOD)mice and NOD background transgenic mice using genetic engineering were used as animal models in our study.PCR and flow cytometry were used to type genotype of experimental animals.Splenocytes were isolated from donor mice before adoptive transfer experiments. Stained with specific monoclonal antibody and determined the type and number of cells to be transferred by flow cytometry,a certain amount of cells were intraperitoneally to recipient mice.Diabetes was monitored by measuring glucose using tail vein blood of recipient mice.Insulitis was asssessed for those recipient mice which did not develop diabetes by immunohistochemistry.Adoptive transferred experiments were carried out first to test whether the diabetogenic T cells existed in young(2 weeks old)NOD mice,and the diabetogenic capacity of T cells increased with the age of donor NOD mice,as well as whether cognate autoantigens were absent in young NOD mice,then to determine whether the expression of costimulatory molecules CD80 on beta cells influence the diabetogenic capability of CD4+ and CD8+ T cells,and to value the effects of costimulatory molecules CD80 on beta cells on naive CD8+ T cells.Spleen cells from NOD.scid.CL4 TCR transgenic mice were labeled with molecular probe of CFSE and transferred to NOD.scid.InsHA mice and NOD.scid mice. Different time post-transfer,HA specific CL4 CD8+ T cells were isolated from the spleen,draining PLN and islets.T cells were stained with mAbs specific for the transgenic TCR,CD69 and CD62L.Proliferation and activation of HA-specific CL4 CD8+ T cells were assessed by flow cytometric analysis.SPSS 11.0 software was used to process the data,test levelα=0.05.Pearson Chi-Square Tests were used for statistical analysis of diabetes incidence induced in recipient mice.Results1 Spleen cells of 14 day-old NOD mice were transferred into adult NOD.scid mice (n=36)and age-matched NOD.scid.Rip.B7 mice(n=35).Diabetes was induced in 68.6%of NOD.scid.Rip.B7 mice by 9 weeks post-transfer while in contrast,no infiltration of lymphocytes in the islets of NOD.scid recipients(as shown in Fig.9). Diabetes was induced in 80%of NOD.scid.Rip.B7 mice by 11 weeks post-transfer (as shown in Fig.13B).In contrast,none of the NOD.scid recipients developed diabetes even by 20 weeks post-transfer.2 Then,spleen cells from different week-old NOD mice were transferred into adult NOD.scid mice in the same number of cells(2×107 cells / recipient).The diabetogenic capacity of T cells in different age of donor NOD mice were observed. As shown in Fig.14～17,none of the NOD.scid recipients(n=36)developed diabetes even by 20 weeks post-transfer when transferred spleen cells from 2 week-old NOD mice.However,when spleen cells from 3 week-old NOD donor mice were transferred, the recipient mice(n=35)began to develope diabetes from 13 weeks post-transfer (5%).There were about 30%of recipient mice developed diabetes by 25 weeks post-transfer.When spleen cells from 4 week-old NOD donor mice were transferred, the recipient mice(n=36)began to develope diabetes from 12 weeks post-transfer (10%)and there were about 50%of recipient mice developed diabetes by 20 weeks post-transfer.When spleen cells from 6 week-old NOD donor mice were transferred, the NOD.scid recipient mice(n=37)began to develope diabetes in earlier time of 5 weeks post-transfer,and there were about 65%of recipient mice developed diabetes by 9 weeks post-transfer,100%by 13 weeks post-transfer.There were significant difference among the diabetes incidence induced by differen age of NOD donor mice by the same time post-transfer(P＜0.001).3 Spleen cells of young NOD mice(14 day-old NOD mice)could not transfer diabetes to NOD.scid recipient mice.Is it duo to the lack of expression of cognate autoantigens of beta cells? We answered the question by adoptive transferred experiments.Spleen cells were isolated from diabetic NOD mice and injected into neonatal(n=30),1 week-old(n=32),2 week-old(n=34),3 week-old(n=32), and 6 week-old(n=36)NOD.scid mice and the development of diabetes monitored (table 2).All recipient groups began to develop diabetes from 5 weeks post transfer and 100%recipient mice developed diabetes by 9 weeks post-transfer.Irrespective of age,diabetes was induced in all recipients with similar time of onset(Fig.13A).This result demonstrated that disease relevant beta cell autoantigens are expressed in neonatal and young NOD.scid mice,at times prior to the initiation of insulitis.There was no significant difference among different age of recipient mice of the diabetes incidence induced by diabetic NOD donor mice by the same time post-transfer(P＞0.05).4 Naive splenic BDC2.5 CD4+ T cells were isolated from 9～10 day-old BDC2.5/NOD.scid mice and adoptively transferred into NOD.scid(n=40)and NOD.scid.Rip.B7 mice(n=40),(5×105 cells / recipient).Both recipient groups began to develop diabetes at 12 days post transfer,and by 15 days post transfer,100% recipient mice in two groups developed diabetes.There was no significant difference between the two recipient groups of the diabetes incidence(P＞0.05).To exclude the possibility that the number of BDC2.5 CD4+ T cells transferred masked the effect of CD80,various numbers of BDC2.5 CD4+ T cells were transferred into NOD.scid and NOD.scid.Rip.B7 recipients.Regardless of the numbers of BDC2.5 CD4+ T cells transferred,diabetes was induced in NOD.scid.Rip.B7 and NOD.scid recipients with a similar frequency and kinetics(Fig. 18A-D).These results demonstrated that expression of CD80 by beta cells had no significant effect on the diabetogenic capacity of CD4+ T cells.5 Activated CD8+ T cells prepared from the spleens of diabetic or 4 week-old NOD.Ragnull.AI4 TCR transgenic mice,7×105 CD8+ T cells were transferred into NOD.scid(n=35)and NOD.scid.Rip.B7 mice(n=40).As demonstrated in table 3, diabetes developed in both NOD.scid and NOD.scid.Rip.B7 recipients,but with markedly different kinetics.All NOD.scid.RipB7 recipients developed overt diabetes by 24 days post-transfer,in contrast,diabetes incidence was only 14.3%in NOD.scid recipients.There were significant difference between the two groups of diabetes incidence(P＜0.001).When 7×104 CD8+ T cells were transferred,all NOD.scid.RipB7 recipients(n=40) developed overt diabetes by 45 days post-transfer,in contrast,diabetes incidence was only 17.1%in NOD.scid recipients(n=35).There were significant difference between the two groups of diabetes incidence(P＜0.001).CD8+ T cells prepared from the spleens of 9～10 day-old NOD.Ragnull.AI4 TCR transgenic mice,7×105 CD8+ T cells were transferred into NOD.scid(n=30)and NOD.scid.Rip.B7 mice(n=32).As demonstrated in Fig.20A,NOD.scid.Rip.B7 recipient mice began to develop diabetes at 31 days post transfer,100%recipient mice developed diabetes by 55 days post transfer.The onset and incidence of diabetes induced by naive CD8+ T cells was comparable to that of CD8+ T cells prepared from diabetic or pre-diabetic AI4 TCR transgenic mice.Insulitis in the NOD.scid recipients was absent at 60 days post-transfer,but at 120 days post-transfer peri-insulitis and insulitis was detected in some recipients(Fig.20B).6 Naive AI4 CD8+ T cells were adoptively transferred into NOD.scid.β2mnull.B7 and NOD.scid.Rip.B7 mice.Diabetes was readily induced in NOD.scid.Rip.B7 mice (Fig.21).It began to develop diabetes at 23 days post transfer,and by 35 days post transfer,all animals in NOD.scid.Rip.B7 recipient mice developed diabetes.In contrast,none of the NOD.scid.β2mnull.Rip.B7 recipient mice developed diabetes even by 20 days post-transfer.7 Splenocytes from NOD.scid.CL4 transgenic mice which express an H2Kd-resticted TCR specific for influenza HA were labeled with CFSE.12 mice of NOD.scid.InsHA, which selectively express HA on bete cells,were injected i.p.with 1×107 CFSE-labeled spleen cells,12 mice of NOD.scid were used as control.By day 4 post-transfer,～50%of CL4 CD8+ T cells isolated from the islets of NOD.scid.InsHA had undergone multiple rounds of division whereas only a few CL4 CD8+ T cells from PLN and spleen showed evidence of proliferation(Fig.22,23). The lack of recovery of CL4 CD8+ T cells from the islets of NOD.scid.InsHA recipients at 5 days post-transfer or later because of the fragility of the islets with heavy infiltration.However,in NOD.scid recipients group,significant T cell proliferation was first detected in the spleen and PLN 7 days post-transfer(Fig.22,23). The small numbers of CL4 CD8+ T cells were found in the islets of NOD.scid control recipients,but did not increase with time.Conclusion1 Spleen cells of young NOD mice contain beta cell-specific T cells,however,the T cell repertoire and/or frequency of diabetogenic T cells are limited.The diabetogenic capacity of T cells increased with the age of NOD mice.The spleen cells from 2-week-old NOD mice succeeded in transferring diabetes to NOD.scid.RipB7 recipients demonstrated that spleen cells of 14 day-old NOD mice contain beta cell-specific T cells.The lack of insulitis and diabetes in NOD.scid recipients,however,also suggested that the T cell repertoire and/or frequency of diabetogenic T cells are limited in young NOD mice.There were recipients in both groups developed diabetes when transferred splenocyte from different-week-old donors,but the incidence in control recipients was far lower from that of in NOD.scid.Rip.B7 mice(P＜0.001).It suggested that the diabetogenic capacity of T cells increased with the age of donor NOD mice.The significant difference between the two groups demonstrated that the expression of costimulatory molecule CD80 on βcells promopt the naive T cells in transferring induction of diabetes.It provided us an evidence of direct interaction between CD8+ T cells and beta cells.2 Eexpression of CD80 on beta cell had no significant effect on the diabetogenic capacity of CD4+ T cellsNaive splenic BDC2.5 CD4+ T cells were isolated from 9-10 day-old BDC2.5/NOD.scid mice and adoptively transferred into 6～8-week-old NOD.scid and NOD.scid.Rip.B7 mice.Diabetes,with identical incidence and kinetics,was observed in both NOD.scid.Rip.B7 and control NOD.scid recipients.Regardless of the numbers of BDC2.5 CD4+ T cells transferred,diabetes was induced in NOD.scid.Rip.B7 and NOD.seid recipients with a similar frequency and kinetics.The result demonstrated that beta cell expression of CD80 had no effect on the diabetogenicity of BDC2.5 CD4+ T cells.3 Beta cell expression of costimulatory molecules CD80 influences the diabetogenic capacity of CD8+ T cells,and CD80 exerts its co-stimulatory function on naive CD8+ T cells during the initiation of islet infiltration.The accelerated onset of diabetes in NOD.scid.Rip.B7 recipients induced by the splenocytes from 4 week-old NOD.Ragnull.AI4 TCR transgenic mice indicates that beta cell expression of costimulatory molecules CD80 enhances the diabetogenic capacity of beta cell-specific CD8+ T cells.However,these naive AI4 CD8+ T cells failed to transfer diabetes in NOD.scid recipients.The incidence of diabetes in NOD.scid.Rip.B7 recipients induced by naive AI4 CDS+ T cells was similar to that of induced by AI4 CD8+ T cells from diabetic or pre-diabetic donors.It suggested that CD8+ T cells directly interaced with beta cells,and CD80 played a costimulatory role on naive CD8+ T cells during the lymphocyte infiltrating islets.4 The initiation of insulitis results from direct interaction between CD8+ T cells and beta cells.Naive AI4 CD8+ T cells were adoptively transferred into NOD.scid.Rip.B7 and NOD.scid.β2mnull.B7 mice that are deficient of MHC class I expression,but still with the expression of CD80 ectopically on beta cells.Diabetes was readily induced in NOD.scid.Rip.B7 mice.In contrast,naive AI4 CD8+ T cells failed to induce diabetes or insulitis in NOD.scid.β2mnull.B7 recipient mice(Fig.21).These results indicated that the loss of MHC classâ… expression on beta cells in NOD.scid.β2mnull.B7 mice rendered incapable of priming naive CD8+ T cells.That is to say,activation of naive CD8+ T cells was in fact the result of a direct interaction with host beta cells expressing MHC classâ… and costimulatory molecule CD80.In summary,a direct interaction between beta cells and CD8+ T cells is required for the initiation of insulitis.5 Naive CD8+ T cells encounter beta cell antigens within the islets.CFSE-labeled CL4 CD8+ T cells were adoptive transferred to NOD.scid.InsHA and NOD.scid mice.By day 4 post-transfer,～50%of CL4 CD8+ T cells isolated from the islets of NOD.scid.InsHA had undergone multiple rounds of division whereas only a few CL4 CD8+ T cells from PLN and spleen showed evidence of proliferation.But in NOD.scid contrast group,significant T cell proliferation was first detected in the spleen and PLN 7 days post-transfer.These data demonstrated that naive CD8+ T cells traffic directly into the islets,encounter cognate autoantigens and initiate insulitis following proliferation and expansion.The innovation1 There has been no report on the role of CD8 + T cells in type 1 diabetes and the mode of its role.Some studies about CD8 T cells and T1D are ongoing abroad, however,there has been no consistent result by now.We explored the autoimmune pathogenesis of type 1 diabetes by studing the role of CD8 + T cells in the triggering of diabetes with adoptive transfer experiments,and found for the first time that the initiation of insulitis results from direct interaction between CD8+ T cells and beta cells.2 We found for the first time that activation of naive CD8+ T cells was in fact the result of a direct interaction between costimulatory molecule CD80 and host beta cells expressing MHC classâ… .3 We found for the first time by CFSE-labelled naive CD8+ T cells that naive CD8+ T cells trafficed directly into the islets and was activated in islets,not in any other lymph organ or tissue like PLN. PART 2 STUDY OF AUTOIMMUNE MECHANISM OF SPORADIC IDIOPATHIC HYPOPARATHYROIDISMObject Idiopathic hypoparathyroidism(IH)includes sporadic IH(SIH),Di Geoge syndrome,familial type 1,familial type 2.Familial type 2 is also called autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy(APECED)or autoimmune polyglandular syndrome type 1(APS-1).The study on autoantibodies of idiopathic hypoparathyroidism(IH)is mainly targeted at the IH patients associated with APS-1.Less study is conducted on autoantibodies of SIH.At least two in three disorders of Addison disease, hypoparathyroidism and chronic skin and mucosal candidiasis disease are referred to as APS 1,that may be companied with other concerned immune diseases.Because the other endocrine components of APS 1(i.e.,APECED)are typical autoimmune diseases,it is natural to assume that this is also true of the HP.While sporadic IH without a family history,nor the autoimmune components of APS-1,the study of APS-1 is no substitute for SIH's.The study on sporadic idiopathic hypoparathyroidism(SIH)is little at home and abroad because this disease is extremely rare.There is no sufficient proof for its autoimmune nature.To explore its autoimmune nature from humoral immunity,we searched for parathyroid autoantibodies in 26 patients with SIH by indirect immunofluorescence techniqes.We also analyzed the expression of lymphocyte subsets in peripheral blood of 10 patients with SIH by flow cytometry and explore its autoimmune nature from cellular immunity.Method1.Patients and control subjects After obtaining informed consent,we enrolled 26 patients with SIH and 60 age-and sex- matched healthy individuals as well as 112 family members of the patients with SIH.None of the patients had a family history suggestive of hypoparathyroidism or adrenal insufficiency or clinical features of mucocutaneous candidiasis infection.The type 1 autoimmune polyglandular syndrome was excluded by demonstrating a normal adrenocorticotrophin(ACTH) value and without clinical features of mucocutaneous candidiasis infection.All sera samples were obtained from subjects after 8-10 hours' fasting during1995 to 2006, kept in -80℃to be detected.Some sera were used to detect calcium,phosphorus, magnesium,intact parathyroid hormone(iPTH),FT3,FT4,TSH,Cortisol,TPOAb, and the rest for detection of anti-parathyroid autoantibodies.Fresh whole blood with ACD anticoagulant were collected from 10 of the 26 patients with SIH,38 of the 112 family members and 12 of the 60 healthy controls,and mononuclear cells were separated for flow cytometry analysis.2.Detection of antibodies against parathyroid Indirect immunofluorescence techniques were used in our study.It was suggested that the antibodies against parathyroid in patients with APECED was a false positive IF results caused by human-specific antimitochondrial antibodies and organ-specific antibodies against parathyroid were extremely rare.To avoid this false positive IF results,we used parathyroid glands from monkey other than from human as substrates.To position the cells of parathyroid glands,we stained the nucleolus with DAPI.The results were firstly observed under fluorescence microscope,if the antibody was positive ina sera titer of 1:10,then repeated the above steps in 1:100 dilution.Then confocal laser scanning microscopy(CLSM)was used to scan and obtain the image.Green fluorescent images(FITC-conjugated antibody)was excitated by the light of 488 nm wavelength,blue fluorescence images(DAPI labeled nuclei)was excitated by the light of 356 nm wavelength(UV).Processing the image with Leica Microsystems Heidelberg GmbH(Leica Confocal Software)analyzing software.3.Detection of Serum calcium,serum phosphorus and serum magnesiumSerum calcium,phosphorus and magnesium were examined respectively with Azoâ…¢arsenium method,ultraviolet direct precipitation method and xylidine chromatometry.4.Detection of Serum FT3,FT4,TSH,Cortisol,TPOAb FT3/FT4 were examined by electrochemiluminescenc immuno-analysis method,iPTH was detected by double antibody sandwich method.5.Analysis of lymphocyte subsets in peripheral blood PBMCs were isolated from the blood with ACD-anticoagulant of patients and healthy donors as well as family members of patients by means of Ficoll density gradient centrifugation.6.Flow cytometryMononuclear cells were isolated from the fresh blood with ACD anticoagulant, stained with antibody fluorescent-labeled.Dual-color flow cytometry was performed using a CellQuest software and Becton Dickinson FACSCalibur instrument. Argon ion laser of 488 nm spectrum as excitation,equipment was calibrated by the use of fluorescent microspheres calibration,adjusted the fluorescence compensation. Isotype-matched controls were used to determine nonspecific background.Cells labeled with one fluorescent antibody and the other isotype control were used to determine the separation threshold of the cytometer channels.Lymphocytes were electronically gated based on their forward scatter(FSC)and side scatter(SSC) characteristics,and then double-positive cells were detected by analyzing at least 10,000 lymphocytes.In these assays,careful color compensation was performed, and their intra-assay reproducibility was corroborated.Obtained cells with CellQuest software.WinMDI2.8 software was used to analyze the data.Results1.General indicator The level of sera iPTH and calcium significantly decreased and sera phosphate significantly increased in 26 patients before treatment compared with the family members and the normal controls.There was no significant difference(P＞0.05)of the indicators above between family members and normal controls.Although there was no significant difference of serum magnesium among the three groups,but the sera magnesium of four patients with SIH were slightly lower than the normal range;The levels of sera FT3,FT4,TSH,Cortisol and TPOAb were all in the normal range of three groups.2.Detection of anti-parathyroid antibody There were 10(8 cases are female)in 26 patients with positive antibodies against parathyroid when sera were diluted in 1:10,the positive rate was 38.46 percent,three were still positive when sera were diluted in 1:100.There were 11 in 112 family members with positive antibodies when sera were diluted in 1:10,the positive rate was 9.82 percent.There were 4 in 60 normal controls with positive antibodies when sera were diluted in 1:10,the positive rate was 6.67 percent.The positive rate in SIH patients were significantly higher than the family members and normal controls(P＜0.01).There was no significant difference between family members and normal controls(P＞0.05). There was no significant difference between patients with positive antibodies and without antibodies in sera calcium,phosphorus,magnesium levels(P＞0.05).There was no significant difference between patients with positive antibodies and without antibodies in gender and age.Although no significantly difference in duration of illness and level of sera iPTH,the patients without antibodies had a longer duration of illness[(4.4±2.07)years]and a lower serum iPTH[(6.0±3.95)pg/ml]than those of in patients with antibodies[(2.6±1.34)years and(9.6±3.77)pg/ml,respectively].3.Detection of lymphocytes phenotype The results of the lymphocyte subsets detected in CD4+,CD8+,ratio of CD4+:CD8+,CD5+,CD19+,ratio of CD5+:CD19+ were similar among the three groups of subjects.There were no significantly difference of the lymphocyte subsets above when compared with each other among three groups.The frequencies of the phenotype of CD4+CD25+ were lower in patients than those of in family members and healthy controls.There was no significant difference between family members and healthy controls(P＞0.05).Conclusion1.Changes of humoral immune function present in patients with sporadic idiopathic hypoparathyroidism.The frequencies of antibody against hypoparathyroid in patients(38.46%)was significantly higher than that of in family members(9.82%)(P＜0.01)and in healthy controls(6.67%)(P＜0.01).It was suggested that humoral immune be activated in patients.In organ-specific autoimmune disease,the patients typically has circulating autoantibodies specific for the affacted organ,often these precede the clinical disease by years and persist for yaers after its onset.This is also true for most of autoimmune components of APS 1,but we did not found in the serum of patients with SIH that autoantibodies against parathyroid had something with the clinical symptoms and the severity of the biochemical abnormalities.2.Changes of cellular immune function present in patients with sporadic idiopathic hypoparathyroidism.CD4+CD25+regulatory T cells(Treg)are the main subset in the peripheral tolerance to self-antigen and the major regulatory T cells responding to foreign antigen.It is important for the maintenance of peripheral tolerance.Some studies showed that both the decrease of number and the dysfunction of CD4 + CD25 + regulatory T cells could lead to autoimmune diseases.We found that the frequencies of CD4 + CD25 + T cells in peripheral blood of SIH patients were lower than the frequencies of family members and healthy controls.It suggested that immunoregulatory cells in patients could not effectively maintain their own tolerance because of the decreased number.The destruction of autoimmune balance in patients made the body easy in pathological immune responses,thus activating immune response to autoimmune diseases.In short,the presence of self-reactive antibodies and the decrease of CD4 + CD25+ regulatory T cells in patients with SIH showed that there were immune function disorders in these patients.In conclusion,serum anti-parathyroid antibody exists in SIH patients demonstrated that humoral immune disorder was present in the disease.Decreased frequency of CD4+CD25+ T regulatory cells support the presence of a cellular immune disturbance in patients with SIH.The innovation1 There has been no report in the world on the expression of CD4+CD25+ T regulatory cells in patients with SIH by now.We explored the pathogenesis of sporadic idiopathic hypoparathyroidism from cellular immunity for the first time.2 We studied the changes of lymphocytes in peripheral blood mononuclear cells of patients with SIH by flow cytometry.There was only one paper about it in the past 20 years in the world.3 We detected anti-parathyroid antibody in the sera of SIH patients by indirect immunofluorescence techniques.No report about this has been seen in China and only little investigation has been conducted in the world.
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