Islet Antigen-specific T Cells Assay And Its Application

Objective:Explore the diagnostic value of combined assay of CD4+T cells stimulated by multiple islet autoantigens with enzyme-linked immunospot (ELISPOT) for classification of type1diabetes.Subjects and Methods:According to inclusion and exclusion,43acute-onset type1diabetic patients with duration less than3years were enrolled in endocrine department ward and outpatient clinic. Forty healthy volunteers without diabetes or any other autoimmune diseases were selected as the controls. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque density gradients. And the numbers of IFN-y secreting CD4+T cells responding to glutamic acid decarboxylase (GAD65), C-peptide (CP) and insulin (INS) were detected by ELISPOT assay. Sensitivities and specificities of ELISPOT with different antigens were compared. GAD-Ab, IA2-Ab and ZnT8-Ab were detected by radioligand assays, and C peptides were measured by radioimmunoassay. According to the status of T cell responses and antibodies, these cases were divided into four groups (T+Ab+, T+Ab-, T-Ab+and T-Ab"). Clinical features including body mass index (BMI), fasting C peptides (FCP), post prandial C peptides (PCP) and other metoblic indexs were compared among these groups.Results:1) T1DM patients positive for CD4+T cells response to GAD65or CP were both significantly higher than that in the controls [GAD65:21.4%(9/42) vs.2.5%(1/40), P＜.05; CP:55.5%(20/36) vs.20.0%(8/40), P<0.05]. T cells positive for INS were not statistically different between T1D and the controls [2.4%(1/42) vs.0.0%(0/39), P>0.05].2) Combined GAD65, CP and INS detection, the positivity was60.5%(26/43) in T1DM patients, which was both higher than GAD65and INS (all P<0.001).3) Among the18patients negative for antibodies,11patients were positive for T cell responses. The positivity was83.7%when combing antibodies and T cells assay, which was significantly higher than T cells (60.5%) or antibodies (58.1%) assay (all P<0.05).4) Significant diferences were found for FCP and PCP between T+group andT-group (FCP:33.5vs.117.6, P<0.05; PCP:63.1vs.218.0, P<0.01). FCP and PCP of T+Ab-group were both lower than TAb-group (FCP:35.7vs.255.7; PCP:42.2vs.339.5, both P<0.05). But no differences of clinical features were found between T+Ab-group and T+Ab+, T-Ab+groups.Conclusion:1) Multiple islet antigens combined CD4+-ELISPOT assays could help to improve the diagnostic sensitivity of autoimmune diabetes.2) Part of T1DM patients with islet-antibody negative present islet specific T cell response positive. Combined islet-antibody with CD4+-ELISPOT assay could help improve the diagnostic sensitivity of autoimmune diabetes.3) T1DM patients with T cell response positive had similar clinical features with antibody positive cases that had worse islet (3cell function than T cell negative patients.