Characteristics And Clinical Value Of Self - Reactive T Cells In Type 1 Diabetes Mellitus Patients

Part I Identification of HLA-A*0201 Restricted T Cell Epitopes for Zn T8 Islet Cell Autoantigens with ELISPOT in Type1 Diabetes Objective: To screen HLA-A*0201 restricted β-cell epitopes derived from zinc transporter eight(Zn T8), and evaluate the value of autoreactive T cells detection in clinical application. Methods: We implemented ELISPOT(Enzyme-linked Immunospot Assay) to detect the responses of interferon-γ secreting CD8+ T cells against islet-specific Zn T8 epitopes in 16 HLA-A2+ type 1 diabetic patients and 10 healthy controls. Results: 16 patients reacted against at least one epitopes of Zn T8. There were average 6(1-6) epitopes inducing positive reactivation of autoreactive T cells in patients. 7 patients reacted more than 2 epitopes. The positive rates of No.2, No.3, No.6 and No.9 epitopes were above 30%. 9 of 10 healthy controls did not react against any epitopes which were significantly different from T1DM(P < 0.001). Conclusions:(1) Zn T8 autoantigens may be one of initiate factors inducing autoimmune attack.(2) The detection of pathogenic T cells played an important role in early diagnosis, differential diagnosis, disease prediction, and evaluation the degree of β cell function depletion.Part II CD8+T-Cell Responses Identify β-Cell Autoimmunity in Human Type 1 Diabetes Objective: To identify autoreactive CD8+T cells in human type 1 diabetes and evaluate the value of autoreactive T cells detection in clinical application. Methods: Taking advantage of a panel of 28 HLA-A2–restricted CD8+T-cell epitopes derived from preproinsulin(PPI), 65 k Da glutamic acid decarboxylase(GAD), islet glucose-6-phosphatase catalytic subunit-related protein(IGRP),insulinoma associated protein 2(IA-2), islet amyloid polypeptide(IAPP), glial ?brillary acidic protein(GFAP),zinc transporter eight(Zn T8), we have implemented an enzyme-linked immunospot(ELISPOT) assay to detect the responses of interferon-γ secreting CD8+ T cells against islet-specific 28 epitopes in 16 HLA-A2+ type 1 diabetic patients, 5 HLA-A2+ questioned type 1 diabetes and 3 HLA-A2+ type 2 diabetes. Results:(1)11 of 12 recent-onset type 1 diabetic patients reacted against at least one epitope. There were average 3(0-26) epitopes inducing positive reactivation of autoreactive T cells in patients. 4 patients reacted more than 10 epitopes. No.4, No.10 epitopes were recognized by ≥50% of recent-onset type 1 diabetics patients.When the analysis is restricted to No.4, No.10, No.25 epitopes, the same type 1 diabetic patients are correctly identi?ed without any loss in sensitivity and speci?city. 3 of 4 long-standing type 1 diabetes reacted against at least one epitope. There were average 1.5(0-4) epitopes inducing positive reactivation.(2)3 of 5 questioned type 1 diabetes reacted against at least one epitopes.(3) None of type 2 diabetes reacted against any epitopes which were significantly different from T1DM(P < 0.05). Conclusion:(1)No.4 and No. 10 are two immunodominant epitopes in recent-onset type 1 diabetes;(2)Positive ELISPOT responses separate type 1 diabetes and type 2 diabetes with high accuracy(87.50% sensitivity, 100% speci?city);(3)Use as few as three immunodominant epitopes can achieve the same high accuracy; Moreover, sensitivity reaches 100% when the ELISPOT assay is complemented by antibody determinations.