AUTOIMMUNE T AND B CELL RESPONSES IN TYPE 1 DIABETES

HLA DR 3、DR 4（0401、0402和0405）、DQ 2和DQ 8转基因小鼠， 非肥胖糖尿病（NOD）背景将用这三种疫苗免疫 主要β胰岛细胞蛋白自身抗原：人前胰岛素原， 人谷氨酸脱羧酶65 Kd和人IA-2。这些老鼠会 对于人CD 4转基因和鼠MHC类也是纯合的 II无效靶向重组第十七染色体（Abeta 0/0）。这将 确保HLA DR上的CD 4阳性T细胞的最佳阳性选择 或DQ限制元件。来自免疫的转基因小鼠的T细胞将被免疫。 与TCR（-）T细胞杂交瘤（BW 5147）融合。Fusion产品将在 选择、亚克隆和用免疫蛋白筛选， 其组成肽，以及单肽，以鉴定肽 每个T细胞杂交瘤的特异性。 然后将这些肽表位提供给合作小组 在华盛顿大学芭芭拉戴维斯糖尿病中心， 威斯康星州医学院，以便他们能够验证是否存在 这些T细胞增殖反应在HLA型糖尿病前期和近期 1型糖尿病患者。来自每种抗原的肽表位， 与所研究的DR或DQ等位基因结合良好，但不引起任何 转基因小鼠的反应，也将用于测试反应， 为了进一步验证肽表位的准确性， 转基因小鼠的分类。 每个肽表位的功能特征将通过以下方法进行测试： 表征细胞因子谱（IL-2，干扰素γ，TNF α， IL-4、IL-10）在转基因小鼠和患者的T细胞中被诱导。 完整蛋白抗原和肽表位。结合尿液 人类研究应该允许设计HLA基因型特异性的 免疫治疗预防 1型糖尿病

HLA DR 3, DR 4 (0401, 0402, and 0405), DQ 2, and DQ 8 transgenic mice on the non-obese diabetic (NOD) background will be immunized with the three principal beta islet cell protein autoantigens: human pre pro-insulin, human glutamic acid decarboxylase 65 Kd, and human IA-2. These mice will also be homozygous for a human CD4 transgene, and for a murine MHC Class II null targeted recombinant seventeenth chromosome (Abeta 0/0). This will ensure optimal positive selection of CD 4 positive T cells on the HLA DR or DQ restricting element. T cells from immunized transgenic mice will be fused with a TCR (-) T cell hybridoma (BW 5147). Fusion products will be selected, subcloned, and screened with the immunizing protein, pools of its component peptides, and single peptides to identify the peptide specificity of each T cell hybridoma. These peptide epitopes will then be provided to the collaborating groups at the Barbara Davis Diabetes Center, the University of Washington, and the Medical College of Wisconsin, so that they can verify the existence of these T cell proliferative responses in HLA type prediabetic and recent onset type I diabetic patients. Peptide epitopes from each antigen which bind well to the DR or DQ allele under study, but do not elicit any response in transgenic mice, will also be used to test for responses in this patient population, to further verify the accuracy of peptide epitope classification in transgenic mice. The functional characteristics of each peptide epitope will be tested by characterizing the cytokine profile (IL-2, interferon gamma, TNF alpha, IL-4, IL-10) elicited in the transgenic mice and in patients' T cells by the intact protein antigen and the peptide epitopes. The combined urine and human studies should permit the design of an HLA genotype specific immunotherapy for the prevention of immunotherapy for the prevention of type I diabetes mellitus.