Kv1.3 channels: functional biomarker and therapeutic target for Type-1 Diabetes

描述（由申请人提供）：自身反应性记忆T淋巴细胞与自身免疫性疾病的发病机制有关。靶向这些细胞而没有全身免疫抑制的安全疗法将具有巨大的医疗价值。使用单细胞膜片钳分析结合共聚焦显微镜和流式细胞术，我们已经证明，疾病相关的T细胞从1型糖尿病（T1 DM），类风湿性关节炎（RA）和多发性硬化症（MS）患者表达水平升高的Kv1.3钾通道，是CCR 7-CD 45 RA效应记忆（TEM）T细胞。我们已经开发了高度特异性的Kv1.3通道抑制剂，这些抑制剂优先抑制细胞因子的产生和自身抗原特异性Kv1.3（高）TEM细胞的增殖，同时保留其他T细胞。此外，Kv1.3抑制剂通过限制β-细胞破坏降低BB-WOR大鼠中自发发展的实验性自身免疫性DM的发生率，并且它们改善MS和RA大鼠模型中的疾病而没有相关毒性。在这项探索性的R21资助中，我们将检验“Kv1.3highTEM表型”是疾病相关自身反应性T细胞的功能性生物标志物的假设，并将证明其对监测高危人群的疾病进展以及跟踪治疗期间的自身免疫反应是有用的。在AIM-1中，我们将检查来自新发T1 DM患者的对GAD 65（274-286）、HRP（247-258）或胰岛素（A1-15）特异性的CD 4 + T细胞和对胰岛素（B10-18）、pplAPP（5-13）和新的HRP表位（152-160）特异性的五聚体分选的CD 8 + T细胞是否表现出Kv 1，3高CCR 7-CD 45 RA-TEM图谱。在AIM-2中，将使用得到最一致结果的多聚体筛选新发T1 DM患者（诊断后<1年）、确诊糖尿病患者（病程>5年）、有发生T1 DM风险的个体（抗胰岛自身抗体阳性）和健康对照受试者的血液样本中是否存在多聚体+Kv1.3高TEM细胞。发现在疾病进展中具有预测性或信息性的标志物将用于（在后续研究中）评估从参与正在进行的免疫干预临床试验的HLA-DRB 1 -0401阳性个体获得的血液样本。在AIM-3中，我们将测试Kv1.3抑制剂是否以相同的有效性抑制细胞因子的产生和高亲和力和低亲和力自身抗原特异性多聚体+细胞的增殖。如果Kv1.3特异性阻断剂被开发为治疗药物，该实验具有重要意义。

DESCRIPTION (provided by applicant): Autoreactive memory T-lymphocytes are implicated in the pathogenesis of autoimmune diseases. Safe therapies that target these cells without generalized immunosuppression would have immense medical value. Using single-cell patch-clamp analysis in conjunction with confocal microscopy and flow cytometry, we have demonstrated that disease-associated T cells from patients with type-1 diabetes mellitus (T1DM), rheumatoid arthritis (RA) and multiple sclerosis (MS) express elevated levels of the Kv1.3 potassium channel and are CCR7-CD45RA- effector memory (TEM) T cells. We have developed highly specific Kv1.3 channel inhibitors and these inhibitors preferentially suppress cytokine production and proliferation of autoantigen-specific Kv1.3(high) TEM cells while sparing other T cells. Moreover, Kv1.3 inhibitors reduce the incidence of spontaneously developing experimental autoimmune DM in BB-WOR rats by limiting beta-cell destruction and they ameliorate disease in rat models of MS and RA without associated toxicity. In this exploratory R21 grant, we will test the hypothesis that the "Kv1.3highTEM phenotype" is a functional biomarker of disease-associated autoreactive T cells and will prove useful to monitor disease progression in people at high risk and also track autoimmune responses during therapy. In AIM-1 we will examine whether CD4+ T cells specific for GAD65 (274-286), IGRP (247-258) or insulin (A1-15) and pentamer-sorted CD8+ T cells specific for insulin (B10-18), pplAPP (5-13) and a novel epitope of IGRP (152-160) from patients with new-onset T1DM exhibit the Kv1,3highCCR7-CD45RA- TEM pattern. In AIM-2, multimers that give the most consistent results will be used to screen blood samples from patients with new onset T1DM (<1 year post diagnosis), established diabetes (>5 year duration), individuals at-risk for developing T1DM (anti-islet autoantibody positive) and healthy control subjects for the presence of multimer+ Kv1.3high TEM cells. Markers that are found to be predictive or informative in disease progression will be used (in subsequent studies) to assess blood samples obtained from HLA-DRB1-0401-positive individuals participating in ongoing clinical trials with immunotherapeutic interventions. In AIM-3 we will test whether Kv1.3 inhibitors suppress cytokine production and proliferatiion of high- and low-avidity autoantigen-specific multimer+ cells with equal effectiveness. This experiment has important implications if Kv1.3-specific blockers are to be developed as therapeutics.