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。以这些细胞为靶点的安全疗法，不会产生普遍的免疫抑制，将具有巨大的医学价值。使用单细胞膜片钳分析结合共聚焦显微镜和流式细胞术，我们已经证明来自1型糖尿病（T1DM）、类风湿性关节炎（RA）和多发性硬化症（MS）患者的疾病相关T细胞表达Kv1.3钾通道水平升高，并且是CCR7-CD45RA效应记忆（TEM） T细胞。我们已经开发出高度特异性的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，同时保护其他T细胞。此外，Kv1.3抑制剂通过限制β细胞破坏减少BB-WOR大鼠自发发生的实验性自身免疫性糖尿病的发生率，并在MS和RA大鼠模型中改善疾病而无相关毒性。在这项探索性的R21资助中，我们将测试“Kv1.3highTEM表型”是疾病相关的自身反应性T细胞的功能性生物标志物的假设，并将证明它有助于监测高风险人群的疾病进展，并在治疗期间跟踪自身免疫反应。在AIM-1中，我们将检测新发T1DM患者的CD4+ T细胞特异性GAD65（274-286）、IGRP（247-258）或胰岛素（A1-15）和五聚体分类CD8+ T细胞特异性胰岛素（B10-18）、pplAPP（5-13）和IGRP新表位（152-160）是否表现出Kv1、3highCCR7-CD45RA- TEM模式。在AIM-2中，给出最一致结果的多聚体将用于筛查来自新发T1DM（诊断后<1年）、已确诊糖尿病（病程≥5年）、T1DM风险个体（抗胰岛自身抗体阳性）和健康对照受试者的血液样本，以检测多聚体+ kv1.3高TEM细胞的存在。在随后的研究中，发现在疾病进展中具有预测性或信息性的标记物将用于评估参与正在进行的免疫治疗干预临床试验的hla - drb1 -0401阳性个体的血液样本。在AIM-3中，我们将测试Kv1.3抑制剂是否同样有效地抑制细胞因子的产生和高、低亲和性自身抗原特异性多时间+细胞的增殖。如果要开发kv1.3特异性阻滞剂作为治疗药物，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.