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。以这些细胞为靶点而不产生全身性免疫抑制的安全疗法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，我们已经证明了来自1型糖尿病(T1 DM)、类风湿性关节炎(RA)和多发性硬化症(MS)患者的疾病相关T细胞表达高水平的Kv1.3钾通道，是CCR7-CD45RA效应记忆(TEM)T细胞。我们已经开发了高度特异的Kv1.3通道抑制剂，这些抑制剂优先抑制自身抗原特异的Kv1.3(高)TM细胞的细胞因子的产生和增殖，而不影响其他T细胞。此外，Kv1.3抑制剂通过限制β细胞的破坏，降低了BB-WOR大鼠自发发展的实验性自身免疫性DM的发生率，并在没有相关毒性的情况下改善了MS和RA大鼠模型的疾病。在这项探索性的R21拨款中，我们将检验这一假设，即“Kv1.3HighTEM型”是疾病相关自身反应性T细胞的功能生物标记物，将被证明有助于监测高危人群的疾病进展，并跟踪治疗期间的自身免疫反应。在AIM-1中，我们将研究新发T1 DM患者的GAD65(274-286)、IGRP(247-258)或胰岛素(A1-15)特异性的CD4+T细胞以及胰岛素(B10-18)、pplAPP(5-13)和IGRP(152-160)的五聚体分选CD8+T细胞是否表现出Kv1.3高CCR7-CD45RA-TEM型模式。在AIM-2中，提供最一致结果的多聚体将用于筛查新发T1 DM患者(确诊后1年)、已确诊糖尿病患者(5年)、T1 DM(抗胰岛自身抗体阳性)高危人群和存在多聚体+Kv1.3高透射电子显微镜细胞的健康对照组受试者的血液样本。被发现对疾病进展具有预测性或信息性的标记物将(在后续研究中)用于评估从参与正在进行的临床试验和免疫治疗干预的人类白细胞抗原-DRB1-0401阳性个体获得的血液样本。在AIM-3中，我们将测试Kv1.3抑制剂是否同样有效地抑制高亲和力和低亲和力自身抗原特异性多聚体+细胞的细胞因子的产生和增殖。如果Kv1.3特异性阻滞剂要被开发成治疗药物，这项实验具有重要的意义。

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.