Manipulating DNA Damage-response Signaling for the Treatment of Type 1 Diabetes

摘要 1型糖尿病(T1D)是儿童和年轻人常见的自身免疫性疾病。T1D礼物 作为免疫介导的胰岛素分泌胰腺破坏所致的急性起病高血糖 β细胞。T1D的中心致病因素是β细胞抗原特异性(ag.-sp.)T细胞。没有经久耐用的 治愈T1D；T1D的唯一和昂贵的治疗方法仍然是每日胰岛素替代。即使有警觉的葡萄糖 监测和控制，T1D患者仍然遭受着一系列危及生命的后遗症，包括宏观和微观的 血管病变、神经病变、肾病、截肢、中风和失明。虽然在以下方面取得了进展 (一)生产和提供胰岛素，(二)监测血糖，(三)识别自身抗原，(四)确定 遗传风险因素，(V)了解潜在的免疫功能障碍，以及(Vi)生产和收获 胰岛细胞用于移植，最棘手的障碍仍然是我们无法移除或控制胰岛 AG.-sp.如果没有T细胞，预防/治愈T1D的承诺很可能会失败。 为了克服这一关键障碍，我们设计了一种方法，从 适应性免疫曲目。事实上，当应用于非肥胖糖尿病(NOD)小鼠时，自发性 新发的T1D，我们观察到(I)缓解期或“蜜月期”显著延长，(Ii)显著 减少β细胞特异性的CD4+和CD8+T细胞，(Iii)显著保存β细胞，和(Iv)高度 显著减少(78%)过渡到显性糖尿病的NOD小鼠的数量。 前提：当T细胞在不同的状态之间切换-幼稚、激活的效应器、静止的和激活的 记忆--它们表现出我们可以精确瞄准的不可避免的特性。对于激活的情况更是如此 效应器CD4+和CD8+T细胞(T细胞)。与它们的同类细胞不同，TEF细胞分裂迅速--以每隔一次的速度分裂 在体内5-6小时-并表现出内在的DNA损伤反应(DDR)，使他们处于 细胞凋亡性死亡我们假设(I)淋巴细胞生物学的这一独特方面导致了基因组压力 在急性激活的淋巴细胞中和(Ii)DDR信号通路的操纵允许选择性 以病理性T细胞为靶点的治疗。与这些假设一致，我们发现老鼠和 人TEF细胞表现出明显的DDR，如DNA损伤，磷酸丝氨酸139 H_2AX(γH_2AX)， 以及ATM、Chk2和P53的磷酸化。此外，我们发现增强P53的新药(通过 抑制MDM2)或损害细胞周期检查点(通过抑制CHK1/2或WEE1)导致选择性 在规定的治疗窗口内给药可消除体内的病理性TJeff细胞。结合在一起 在这些化合物中--我们称之为“P53增强与检查点消除”(PPCA)--显示出清晰的 治疗益处，靶向病理性T细胞，但不是幼稚的、调节性的或静止的记忆T细胞 池，并具有适度的非免疫毒性特征。这些结果，最近发表，(PNAS 2017， PMC5474825)为一种高度选择性的免疫疗法提供了一种新的、易处理的临床策略 是(I)对CD4+和CD8+自身反应性T细胞有特异性，(Ii)最小或无遗传毒性，以及(Iii) 其耐受性明显好于目前的方法。重要的是，这种方法不会改变组织驻留 Treg细胞数量；事实上，我们的数据表明，PPCA重置了调节平衡，有利于Treg控制 抗β细胞免疫。 根据我们的初步和已公布的数据，我们提出了三个相互关联的假设：(I) PPCA有一个独特的作用机制，它消除了TJeff细胞，同时保留了Treg细胞，从而重新 建立局部的监管平衡；(Ii)PPCA可以针对自体和异体T细胞的控制 细胞，从而允许对胰岛的持续移植耐受，以及(Iii)PPCA可以优先 目标胰岛股份公司。在保留记忆隔间的情况下，激活了患有T1D的人的T细胞。

Abstract Type 1 diabetes (T1D) is a common autoimmune disease in children and young adults. T1D presents as acute onset hyperglycemia resulting from the immune-mediated destruction of insulin-producing pancreatic beta cells. The central pathogenic driver of T1D is the beta cell antigen-specific (ag.-sp.) T cell. There is no durable cure for T1D; the sole and costly treatment for T1D remains daily insulin replacement. Even with vigilant glucose monitoring and control, T1D patients still suffer a host of life-threatening sequalae including macro- and micro- vasculopathies, neuropathy, nephropathy, amputations, stroke, and blindness. While progress has been made in (i) producing and delivering insulin, (ii) monitoring blood glucose, (iii) identifying autoantigens, (iv) defining genetic risk factors, (v) understanding underlying immune dysfunction, and (vi) producing and harvesting pancreatic islet cells for transplant, the most intractable barrier remains our inability to remove or control islet ag.-sp. T cells, without which the promise of preventing/curing T1D will likely fail. To surmount this critical barrier, we devised the means to eliminate diabetogenic T cells from the adaptive immune repertoire. In fact, when applied to non-obese diabetic (NOD) mice with spontaneous new-onset T1D, we observe (i) a striking prolongation of the remission or “honeymoon” period, (ii) a significant reduction in beta cell-specific CD4+ and CD8+ T cells, (iii) a significant preservation of beta cells, and (iv) a highly significant reduction (78%) in the number of NOD mice that transit to overt diabetes. The premise: As T cells toggle between distinct states – naïve, activated effector, quiescent and activated memory – they exhibit ineluctable properties that we can precisely target. This is particularly true of activated effector CD4+ and CD8+ T cells (Teff). Unlike their counterparts, Teff cells divide rapidly – at a rate of once every 5-6 hours in vivo – and exhibit an intrinsic DNA damage response (DDR) that places them on the edge of apoptotic cell death. We hypothesize (i) that this unique aspect of lymphocyte biology lead to genomic stress in acutely activated lymphocytes and (ii) that manipulation of DDR signaling pathways allows for selective therapeutic targeting of pathological T cells. Consistent with these hypotheses, we find that both mouse and human Teff cells display a pronounced DDR, as evidenced by DNA damage, phospho-ser139 H2AX (γH2AX), and phosphorylation of ATM, CHK2, and p53. Moreover, we find that novel drugs that potentiate p53 (via inhibition of MDM2) or impair cell cycle checkpoints (via inhibition of CHK1/2 or WEE1) lead to the selective elimination of pathological Teff cells in vivo when given during a prescribed therapeutic window. In combination of these compounds – which we termed “p53 potentiation with checkpoint abrogation” (PPCA) – display clear therapeutic benefit, targeting pathological T cells but does not naive, regulatory, or quiescent memory T-cell pools, and has a modest nonimmune toxicity profile. These results, recently published, (PNAS 2017, PMC5474825) suggest a novel and tractable clinical strategy for a highly selective form of immune therapy that is (i) specific for both CD4+ and CD8+ auto-reactive Teff cells, (ii) minimally or non-genotoxic, and (iii) markedly better tolerated than current approaches. Importantly, this approach does not alter tissue-resident Treg cell numbers; in fact, our data suggest that PPCA resets the regulatory balance in favor of Treg control of anti-beta cell immunity. Based on our preliminary and published data, we propose three inter-related hypotheses: (i) that PPCA has a distinct mechanism of action that eliminates Teff cells while sparing Treg cells, thereby re- establishing a localized regulatory balance; (ii) that PPCA can target the control of both auto- and allogeneic T cells, thereby allowing for sustained transplantation tolerance to islets, and (iii) that PPCA can preferentially target islet ag.-sp. activated human T cells in individuals with T1D while sparing the memory compartment.