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

摘要 1型糖尿病（T1 D）是儿童和年轻人常见的自身免疫性疾病。T1 D呈现 由于免疫介导的对产生胰岛素的胰腺细胞的破坏而导致急性发作的高血糖症， β细胞T1 D的中心致病驱动因素是β细胞抗原特异性（ag.- sp.）T细胞。没有持久的 治疗T1 D; T1 D的唯一和昂贵的治疗仍然是每日胰岛素替代。即使有警惕的葡萄糖 尽管监测和控制，T1 D患者仍然遭受许多危及生命的后遗症，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： (i)产生和递送胰岛素，（ii）监测血糖，（iii）鉴定自身抗原，（iv）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 胰岛细胞的移植，最棘手的障碍仍然是我们无法删除或控制胰岛 ag.- sp. T细胞，没有它，预防/治愈T1 D的承诺可能会失败。 为了克服这一关键障碍，我们设计了一种方法来消除致糖尿病T细胞， 适应性免疫系统事实上，当应用于非肥胖糖尿病（NOD）小鼠时， 在新发T1 D中，我们观察到（i）缓解期或“蜜月期”显著延长，（ii） β细胞特异性CD 4+和CD 8 + T细胞的减少，（iii）β细胞的显著保存，和（iv）β细胞特异性CD 4+和CD 8 + T细胞的高度减少。 NOD小鼠转变为明显糖尿病的数量显著减少（78%）。 前提：当T细胞在不同的状态之间切换-幼稚，激活效应，静止和激活 记忆-它们表现出我们可以精确定位的不可避免的特性。这是特别真实的激活 效应CD 4+和CD 8 + T细胞（Teff）。与它们的同类不同，Teff细胞分裂迅速--每隔一周分裂一次。 5-6小时-并表现出内在的DNA损伤反应（DDR），使它们处于 细胞凋亡我们假设（i）淋巴细胞生物学的这一独特方面导致基因组应激 在急性活化淋巴细胞中，和（ii）DDR信号传导途径的操纵允许选择性地 治疗靶向病理性T细胞。与这些假设一致，我们发现小鼠和 人Teff细胞显示出明显的DDR，如DNA损伤、磷酸化丝氨酸139 H2 AX（γ H2 AX） 以及ATM、CHK 2和p53的磷酸化。此外，我们发现，新的药物，加强p53（通过 MDM 2的抑制）或受损的细胞周期检查点（通过CHK 1/2或WEE 1的抑制）导致选择性 当在规定的治疗窗期间给予时，在体内消除病理性Teff细胞。组合 这些化合物-我们称之为“p53增强与检查点废除”（PPCA）-显示明确的 治疗益处，靶向病理性T细胞，但不靶向幼稚、调节或静止记忆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， PMC 5474825）提出了一种用于高度选择性形式的免疫治疗的新颖且易处理的临床策略， （i）对CD 4+和CD 8+自身反应性Teff细胞都具有特异性，（ii）具有最低限度的遗传毒性或无遗传毒性，和（iii） 比目前的方法耐受性更好。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发表的数据，我们提出了三个相互关联的假设：（i）， PPCA具有独特的作用机制，消除Teff细胞，同时保留Treg细胞，从而重新激活Teff细胞。 建立局部调节平衡;（ii）PPCA可以靶向控制自体和同种异体T细胞 细胞，从而允许对胰岛的持续移植耐受，和（iii）PPCA可以优先 塔吉特胰岛股份公司在T1 D患者中，sp.激活了人类T细胞，同时保留了记忆区。

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.