Molecular Mechanisms and Treatment Of Autoimmunity In Man And Animal Models

在这个项目中，我们正在研究免疫失调疾病的新诊断和治疗方法的发展。首先，我们对三种具有共同发病机制的免疫性疾病进行了缺失治疗：多发性硬化症（MS）、I型糖尿病，以及在血友病治疗中开发针对FVIII的抑制抗体。我们的方法的关键特征是抗原本身将被用来编程特定同源T细胞通过细胞凋亡死亡，通过T细胞的内部调节程序称为再刺激诱导细胞死亡（RICD）。这种死亡是克隆特异性的，代表了一种通过使用特异性抗原过度接种来消除致病T细胞的方法。我们希望在临床试验中使用抗原来治疗自身免疫性疾病，并选择了针对MS的抗原药物，证据表明髓磷脂蛋白抗原是自身免疫性攻击的靶点，但如何特异性决定进展性和复发-缓解型MS的疾病结局尚不清楚。通过编程使识别这些抗原的T细胞死亡，可以证明消除这些细胞对疾病的影响。我们还在研究新的高灵敏度诊断测试，以检测针对这些抗原的反应性T细胞，以确定这些抗原是否可以提供自身免疫攻击的早期预警系统。我们已经开始研究含有可能参与MS的抗原的重组分子，目标是与一家大型制药公司建立合作研究与开发协议（CRADA），以临床测试这种形式的治疗。在过去的一年里，我们有幸被选为NIH NCATS和辉瑞治疗创新中心新协议的牵头项目，实现了这一目标。我们的研究表明，使用一种易于表达的56 kD重组治疗性耐受性蛋白MMPt（包括髓鞘碱性蛋白（MBP）、髓鞘寡脱细胞糖蛋白（MOG）和蛋白脂质蛋白（PLP）的免疫原片段）对T细胞进行抗原特异性缺失，可以安全有效地改善多发性硬化症（MS）的临床前模型。我们发现T细胞被特异性地从脊髓中清除，炎症细胞浸润，细胞因子的产生减少。我们计划根据T细胞对药物的反应性对患者进行分层，然后以重复的方式给药，以去除自身反应性致病T细胞，而不会引起一般的免疫抑制，这是一种广泛适用于免疫疾病的治疗策略。通过短期内联合使用免疫抑制剂，可实现对疾病恶化的安全性，这种抑制剂可阻断幼稚T细胞的激活，但不影响缺失耐受性。我们预计将在2016年秋季正式启动CRADA工作。

In this project, we are investigating the development of new diagnostic and therapeutic approaches to immune dysregulatory diseases. First we have pursued deletional therapy in three immunological diseases that share a common pathogenesis: multiple sclerosis (MS), type I diabetes, and the development of inhibitory antibodies against FVIII in the treatment of hemophilia. The key feature of our approach is that the antigen itself will be used to program the specific cognate T cells to die through apoptosis via an internal regulatory program of T cells termed restimulation-induced cell death (RICD). The death is clonally specific and represents a way to eliminate disease-causing T cells by hyper-vaccinating with their specific antigen. We would like to use antigen to treat autoimmune diseases in a clinical trial and have chosen to focus on an antigenic drug against MS. Evidence suggests that myelin proteins antigens are targets of the autoimmune attack, but how the specificities determine disease outcome in progressive and relapsing-remitting MS is unclear. By programming the T cells that recognize such antigens to die, the effect of eliminating these cells on the disease can be demonstrated. We are also studying new highly sensitive diagnostic tests to detect reactive T cells against these antigens to determine if these can provide an early warning system of autoimmune attack. We have initiated studies of recombinant molecules containing antigens potentially involved in MS with the goal of establishing a Cooperative Research and Development Agreement (CRADA) with a large pharmaceutical company to clinically test such a form of therapy. We have been fortunate in achieving this goal in the past year through being selected as the lead project in a new agreement between the NIH NCATS and the Center for Therapeutic Innovation of Pfizer. Our studies have shown that antigen-specific deletion of T cell using an easily expressed 56 kD recombinant therapeutic tolerogenic protein, MMPt, comprising immunogenic segments of myelin basic protein (MBP), myelin oligodedrocyte glycoprotein (MOG) and proteolipid protein (PLP), can safely and effectively ameliorate disease in preclinical models for multiple sclerosis (MS). we find that T cells are specifically eliminated from the spinal cord and the inflammatory cell infiltrate and cytokine production is decreased. We plan to stratify patients based on T cell reactivity to the drug and then administer the drug in a repeated fashion to remove autoreactive disease-causing T cells without causing a general immunosuppression a widely applicable therapeutic strategy for immune diseases. Safety against disease exacerbation can be achieved by co-administration of immunosupressants in short term which blocks naive T cell activation but does not affect deletional tolerance. We expect to formally launch the CRADA work in fall, 2016. In order to understand the molecular underpinnings of this regular mechanism, we have carried out extensive molecular investigations of how TCR stimulation directs cells to a death pathway instead of simply activation. We have found that cycling T cell blasts become susceptible to restimulation-induced cell death (RICD) by redirecting the NF-B-activating pathway. We carried out a chemical genetics screen that revealed a critical role for PI-3K directly a novel pathway to the induction of I-kappaB kinase (IKK) in transducing a death signal independently of Fas or other tumor necrosis factor superfamily receptors. Interestingly, NF-B itself and new transcription/translation were found to be dispensable for RICD and that activated but not nave T cells are susceptible to death. This is apparently due to the fact that the caspase-3 dimer is partially cleaved but not fully processed in activated cycling T cells blasts suggesting the molecular pathway has been engaged. TCR restimulation then introduces the full processing of caspase-3. Further experiments are being directed at determining the precise substrate of IKK. This may shed light on a key molecular process that will be useful for antigen-induced treatment of autoimmune diseases and, potentially, immunotherapy for cancer. We have also discovered a genetic cause of deficient RICD involving a new class of disorders affecting the regulation of phosphoinositide-3 kinase (PI-3K),a key regulator of cell proliferation in both normal and malignant cells. We have discovered patientswho are heterozygous for mutations in the leukocyte-restricted PIK3CD gene encoding the p110delta catalytic PI3K subunit and suffer from a unique disorder we have termed "p110delta activating mutations causing Senescent T cells, Lymphadenopathy, and Immunodeficiency (PASLI) disease. The p110delta catalytic subunit of phosphoinositide 3-kinase (PI3K) has been found to be selectively expressed in leukocytes and critical for lymphocyte differentiation, growth, and survival by pharmacologic and genetic inactivation in experimental animals. We discovered the first germline, heterozygous, dominant, gain-of-function mutations in the p110delta catalytic subunit of PI3K in 9 patients from 7 unrelated families. These patients' clinical presentation comprised sinopulmonary infections, EBV viremia, lymphadenopathy, nodular lymphoid hyperplasia at mucosal surfaces, and lymphoma. Our laboratory studies showed that patient T cells exhibited defective response to antigen receptor stimulation despite constitutive activation of the PI3K signalling system. We found that patients were lacking in long-lived central memory T cells and instead exhibited A surfeit of short-lived effector/TEM cells. As expected from a TEM phenotype, proliferation and IL-2 secretion were diminished while effector functions, including granzyme expression, IFN-gamma secretion, and degranulation, were elevated. TCR signaling was intact; however, hyper-activation of mTOR caused changes in cellular metabolism that were characteristic of terminal differentiation and senescence. Importantly, treatment of patients with The FDA-approved drug rapamycin to inhibit mTOR activity in vivo partially restored appearance of naive T cells in the peripheral blood, decreased the number of senescent T cells, and largely rescued the T cell activation defects. we next wanted to test specific p110delta enzyme inhibitors in PASLI patients and we have entered into a Cooperative Research and Development Agreement with Novartis to carry out a clinical trial testing their inhibitor of p110delta, a compound called CDZ173, in the NIH clinical Center. Preliminary evidence shows that CDZ173 is a potent and selective inhibitor of p110delta and can inhibit the wild-type as well as overactive forms of the enzyme. A clinical trial is currently ongoing at NIH. PI3K exists in the cell as a holocomplex of a p110 catalytic subunit bound to a various regulatory subunits: p85alpha, p55alpha, and p50alpha, which are important for p110 stability, inhibition, and recruitment to signaling locations on inner leaflet of the membrane. We recently discovered 4 patients with a PASLI-like disease with heterozygous splice site mutations in PIK3R1, a ubiquitously expressed gene encoding the PI3K regulatory subunits. Similar to other PASLI patients, PIK3R1 mutant patients suffer from recurrent sinopulmonary infections and lymphoproliferation, have increased PI3K signaling, and have expansion and skewing of peripheral blood CD8+ T cells towards a terminally differentiated, senescent effector T cell phenotype. The PIK3R1 splice mutation resulting in the deletion of 42 amino acids in a critical region of the protein. The mutant proteins are expressed, albeit at low levels, in patient T cells and are associated with increased PI3K signaling. These results suggest that this new group of patients may also benefit from CDZ173.