B-Lymphocyte Immunotherapy in Islet Transplantation

描述（由申请人提供）：糖尿病控制和并发症试验（DCCT）证实，1型糖尿病（T1D）患者可以通过维持接近正常的血糖控制来预防糖尿病的微血管并发症。这一开创性的结果为T1D患者通过胰腺或孤立胰岛移植进行细胞替代提供了强有力的推动力。最近“埃德蒙顿协议”的成功是胰岛移植领域的一个重大进展。然而，尽管最初达到了胰岛素独立状态，但β细胞功能的逐渐丧失最终导致大多数受体糖尿病复发。因此，仅针对T淋巴细胞室的埃德蒙顿方法似乎不足以预防免疫排斥和复发性抗-细胞自身免疫。尽管T淋巴细胞在这两个过程中起着关键作用，但已经确定B淋巴细胞对β细胞衍生的同种异体抗原和自身抗原表位也会产生伴随的特异性反应。在啮齿动物研究中，我们证明了B淋巴细胞功能的中断减少了T细胞介导的胰岛同种异体移植物的破坏。因此，我们假设针对B和T淋巴细胞区室的诱导免疫治疗方案将促进对胰岛异体移植物的免疫耐受。我们的临床前研究在非人灵长类动物（NHPs）中验证了这一前提，并表明联合使用抗b和t淋巴细胞抗体（即利妥昔单抗和胸腺球蛋白）可以在不需要钙调磷酸酶抑制剂（CNI）药物维持治疗的情况下导致长期的胰岛同种异体移植存活。在目前的应用中，我们建议确定联合B淋巴细胞和T淋巴细胞定向免疫治疗对促进T1D患者对胰岛同种异体移植的免疫耐受的疗效。我们将启动一项临床试验，该试验将：1)基于我们在“埃德蒙顿方案”中的经验，将B淋巴细胞特异性单克隆抗体利妥昔单抗纳入其诱导方案；2)评估包括胸腺球蛋白和利妥昔单抗在内的联合诱导方案的疗效，随后使用雷帕霉素进行无cni维持单药治疗。重要的是，后一种方案与我们临床前NHP研究中使用的方案相似，将允许将微量白蛋白尿T1D患者纳入胰岛移植试验。将进行一系列前瞻性体内代谢研究，专门评估移植后的β细胞功能和分泌能力，1)包括利妥昔单抗，2)消除CNI药物。我们的机制研究旨在验证利妥昔单抗免疫治疗为重建B淋巴细胞库提供了一个“耐受性窗口”的假设，在此期间，具有过渡性表型的同种和自身反应性克隆受到负选择的影响。胰岛细胞移植后，我们将：1)监测针对HLA抗原的同种抗体和针对胰岛抗原的自身抗体的发展，2)利用抗体CDR3谱型分析免疫球蛋白库的克隆持久性和异质性，3)利用ELISpot调查与胰岛β细胞反应的自身抗原特异性T细胞的细胞因子谱，4)对循环淋巴细胞进行免疫分型和功能分析，以评估淋巴细胞分化和能量的整体变化。总的来说，拟议的研究将确定针对B和T淋巴细胞区室的平衡免疫治疗方案是否促进对胰岛异体移植物的免疫耐受状态，同时避免慢性免疫抑制的需要。

DESCRIPTION (provided by applicant): The Diabetes Control and Complications Trial (DCCT) established that the microvascular complications of diabetes can be prevented by maintaining near normal glycemic control in patients with type 1 diabetes (T1D). This seminal outcome has provided a strong impetus for developing effective tolerogenic strategies for a cell replacement via pancreas or isolated islet transplantation in T1D patients. The recent success of the "Edmonton protocol" was a major advance in the field of islet transplantation. However, despite initial achievement of an insulin independent state, a progressive loss of beta cell function culminates in the recurrence of diabetes in a majority of these recipients. Thus, the Edmonton approach, which targets the T lymphocyte compartment alone, seems insufficient in preventing immunological rejection and recurrent anti-beta cell autoimmunity. Despite the critical role of T lymphocytes in these two processes, it is established that a concomitant and specific B lymphocyte response against beta-cell derived allo- and auto-antigenic epitopes also occurs. In rodent studies we demonstrated that interruption of B lymphocyte function curtails the T cell mediated destruction of islet allografts. Thus, we hypothesize that an induction immunotherapy regimen which targets both the B- and T lymphocyte compartments, will promote immunological tolerance to islet allografts. Our preclinical studies test this premise in non-human primates (NHPs) and indicate that the combined use of anti-B and T-lymphocyte antibodies (i.e., Rituximab and Thymoglobulin) results in long-term islet allograft survival without the need for maintenance therapy with a calcineurin inhibitor (CNI) agent. In the present application we propose to determine the efficacy of combined B- and T- lymphocyte directed immunotherapy for promoting immunological tolerance to islet allografts in T1D patients. We will initiate a clinical trial which will: 1) build upon our experience with the "Edmonton protocol", by incorporating the B lymphocyte specific monoclonal antibody, Rituximab, into its induction regimen and 2) assess the efficacy of a combined induction regimen including Thymoglobulin and Rituximab followed by CNI-free maintenance monotherapy with Rapamycin. Importantly, the latter protocol, which parallels that used in our preclinical NHP studies, will permit the inclusion of T1D patients with microalbuminuria into islet transplantation trials. A series of prospective in vivo metabolic studies will be undertaken to specifically evaluate beta cell function and secretory capacity following transplantation that 1) includes Rituximab and 2) eliminates CNI agents. Our mechanistic studies are designed to test the hypothesis that Rituximab immunotherapy provides a "tolerogenic window" for the reconstituting B lymphocyte repertoire, during which allo- and auto-reactive clones with a transitional phenotype are subject to negative selection. Following islet cell transplantation we will: 1) monitor the development of alloantibodies to HLA antigens and autoantibodies to islet antigens, 2) analyze the immunoglobulin repertoire for clonal persistence and heterogeneity using antibody CDR3 spectratyping, 3) survey the cytokine profiles of autoantigen-specfic T cells reactive to islet beta-cells by ELISpot, and 4) perform immunophenotyping and functional assays of circulating lymphocytes to assess global alterations in lymphocyte differentiation and energy. Overall, the proposed studies will determine whether a balanced immunotherapy regimen targeting the B- and T- lymphocyte compartments promotes a state of immunological tolerance to islet allografts, while obviating the need for chronic immunosuppression.