B-Lymphocyte Immunotherapy in Islet Transplantation

描述(申请人提供)：糖尿病控制和并发症试验(DCCT)证实，通过对1型糖尿病(T1D)患者保持接近正常的血糖控制，可以预防糖尿病的微血管并发症。这一开创性的结果为开发有效的耐受性策略提供了强大的推动力，可用于T1D患者的经胰腺或孤立胰岛移植的细胞替代。最近“埃德蒙顿方案”的成功是胰岛移植领域的一大进步。然而，尽管最初实现了胰岛素非依赖状态，但在大多数受者中，β细胞功能的进行性丧失最终导致糖尿病的复发。因此，仅针对T淋巴细胞室的Edmonton方法似乎不足以预防免疫排斥反应和反复出现的抗β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，但已经证实，伴随的、特异性的B淋巴细胞对β细胞来源的同种和自身抗原表位的反应也会发生。在啮齿动物研究中，我们证明了B淋巴细胞功能的中断会抑制T细胞介导的同种异体胰岛移植物的破坏。因此，我们假设，同时针对B和T淋巴细胞室的诱导免疫治疗方案将促进对同种异体胰岛移植的免疫耐受。我们的临床前研究在非人类灵长类动物(NHP)中测试了这一前提，并表明联合使用抗B和T淋巴细胞抗体(即利妥昔单抗和胸球蛋白)可以导致同种异体胰岛移植物长期存活，而不需要钙调神经磷酸酶抑制剂(CNI)的维持治疗。在目前的应用中，我们建议确定联合B和T淋巴细胞定向免疫疗法在促进T1D患者同种异体胰岛移植免疫耐受方面的有效性。我们将启动一项临床试验，该试验将：1)在“埃德蒙顿方案”经验的基础上，将B淋巴细胞特异性单抗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，然后用雷帕霉素进行CNI非维持性单一疗法。重要的是，后一种方案与我们临床前NHP研究中使用的方案类似，将允许将有微量白蛋白尿的T1D患者纳入胰岛移植试验。将进行一系列前瞻性的体内代谢研究，以具体评估移植后的β细胞功能和分泌能力，其中1)包括利妥昔单抗，2)消除CNI药物。我们的机制研究旨在检验这样一种假设，即利妥昔单抗免疫疗法为重建的B淋巴细胞库提供了一个“耐受窗口”，在此期间，具有过渡性表型的同种和自身反应克隆受到负面选择。在胰岛细胞移植后，我们将：1)监测抗人类白细胞抗原同种异体抗体和自身抗体的发展；2)用抗体CDR3分型分析免疫球蛋白的克隆持久性和异质性；3)用ELISpot检测与胰岛β细胞反应的自身抗原特异性T细胞的细胞因子谱；4)进行循环淋巴细胞的免疫表型和功能分析，以评估淋巴细胞分化和能量的整体变化。总体而言，拟议的研究将确定针对B-和T-淋巴细胞室的平衡免疫疗法是否能促进对同种异体胰岛移植物的免疫耐受状态，同时消除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.