Stem Cell Therapies for Primary Immune Deficiency

该项目的总体目标是开发新的干细胞移植和基因治疗方法，以在严重联合免疫缺陷（SCID）和其他原发性免疫缺陷患者中产生完整的免疫系统。对严重免疫缺陷SCID患者的干细胞和祖细胞的操作为揭示干细胞植入、淋巴生成和免疫重建的机制提供了独特的生物学和临床环境。该项目的中心假设是造血移植的年龄和宿主环境影响SCID患者免疫系统的最终重建。每个项目都将从不同和互补的角度检验这一假设。项目1 （Crooks）的目标是描述胸腺血管生态位在骨髓移植（BMT）后新生儿胸腺归巢调节中的作用。项目2 （Dorshkind）将提供有关胎儿B细胞发育和体液免疫反应发展的新信息，特别关注B-1 B细胞的个体发生。这一信息将与理解BMT和基因治疗后体液免疫的恢复高度相关。该项目汇集了来自加州大学洛杉矶分校经验丰富的合作研究团队，并通过行政核心促进了互动。两个科学核心将通过提供细胞和组织分离和分析以及移植动物模型方面的专业知识来支持这些项目。这些项目和核心将为SCID（最致命的原发性免疫缺陷）干细胞治疗的基础和临床研究提供协同和重点。

The overall goal of this Program is to develop novel stem cell transplantation and gene therapy approaches to produce an intact immune system in patients with Severe Combined Immune Deficiency (SCID) and other Primary Immune Deficiencies. The manipulation of stem and progenitor cells in the profoundly immune deficient patient with SCID provides a unique biological and clinical setting to unravel mechanisms of stem cell engraftment, lymphopoiesis and immune reconstitution. The central hypothesis of the Program is that the age of the hematopoietic graft and the host environment influences the ultimate reconstitution of the immune system in the patient with SCID. Each of the Projects will test this hypothesis from a different and complementary perspective. The goal of Project 1 (Crooks) is to delineate the role of the thymic vascular niche in the regulation of homing to the neonatal thymus after bone marrow transplantation (BMT). Project 2 (Dorshkind) will provide new information regarding fetal B cell development and the development of the humoral immune response, with a specific focus on the ontogeny of B-1 B cells. This information will be highly relevant to understanding the restoration of humoral immunity following BMT and gene therapy. The Program brings together an experienced and collaborative team of investigators from UCLA, with interactions facilitated through an Administrative Core. Two scientific cores will support the Projects by providing specialized expertise in cell and tissue isolation and analysis, and in animal models of transplantation. These Projects and Cores will provide synergy and focus for basic and clinical studies in Stem Cell therapies for SCID, the most lethal form of Primary Immune Deficiency. Project 1: Thymic Reconstitution and the Vascular Niche (Gay Crooks, M.D.) Revised Abstract Section (No Change) Studies of age-related changes in thymic function and reconstitution after bone marrow transplant (BMT) have focused on the epithelial component of the thymic microenvironment; little attention has been paid to the influence of the endothelial compartment of the thymus in this process. The goal of this proposal is to delineate the role of the thymic vascular niche in the regulation of homing of bone marrow cells to thymus after transplantation. Clinical data from patients with Severe Combined Immune Deficiency (SCID) and our own data in experimental immune deficient murine models, show that when non-conditioned BMT is performed specifically in the neonatal period, rapid thymic engraftment results in donor lymphopoiesis, with little or no contribution of donor cells to non-lymphoid lineages. These findings demonstrate that although microenvironmental signals for T cell differentiation exist throughout postnatal life, different host mechanisms for thymic engraftment may exist in neonatal and adult hosts. We propose that in the neonatal setting, bone marrow can home to the thymus directly without the need for an intermediate stage of marrow engraftment. We will use the striking dichotomy between neonatal and adult thymic engraftment patterns to define the endothelial mechanisms regulating these differences. Data from murine allogeneic models will be translated to the human setting by testing our findings in a similar xenogeneic BMT model and by analysis of human thymus samples at different stages of postnatal development. Project 2: Identification of Human Cord Blood B Lineage Cells (Kenneth Dorshkind, Ph.D.) Revised Abstract Section Studies of murine B cell development have demonstrated the existence of two distinct waves of cell production. The first initiates during embryogenesis and results in the production of progenitors destined to generate B-1 B cells that are part of the innate immune system and which are particularly efficient at recognizing encapsulated bacteria such as S. pneumoniae. The second major wave generates B-2 B cells that are produced in the bone marrow throughout life and function in adaptive immunity. The central hypothesis of this application continues to be that successful reconstitution of humoral immunity in humans following bone marrow transplantation is dependent upon the recapitulation of these two distinct ontogenic programs. However, other than the fact that B cells emerge in the embryo, nothing is known about human fetal B cell development. Studies to characterize novel human cord blood progenitors that include an intermediate that co-expresses B and myeloid lineage determinants will be performed. The information that will be obtained is relevant to restoration of humoral immunity following bone marrow transplantation, the treatment of immunodeficiency diseases, and the recovery of the immune response following various myeloablative treatments. Administrative Core A Gay Crooks Revised Abstract Section Dr Gay Crooks is the Principal Investigator of the Program Project Grant and Director of the Administrative Core. The Administrative Core will be an essential mechanism to enable all the scientific components of the Program to interact by providing the structure and financial support for regular planning and evaluation of the research. Regular communication between investigators will be critical for the success of the Program. The Core will be responsible for ensuring that the investigators act as a cohesive and interactive group by providing the planning and leadership required for regular and frequent interactions between projects and cores. The Administrative Core will also be responsible for supervising budgetary and regulatory aspects of the Program, and providing the logistical support for transporting samples between institutions. Program interactions and oversight will be facilitated by both an Internal Advisory Board and an External Advisory Board composed of experts in the fields of Primary Immune Deficiency, Bone marrow Transplantation and basic immuno-biology. Core B: Cell Isolation and Analysis Core Director: Linda Baum, MD, PhD Revised Abstract Section The Cell Isolation and Analysis Core (Core B) will consist of two components, flow cytometry and histology, and will provide technical and advisory support for both Projects. The Core will aid investigators in experimental design for cell isolation and in analysis of flow cytometry data from murine and human samples. Core B will be administered by Dr Linda Baum. The core will provide FACS analysis and sorting and preparation of tissue sections and interpretation of specific tissue samples. FACSAria, and BD LSRII flow cytometry machines will be available for cell isolation and analysis. The histology component will support Projects 1 and 2 by preparing whole mounts and tissue sections by cryostat and microtome, and performing staining with standard or immunofluorescent techniques. Core C: Animal Core Director: Donald B. Kohn, MD Revised Abstract Section The focus of the Animal Core Laboratory (ACL) is to provide immunodeficient mice for the Program projects. Chimeric mouse human hybrids have become an important research tool for the evaluation of reconstitution and differentiation potential of hematopoietic and immunologic progenitor populations. The ACL will provide the following services for the projects in the Program: 1) establishment and maintenance of breeding colonies of Immunodeficient mice 3) provision of pups and adult for experiments 3) assistance with transplantation of immune deficient mice with human cells 4) health monitoring and day-to-day care of immune deficient mice, and 5) training of research personnel on the care and techniques used with immunodeficient mice. The Core will be housed within the animal care facility at UCLA under the direction of Dr. Donald Kohn.