Strategy for Rescuing Primary Thymic Stromal Failure

描述(申请人提供)：原发T淋巴细胞(T细胞)免疫缺陷主要归因于胸腺发育缺陷，因为胸腺为发展和维持广泛反应和自我限制的T细胞以及不同的外周T细胞谱系提供了独特的微环境。由胸腺器官发生和胸腺基质细胞(TSC)发育障碍引起的原发T细胞免疫缺陷，如可能由TBX1基因突变引起的DiGeorge综合征(DGS)和由Foxn1基因突变引起的人类裸体(HN)，可以通过使用体外三维TSC网络再生新生异位胸腺来挽救。然而，安全、稳定和有效的供者胸腺的来源成为一个问题，因为心胸手术期间胸腺切除将导致供者免疫系统的严重问题。为了获得安全、稳定和有效的TSC网络，我们建议在小鼠模型中使用不同的基质细胞在肾被膜下生成新生异位胸腺。我们将测试Notch配体和Foxn1过表达的正常或TBX1突变小鼠的皮肤成纤维细胞和角质形成细胞，以生成用于DGS治疗的功能性从头胸腺，这在我们的目标1中详述。我们将通过我们的新的Foxn1Neo/Neo和Foxn1Neo/+小鼠通过G418选择培养从可能的胸腺上皮祖细胞中鉴定出特异性的表型标记；然后根据特定的胸腺上皮祖细胞表型标志物，从野生型胸腺中分离和扩增胸腺上皮祖细胞，以检验我们目标2中详细描述的HN治疗策略。从本研究中获得的全面知识将为将来将该研究扩展到人类对象奠定基础，并导致针对原发胸腺器官发生和/或TSC发育障碍引起的T细胞免疫缺陷的实用策略的发展。公共卫生相关性：我们将测试各种基质细胞，包括Notch Ligand和Foxn1过表达的皮肤成纤维细胞和来自野生型或TBX1突变小鼠的角质形成细胞，并分离和扩增来自野生型小鼠的可能的胸腺上皮祖细胞，以在小鼠模型中产生治疗DiGeorge综合征和人类裸体治疗的功能性新生胸腺。

DESCRIPTION (provided by applicant): Primary T-lymphocyte (T-cell) immunodeficiency is mainly attributable to defects in thymus development because the thymus provides a unique microenvironment for development and maintenance of broadly reactive and self-restricted T cells and a diverse peripheral T-cell repertoire. Primary T-cell immunodeficiency caused by thymic organogenetic and thymic stromal cell (TSC) developmental failure, such as DiGeorge syndrome (DGS) which is likely caused by a mutation in the Tbx1 gene and human nude (HN) which is caused by a mutation in the Foxn1 gene, can be rescued by regenerating a de novo ectopic thymus using an ex vivo three-dimensional TSC network. However, the source of a safe, stable, and effective donor thymus becomes a problem because thymectomy during cardiothoracic surgery will cause profound problems in the donor's immune system. In order to obtain a safe, stable, and effective TSC network, we propose generating a de novo ectopic thymus under the kidney capsule by using various stromal cells in a mouse model. We will test engineered Notch ligand and Foxn1 over-expressing skin fibroblasts and keratinocytes from normal or Tbx1 mutant mice for generating a functional de novo thymus for DGS therapy detailed in our Aim 1. We will identify specific phenotypic markers from putative thymic epithelial progenitors through our novel Foxn1Neo/Neo and Foxn1Neo/+ mice by G418-selection culture; and then isolate and expand the putative thymic epithelial progenitors from wild-type thymus based on the specific thymic epithelial progenitor phenotypic markers for testing the strategy of HN therapy detailed in our Aim 2. The comprehensive knowledge obtained from the proposed studies will lay the groundwork to extend the study to human subjects in the future, and lead to the development of practical strategies against T-cell immunodeficiency caused by primary thymic organogenetic and/or TSC developmental failure. PUBLIC HEALTH RELEVANCE: We will test various stromal cells, including Notch ligand and Foxn1 over-expressing skin fibroblasts and keratinocytes from wild-type or Tbx1 mutant mice and isolate and expand the putative thymic epithelial progenitors from wild-type mice, for generating a functional de novo thymus for DiGeorge syndrome and human nude therapies in a mouse model.