Synthetic, Thymus-like 3D Niche for T Cell Generation from Stem Cells

描述（由申请人提供）：目前在T细胞发育文献中已充分确立，以高度受控和顺序的方式存在于胸腺小生境中的两种关键信号在产生功能性T细胞中起关键作用：（i）δ样配体- Notch受体信号传导和（ii）主要组织相容性复合物（MHC）- T细胞受体（TcR）信号传导。在胸腺中控制Notch信号传导的Delta样配体和指导TcR信号传导的MHC分子存在于基质细胞和抗原呈递细胞的表面上，发育中的干细胞/祖细胞与其直接相互作用。已经显示，至少Notch信号传导过程可以使用展示Notch配体的逆转录病毒转染的基质细胞在体外模拟。最近，我们报道了表面固定Notch配体（δ样配体4，DLL 4）的微珠可以有效地将造血祖细胞引导至早期T细胞谱系。此外，我们现在已经证明，使用表面固定的Notch配体介导的信号传导和外源性可溶性抗原负载的MHC四聚体介导的TcR信号传导的组合，小鼠胚胎干（ES）细胞和小鼠诱导的多能干（iPS）细胞都可以被引导至T细胞途径并分化为对相同抗原特异性的功能性CD 8 + T细胞。然而，迄今为止，包括我们自己在内的大多数努力都集中在模拟2D环境中的这些特定于利基的信号。基于我们在这些2D系统中的有希望的数据，我们假设可以在培养的ES或iPS细胞中有效触发Delta-Notch和MHC-TcR信号传导途径的3D组织样微环境将提供T细胞生成效率的显著改善。我们的目标是设计这种合成的3D T细胞小生境（人工胸腺样微环境），将多能干细胞定向为治疗性抗原特异性T细胞。本探索性研究的具体目标是：目的1：开发和表征反蛋白石水凝胶支架功能化与T细胞分化所必需的配体。目的2：利用Notch配体功能化的反蛋白石水凝胶支架有效地将小鼠ES和iPS细胞分化为早期T细胞。目标3：为了使用负载抗原的MHC功能化反蛋白石水凝胶从目标2中获得的早期T细胞群有效地产生抗原特异性功能性CD 8 + T细胞，并评估其抗原特异性和对自身抗原的交叉反应性。 公共卫生相关性：这个为期两年的探索性项目的目标是开发三维胸腺样微环境，以从小鼠胚胎或诱导多能干细胞产生功能性抗原特异性T细胞。我们的方法是通过创建以有效方式呈现特异性T细胞分化信号的支架来模拟胸腺小生境。如果成功，这可能导致使用干细胞衍生细胞的过继性T细胞疗法。

DESCRIPTION (provided by applicant): It is now well established in the T cell development literature that two key signals, presented in the thymic niche, in a highly controlled and sequential manner, play crucial roles in generating functional T cells: (i) Delta like ligands - Notch receptor signaling and (ii) Major Histocompatibility Complex (MHC) - T cell receptor (TcR) signaling. The Delta like ligands that control Notch signaling and the MHC molecules that direct TcR signaling in the thymus are present on the surface of stromal cells and antigen presenting cells, with which the developing stem/progenitor cells directly interact. It has been shown that at least the Notch signaling process can be mimicked in vitro using retrovirally transfected stromal cells displaying Notch ligands. Recently, we have reported that microbeads with surface-immobilized Notch ligands (delta-like ligand 4, DLL4) can efficiently direct hematopoietic progenitor cells to the early T cell lineage. In addition, we have now demonstrated that, using a combination of surface-immobilized Notch ligand-mediated signaling and exogenous, soluble antigen-loaded MHC tetramer-mediated TcR signaling, both mouse embryonic stem (ES) cells and mouse induced pluripotent stem (iPS) cells can be directed to the T cell pathway and differentiated into functional, CD8+ T cells specific for the same antigen. However, most efforts to date, including our own has focused on mimicking these niche-specific signals in 2D environments. Based on our promising data in these 2D systems, we hypothesize that a 3D tissue-like microenvironment that can efficiently trigger Delta- Notch and MHC-TcR signaling pathways in cultured ES or iPS cells would provide significant improvement in the efficiency of T cell generation. Our goal here is to engineer such synthetic, 3D T cell niches (artificial thymus-like microenvironment) to direct pluripotent stem cells into therapeutic, antigen-specific T cells. The specific aims of this exploratory research are: Aim 1: To develop and characterize inverse opal hydrogel scaffolds functionalized with ligands essential for T cell differentiation. Aim 2: To efficiently differentiate mouse ES and iPS cells into early T cells using Notch ligand-functionalized inverse opal hydrogel scaffolds. Aim 3: To efficiently generate antigen-specific, functional CD8+ T cells from the early T cell population obtained in Aim 2, using antigen-loaded, MHC-functionalized inverse opal hydrogels and evaluate their antigen specificity and cross-reactivity to self antigens. PUBLIC HEALTH RELEVANCE: The goal of this two year exploratory project is to develop a three dimensional thymus- like microenvironment to generate functional, antigen-specific T cells from mouse embryonic or induced pluripotent stem cells. Our approach is to mimic the thymic niche by creating scaffolds that present specific T cell differentiation signals in an efficient manner. If successful this could lead to adoptive T cell therapy using stem cell derived cells.