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

描述（由申请人提供）：目前，在T细胞发育文献中已经确定，胸腺生态位中以高度控制和顺序的方式呈现的两个关键信号在生成功能性T细胞中起着至关重要的作用：(i) δ样配体- Notch受体信号传导和（ii）主要组织相容性复合体(MHC) - T细胞受体（TcR）信号传导。控制Notch信号传导的δ样配体和胸腺中指导TcR信号传导的MHC分子存在于基质细胞和抗原提呈细胞表面，与发育中的干细胞/祖细胞直接相互作用。研究表明，至少Notch信号传导过程可以在体外用显示Notch配体的逆转录病毒转染的基质细胞模拟。最近，我们报道了带有表面固定化Notch配体（delta-like ligand 4, DLL4）的微珠可以有效地将造血祖细胞引导到早期T细胞谱系。此外，我们现在已经证明，使用表面固定化Notch配体介导的信号和外源性可溶性抗原负载MHC四聚体介导的TcR信号的组合，小鼠胚胎干细胞（ES）和小鼠诱导多能干细胞（iPS）细胞都可以被引导到T细胞途径并分化为对相同抗原特异性的功能性CD8+ 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环境中的这些特定利基信号上。基于我们在这些2D系统中的有希望的数据，我们假设一个3D组织样微环境，可以有效地触发培养的ES或iPS细胞中的Delta- Notch和MHC-TcR信号通路，将显著提高T细胞生成的效率。我们的目标是设计这种合成的3D T细胞龛（人工胸腺样微环境），将多能干细胞引导成治疗性的抗原特异性T细胞。本探索性研究的具体目的是：目的1：开发和表征T细胞分化所需配体功能化的反蛋白石水凝胶支架。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。目的3：利用抗原负载、mhc功能化的反蛋白石水凝胶，从Aim 2中获得的早期T细胞群中高效生成抗原特异性、功能性CD8+ T细胞，并评估其抗原特异性和对自身抗原的交叉反应性。

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.