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

描述(申请人提供)：现已在T细胞发展文献中证实，两个关键信号，以高度受控和顺序的方式在胸腺壁龛中呈现，在产生功能性T细胞中起关键作用：(I)Delta样配体-Notch受体信号和(Ii)主要组织相容性复合体(MHC)-T细胞受体(TCR)信号。控制Notch信号的Delta样配体和指导胸腺TCR信号的MHC分子存在于基质细胞和抗原提呈细胞的表面，发育中的干/祖细胞直接与其相互作用。已有研究表明，至少可以通过逆转录病毒转染法在体外模拟显示Notch配体的基质细胞的Notch信号过程。最近，我们报道了表面固定的Notch配体(Delta-like Ligand 4，DLL4)微球可以有效地将造血祖细胞定向到早期T细胞谱系。此外，我们现在已经证明，使用表面固定的Notch配体介导的信号和外源的、可溶性抗原负载的MHC四聚体介导的TCR信号的组合，小鼠胚胎干细胞(ES)和小鼠诱导的多能干细胞(IPS)都可以被定向到T细胞途径，并分化为针对同一抗原的功能性CD8 T细胞。然而，到目前为止，包括我们自己在内的大多数努力都集中在在2D环境中模拟这些特定于小众的信号。基于我们在这些2D系统中的可喜数据，我们假设，在培养的ES或iPS细胞中，能够有效地触发Delta-Notch和MHC-TCR信号通路的3D组织样微环境将显著提高T细胞的生成效率。我们在这里的目标是设计这种人工的3D T细胞利基(人工胸腺样微环境)，将多能干细胞引导成治疗性的、抗原特异性的T细胞。这项探索性研究的具体目的是：目标1：开发和表征具有T细胞分化所必需的配体的反蛋白石水凝胶支架。目的：利用Notch配体功能化的反蛋白石水凝胶支架高效诱导小鼠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获得的早期T细胞群中高效地产生抗原特异的、有功能的CD8T细胞，并评价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一个类似胸腺的三维微环境，以从小鼠胚胎或诱导的多能干细胞中产生具有功能的、抗原特异性的T细胞。我们的方法是通过创建以有效方式呈现特定T细胞分化信号的支架来模拟胸腺利基。如果成功，这可能会导致使用干细胞来源的T细胞过继治疗。

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.