T Cell Differentiation

本项目的主要重点是阐明在发育T细胞中控制细胞命运决定的机制。前体T细胞在胸腺中经历一个测试过程，以确保表达无用或自我反应性T细胞抗原受体（TCR）的细胞不会成熟（阳性和阴性选择）。这些选择过程需要TCR参与自身mhc抗原，但这些相互作用的不同方面决定了细胞的生存或死亡。TCR信号也促进其他分化事件，以及谱系的发展。加入T细胞谱系的早期前体胸腺细胞必须指定α - β (ab)或γ - δ (gd) T细胞命运。通过适当的TCR-MHC相互作用，在ab途径中发育的T细胞将采用CD4辅助细胞或CD8细胞毒性T细胞的命运。我们工作的一个主要目标是了解TCR信号如何与其他发育线索协同作用，与谱系承诺过程相关联。在我们之前的工作中，我们已经证明TCR信号的数量差异可以指导胸腺细胞发育过程中的许多细胞命运决定。我们发现，TCR激活所需的某些激酶的缺陷，有利于CD8而不是CD4的命运。此外，限制胸腺细胞迁移以及TCR和MHC之间相关的相互作用也有利于CD8的命运，这表明TCR信号传导的动力学可能在谱系承诺中发挥作用。同样，我们发现TCR信号的数量差异影响gd与ab谱系的决定。增强TCR信号的突变有利于gd，而牺牲了ab谱系的发育。通过高度保守的跨膜受体Notch传递的信号也影响发育中的T细胞的命运决定。我们发现一种构成活性形式的Notch可以覆盖通常由特定TCR信号施加的偏差，这表明这两个信号系统可能协同作用来指定细胞命运。在某些系统中，Notch活性可以通过配体结合或与其他蛋白质如Numb和Fringe结合来调节，这些蛋白质抑制对Notch配体的反应。在这方面，我们观察到Numb在发育中的CD4和CD8胸腺细胞中受到不同的调节。由于Numb有可能与TCR信号通路的组分相互作用，因此该蛋白是连接TCR信号与Notch的主要候选蛋白。在了解Notch信号如何在胸腺中被调节的其他努力中，我们研究了早老素，一种产生Notch活性形式所需的蛋白质。我们开发了一种新的系统来评估Notch在体内的功能，该系统可以解决Notch受体/配体冗余、缺失突变相关的早期致死率以及与转基因异位和过表达相关的问题。我们使用了一种转基因，表达了早老素的显性阴性形式，它在T细胞发育中产生深刻的阻滞，同时促进B细胞的异位发育。该蛋白似乎主要作用于早期胸腺前体，也增强NK发育，同时减弱gd发育。最有可能的是，早老素的显性阴性形式通过Notch起作用，因为这些功能缺陷可以通过编码Notch活性形式的转基因的共同表达来补偿。我们的研究结果进一步支持了这一观点，即淀粉样前体蛋白（APP）家族成员（早老素介导活性的另一个主要底物）在胸腺细胞中不表达，并且这些蛋白的缺失突变对胸腺发育没有影响。因此，Notch似乎在胸腺发育的几个阶段调节谱系决定，包括T/B谱系承诺。

The major focus of this project is to elucidate the mechanisms controlling cell fate decisions in developing T cells. Precursor T cells undergo a testing process in the thymus to ensure that cells expressing useless or self-reactive T cell antigen receptors (TCR) do not mature (positive and negative selection). These selection processes require TCR engagement of self-MHC antigens, but different aspects of these interactions determine whether the cells will live or die. TCR signals also promote other differentiation events, as well as the development of lineages. Early precursor thymocytes that commit to the T cell lineage must specify an alpha-beta (ab) or gamma-delta (gd) T cell fate. With the appropriate TCR-MHC interactions, T cells developing in the ab pathway will adopt a CD4 helper or CD8 cytotoxic T cell fate. A major goal in our work is to understand how TCR signals, acting in concert with other developmental cues, are linked to the process of lineage commitment. In our previous work, we have shown that quantitative differences in TCR signaling can instruct many cell fate decisions in developing thymocytes. We have found that deficiencies of certain kinases, required for TCR activation, favor a CD8 over a CD4 fate. Moreover, limiting thymocyte migration and the associated interactions between TCR and MHC also favors the CD8 fate, indicating that the kinetics of TCR signaling could play a role in lineage commitment. Similarly, we find that quantitative differences in TCR signaling influence the gd versus ab lineage decision. Mutations that enhance TCR signaling favor gd at the expense of ab lineage development. Signals through the highly conserved transmembrane receptor, Notch, also influence cell fate decisions in developing T cells. We find that a constitutively active form of Notch can override the bias normally imposed by specific TCR signals, suggesting that these two signaling systems may act in concert to specify cell fate. In some systems, Notch activity can be regulated by ligand binding or by association with other proteins like Numb and Fringe that inhibit responses to Notch ligands. In this regard, we observe that an isoform of Numb is differentially regulated in developing CD4 and CD8 thymocytes. Since Numb has the potential to interact with components of the TCR signaling pathway, this protein is a prime candidate for linking TCR signals to Notch. In other efforts to understand how Notch signaling is regulated in the thymus, we have studied Presenilins, proteins that are required for generating the active form of Notch. We have developed a novel system for assessing Notch function in vivo that gets around the problems of redundancy in Notch receptors/ligands, early lethality associated with deletion mutants, and those associated with ectopic and over expression of transgenes. We have used a transgene, expressing a dominant negative form of presenilin, which produces a profound block in T cell development, while promoting the ectopic development of B cells. This protein appears to act predominantly in an early thymic precursor, also enhancing NK development while attenuating gd development. It is most likely that this dominant negative form of Presenilin is acting through Notch since these functional defects can be compensated by the co-expression of a transgene encoding an active form of Notch. This belief is further supported by our findings that Amyloid Precursor Protein (APP) family members (the other major substrate for Presenilin-mediated activity) are not expressed by thymocytes and that deletion mutants of these proteins have no effect on thymic development. Thus, Notch appears to regulate lineage decisions at several stages of thymic development, including T/B lineage commitment.