Mechanisms of positive and negative thymic selection of CD8 T cells

 描述(由申请人提供)：TCR与胸腺中的自体肽-MHC复合体的相互作用控制着阳性选择、谱系承诺和阴性选择的关键发育检查点，这些点对于产生功能上的CD8T细胞库是必不可少的。这些不同步骤背后的机制仍然不完全清楚。最初的假设是，MHC I呈现的自体肽在所有选择检查点都是相同的。然而，一个令人兴奋的发现是，皮质胸腺上皮细胞(CTEC)表达一个独特的蛋白酶体活性部位亚单位ss5t，该亚单位结合到蛋白酶体中生成胸腺蛋白酶体。SS5T的基因失活减少了CD8T细胞的发育。CTEC是呈递多肽-MHC复合体进行正向选择的关键细胞，该领域的主流观点认为胸腺蛋白酶体的作用是产生更适合于这一选择步骤的多肽。另一种不受欢迎的模式是，胸腺蛋白酶体不需要为正选择产生特殊的多肽，而是产生不同于负选择的多肽，从而防止负选择抵消正选择。到目前为止，研究人员使用了部分基因“敲除”来试图阐明胸腺蛋白酶体在正选择和负选择中的作用。我们已经建立了一个更完整的遗传模型，在该模型中，小鼠缺乏所有专门的蛋白酶体，并被证明在CD8 T细胞发育中具有明显更强的阻滞性。与早期的模型相比，这些小鼠缺乏cTECs中的所有三个胸腺蛋白酶体亚单位，也完全不能在阳性和阴性选择步骤之间切换多肽。我们建议将我们的新模型与其他最先进的遗传模型、培养系统和质谱学结合起来，以阐明胸腺选择的潜在机制。我们的初步数据显示了可行性，并表明目前青睐的模型可能是不正确的。这项建议有3个目的，就是检验这些，阐明这些问题。

 DESCRIPTION (provided by applicant): TCR interactions with self-peptide-MHC complexes in the thymus control the key developmental check points of positive selection, lineage commitment and negative selection that are essential for the generation of a functional CD8 T cell repertoire. The mechanisms underlying these various steps are still incompletely understood. It was originally assumed that the MHC I-presented self-peptides were identical at all the selection check points. However, an exciting discovery was that cortical thymic epithelial cells (cTEC) express a unique proteasome active site subunit, ss5t, which incorporates into proteasomes to generate thymoproteasomes. Genetic inactivation of ss5t reduces the development of CD8 T cells. cTECs are the key cells that present peptide-MHC complexes for positive selection and the prevailing opinion in the field is that the role of thymoproteasomes is to generate peptides that are more optimal for this selection step. An alternate and disfavored model is that thymoproteasomes are not required to generate special peptides for positive selection but rather to make different peptides from those involved in negative selection and thereby prevent negative selection from cancelling out positive selection. So far investigators have used partial genetic "knock outs" to try to elucidate the role of thymoproteasomes in positive and negative selection. We have generated a more complete genetic model in which mice lack all specialized proteasomes and turn out to have a significantly stronger block in CD8 T cell development. Compared to earlier models, these mice lack all three thymoproteasome subunits in cTECs are also completely unable to switch peptides between the positive and negative selection steps. We propose to use our novel model together with other state of the art genetic models, culture systems, and mass spectrometry to elucidate the underlying mechanisms of thymic selection. Our preliminary data show feasibility and suggest that the currently favored model may be incorrect. This proposal has 3 aims to test these elucidate these issues.