MECHANISMS OF SIGNALLING VIA MHC CLASS II MOLECULES

除了其抗原提呈功能外，MHC II类分子 传递调节免疫细胞功能的信号。我们有 显示MHC II类分子是细菌的受体 超抗原，如TSST-1和MHC II类分子的结合 TSST-1诱导单核细胞合成IL-1和TNF-α并传递 B细胞的增殖和分化信号。MHC II类 信号涉及SRC型酪氨酸激酶(HCK和FGR)的激活 单核细胞和B细胞中的Lyn)，磷脂酶C-γ的激活， Ca+2流出、蛋白激酶C和细胞核的激活 转录因子NF-kappaB和AP-1。MHC的一个重要后果 第二类信号是诱导B7，即 共刺激T细胞分子CD28/CTLA4的反向受体。 有证据表明，小鼠MHC的胞质结构域 II类分子对抗原提呈和B7很重要 表情。我们建议进行详细的结构/功能分析 人类MHC II类分子的信号特性，以及 明确第II类分子介导的B7表达激活的机制 同源T-B细胞相互作用的过程。 在目标1中，我们将研究MHC类的细胞质结构域的作用 II分子在信号中的作用。我们将把截短的DRAlpha和DRbeta 链、嵌合CD8/DRpha和CD8/DRbeta、链和突变的DRpha 和DRbeta分子进入II-B类细胞系并分析抗原 提呈、同型黏附、蛋白酪氨酸磷酸化和B7 根据需要，我们将使用类似的方法来 检查跨膜区和细胞外结构域的作用 信令中的DRAlpha和DRbeta结构域。 在目标2中，我们建议鉴定MHC II类相关蛋白。我们会 以融合蛋白的形式过表达DRAlpha和DRbeta胞浆结构域 并用它们通过免疫沉淀来鉴定相关蛋白质， Western blotting和表达文库筛选。 在目标3中，我们将研究转录激活的机制 MHC II类配体对B7的影响。我们还将剖析两者之间的相互作用 MHC-II和CD4O介导的信号诱导B7表达 在涉及相互作用的同源T-B细胞相互作用过程中 TCR与II类/多肽复合体之间的相互作用 活化T细胞表面的B细胞抗原CD4O及其配体。在这些 实验中我们将利用CD4O配体缺乏的同种异体反应性T细胞 来自高IgM综合征患者的细胞，我们将使用来自 II类基因敲除小鼠和CD4O基因敲除小鼠定义相关基因 第II类和CD4O类信号对B7表达的贡献。

In addition to their antigen presenting function, MHC class II molecules transduce signals that regulate the function of immune cells. We have shown that MHC class II molecules serve as receptors for bacterial superantigens such as TSST-1 and that engagement of MHC class II molecules by TSST-1 induces IL-1 and TNF-alpha synthesis in monocytes and delivers proliferation and differentiation signals to B cells. MHC class II signalling involved activation of src type tyrosine kinases (hck and fgr in monocytes and lyn in B cells), activation of phospholipase C-gamma, Ca+2 fluxes, activation of protein kinase C and of the nuclear transcription factors NF-kappaB and AP-1. An important consequence of MHC class II signalling is the induction of expression of B7, the counterreceptor of the costimulatory T cell molecules CD28/CTLA4. There is evidence to suggest that the cytoplasmic domains of murine MHC class II molecules is important for antigen presentation and B7 expression. We propose to carry out a detailed structure/function analysis of the signalling properties of human MHC class II molecules, and to define the mechanisms of class II mediated activation of B7 expression in the course of cognate T-B cell interactions. In Aim 1, we will examine the role of the cytoplasmic domain of MHC class II molecules in signalling. We will transfect truncated DRalpha and DRbeta chains, chimeric CD8/DRalpha and CD8/DRbeta, chains, and mutated DRalpha and DRbeta molecules into class II- B cell lines and analyze antigen presentation, homotypic adhesion, protein tyrosine phosphorylation and B7 expression in these cells We will use, as required, a similar approach to examine the role of the transmembrane domains and of the extracellular domains DRalpha and DRbeta in signalling. In Aim 2, we propose to identify MHC class II-associated proteins. We will overexpress the DRalpha and DRbeta cytoplasmic domains as fusion proteins and use them to identify associated proteins by immunoprecipitation, Western blotting and screening of expression libraries. In Aim 3, we will investigate the mechanisms of transcriptional activation of B7 by MHC Class II ligands. We will also dissect the interplay between MHC class II and CD4O mediated signals in the induction of B7 expression in the course of cognate T-B cell interactions which involve interaction between TCR and class II/peptide complexes as well as interaction between the B cell antigen CD4O and its ligand on activated T cells. In these experiments we will make use of CD4O ligand deficient alloreactive T cells from patients with the HyperIgM syndrome and we will use B cells from class II knockout and CD4O knockout mice to define the relative contribution of class II and CD4O signals to B7 expression.