MECHANISMS OF SIGNALLING VIA MHC CLASS II MOLECULES

除了它们的抗原呈递功能，MHC II类分子 调节免疫细胞功能的信号。我们有 表明MHC II类分子作为细菌的受体， 超抗原如TSST-1和MHCII类分子接合 通过TSST-1诱导单核细胞中IL-1和TNF-α的合成， 增殖和分化信号传递给B细胞。MHC II类 信号转导涉及SRC型酪氨酸激酶（HCK和FGR）的活化 单核细胞和B细胞中林恩），磷脂酶C-γ的活化， Ca+2通量、蛋白激酶C和细胞核的活化 转录因子NF-κ B和AP-1。MHC的一个重要后果 II类信号传导是B7表达的诱导，即 共刺激T细胞分子CD 28/CTLA 4的反受体。 有证据表明，小鼠MHC的胞质结构域 II类分子对于抗原呈递和B7 表情我们建议进行详细的结构/功能分析 人MHC II类分子的信号传导特性， 定义II类介导的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类的胞质结构域在细胞内的作用。 II分子在信号传导中的作用。我们将检测截短的DR α和DR β， 嵌合CD 8/DR α和CD 8/DR β链和突变的DR α 和DR β分子导入II- B类细胞系，并分析抗原 呈递、同型粘附、蛋白酪氨酸磷酸化和B7 我们将根据需要使用类似的方法， 检查跨膜结构域和细胞外膜的作用， DR α和DR β结构域。 在目标2中，我们提出鉴定MHC II类相关蛋白。我们将 过表达DR α和DR β胞质结构域作为融合蛋白 并利用它们通过免疫沉淀来鉴定相关蛋白质， 蛋白质印迹和表达文库的筛选。 在目标3中，我们将研究转录激活的机制， MHC II类配体对B7的抑制作用。我们还将剖析 MHC II类分子和CD 4 O介导的信号在诱导B7表达中的作用 在同源T-B细胞相互作用的过程中， TCR和II类/肽复合物之间的相互作用以及 活化T细胞上的B细胞抗原CD 40及其配体。在这些 实验中，我们将利用CD 4 O配体缺陷的同种异体反应性T细胞， 从高IgM综合征患者身上，我们将使用B II类基因敲除和CD 4 O基因敲除小鼠，以确定相对 II类和CD 4 O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.