GIF regulation of B cells and CD4 cells

描述（申请人提供）：GIF是在IgE合成研究过程中发现的一种13kda的细胞因子。它由基因组中的单个基因编码，其核苷酸序列与巨噬细胞迁移抑制因子（MIF）的核苷酸序列相同。MIF(GIF)-/-小鼠对利什曼原虫的易感性增强。这些小鼠不能发生实验性结肠炎。这些发现支持了GIF与先天免疫系统失活有关的观点。GIF蛋白在造血细胞和非造血细胞的细胞质中表达。从T细胞分泌的GIF在C60时被半胱氨酸化，而同一细胞的细胞质中含有的GIF则未被修饰。C60位点的修饰是该细胞因子与靶细胞结合、抑制bcr介导的抗原摄取和抑制CD4细胞分泌IL-4的能力所必需的。结合实验表明，在T细胞和B细胞被激活后，GIF受体在这些细胞上表达，但在巨噬细胞和树突状细胞系上不表达。化学交联实验表明，GIF与一个50-52 kDa的细胞表面蛋白结合。GIF-/-小鼠对t依赖性抗原的抗体反应增强。与野生型细胞相比，GIF-/- CD4细胞向Th2表型极化。为了阐明这种细胞因子在t依赖性和非依赖性体液反应中的作用，我们将用各种抗原和佐剂免疫与BALB/c和B6杂交的GIF+/+和-/-小鼠。在GIF+/+和-/-背景下，利用TCR Tg小鼠和BCR Tg小鼠在体内和体外分析GIF在同源T-B相互作用中的作用。我们将分析GIF调节bcr介导的抗原呈递的生化机制。TCR Tg GIF+/+和-/-小鼠将比较调节Th分化的信号。这些小鼠也将被用来确定先天免疫参与调节Th分化。gif依赖性Th发育的功能相关性将在过敏性气道炎症小鼠模型中得到解决。最后，利用c60修饰的rGIF，对GIF受体进行分子表征。这些实验将有助于确定该细胞因子是否直接调节先天免疫或抗原特异性免疫，以及T细胞分泌的形式或细胞质中包含的形式是否在其功能中起重要作用。这个项目将填补我们在细胞因子生物学知识方面的一个关键空白。

DESCRIPTION (provided by applicant): GIF is a 13-kDa cytokine discovered in the course of the study on IgE synthesis. It is encoded by a single gene in the genome and its nucleotide sequence is identical is that published for macrophage migration inhibitory factor (MIF). MIF(GIF)-/- mice show enhanced susceptibility to Leishmania major. These mice are incapable of developing the experimental colitis. These findings support the notion that GIF is involved inactivating the innate immune system. GIF protein is expressed in the cytosol of hematopoietic and nonhematopoietic cells. GIF secreted from T cells is cysteinylated at C60, whereas that contained in the cytosol of the same cells is unmodified. The modification at C60 is required for the capability of this cytokine to bind to target cells, to inhibit BCR-mediated uptake of antigen, and to inhibit IL-4 secretion from CD4 cells. Binding assays demonstrated that GIF receptors are expressed on T and B cells after these cells are activated, but not on macrophage and dendritic cell lines. Chemical crosslinking experiments suggest that GIF binds to a 50-52 kDa cell surface protein. GIF-/- mice show enhanced antibody responses to T-dependent antigens. GIF-/- CD4 cells were polarized toward a Th2 phenotype as compared with wild type cells. To clarify the role of this cytokine in T-dependent and -independent humoral responses, GIF+/+ and -/- mice crossed to BALB/c and those to B6 will be immunized with various antigens and adjuvants. The role of GIF in cognate T-B interaction will be analyzed in vitro and in vivo using TCR Tg mice and BCR Tg mice on GIF+/+ and -/- backgrounds. The biochemical mechanisms by which GIF regulates BCR-mediated antigen presentation will be analyzed. TCR Tg GIF+/+ and -/- mice will be compared for the signals that regulate Th differentiation. These mice will also be used to determine the involvement of the innate immunity in the regulation of Th differentiation. The functional relevance of GIF-dependent Th development will be addressed in the mouse model of allergic airway inflammation. Finally, using C60-modified rGIF, GIF receptor will be molecularly characterized. These experiments will be useful to determine whether this cytokine directly regulates innate immunity or antigen-specific immunity and whether the form secreted from T cells or that contained in the cytosol plays an important role in its function. This project will fill a critical gap in our knowledge in cytokine biology.