GIF regulation of B cells and CD4 cells

描述（由申请人提供）：一种称为GIF或MIF的细胞因子的功能长期以来一直不清楚。然而，最近的研究表明，这种细胞因子在C60处的翻译后修饰是与受体结合以及抑制B细胞IG转换、BCR介导的抗原摄取和CD 4细胞分泌IL-4的能力所必需的。GIF-/-小鼠表现出增强的抗体反应的T依赖性抗原，无论IG同种型，但正常的反应的T-非依赖性抗原。与野生型细胞相比，来自GIF-/-小鼠的CD 4细胞向Th 2表型分化。在静止的小鼠T和B细胞中检测不到GIF受体，但在体外刺激后，这些细胞上的受体被诱导。GIF受体似乎是一个50-52 kDa的细胞表面蛋白。为了确定GIF的生物学效应是否是通过GIF与其受体的相互作用介导的，将制备抗受体mAb并克隆编码受体的cDNA。为了确定该细胞因子的靶细胞，将使用来自各种造血和非造血组织的细胞和mRNA样品描绘受体的表达。为了阐明GIF在调节T-B协作中的B细胞活化中的作用，将在GIF+/+或-/-背景下用HEL-Ova缀合物体外刺激来自MD 4 BCR Tg小鼠的HEL-B特异性B细胞和来自DO11.10 TCR Tg小鼠的Ova特异性CD 4细胞。在同源T-B相互作用过程中分析GIF的分泌、其受体的诱导以及与B细胞活化相关的事件。将分析GIF对BCR依赖性抗原摄取、运输和加工的影响。为了确定GIF对CD 4细胞的细胞因子产生和Th分化的作用，将分析T细胞活化时GIF受体的表达和GIF的分泌。调节Th分化的GIF的来源将通过使用来自GIF+/+和-/- DO11.10 TCR Tg小鼠和骨髓嵌合体构建体的CD 4细胞的过继转移实验来确定。GIF抑制Th 2分化和/或诱导Th 1分化的机制将结合Stat磷酸化和转录因子进行分析。GIF在调节Th 2型炎症中的作用将在抗原诱导的支气管哮喘的小鼠模型中评估。这些实验将确定这种细胞因子在CD 4 T细胞介导的免疫应答中的作用。

DESCRIPTION (provided by applicant): The function of a cytokine called GIF or MIF has been unclear for a long time. However, recent studies have demonstrated that a posttranslational modification of this cytokine at C60 is required for the binding to the receptor and for the capability to inhibit B cell Ig switch, BCR-mediated antigen uptake, and IL-4 secretion from CD4 cells. GIF-/- mice displayed enhanced antibody responses to a T-dependent antigen regardless of Ig isotype but normal responses to a T-independent antigen. CD4 cells from GIF-/- mice differentiated toward a Th2 phenotype as compared with wild type cells. GIF receptor was undetectable in resting mouse T and B cells, but after in vitro stimulation the receptor was induced on these cells. GIF receptor appeared to be a 50-52 kDa cell surface protein. To determine whether the biological effects of GIF is mediated through the interaction of GIF and its receptor, anti-receptor mAb will be generated and the cDNA encoding the receptor will be cloned. To define the target cells for this cytokine, the expression of the receptor will be delineated using cells and mRNA samples from various hematopoietic and nonhematopoietic tissues. To clarify the role of GIF in regulating B cell activation in T-B collaboration, HEL-specific B cells from MD4 BCR Tg mice and Ova-specific CD4 cells from DO11.10 TCR Tg mice, either on GIF+/+ or -/- background, will be stimulated in vitro with HEL-Ova conjugate. The secretion of GIF, the induction of its receptor, and the events associated with B cell activation will be analyzed in the course of the cognate T-B interaction. The effects of GIF on BCR-dependent antigen uptake, trafficking, and processing will be analyzed. To determine the role of GIF on cytokine production and Th differentiation of CD4 cells, the expression of GIF receptor and the secretion of GIF upon T cell activation will be analyzed. The source of GIF that regulates Th differentiation will be determined by adoptive transfer experiments using CD4 cells from GIF+/+ and -/- DO11.10 TCR Tg mice and bone marrow chimera constructions. The mechanism by which GIF inhibits Th2 differentiation and/or induces Th1 differentiation will be analyzed with regard to Stat phosphorylation and transcription factors. The role of GIF in regulating Th2-type inflammation will be evaluated in the mouse model of antigen-induced bronchial asthma. These experiments will establish the role of this cytokine in CD4 T cell-mediated immune responses.