GIF regulation of CD4 T-dependent immune responses

GIF 调节 CD4 T 依赖性免疫反应

• 批准号: 6333331
• 负责人: KATSUJI SUGIE
• 金额: $ 23.5万
• 依托单位: LA JOLLA INSTITUTE FOR IMMUNOLOGY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-05-01 至 2002-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6333331
• 关键词: B cell receptor; B lymphocyte; T cell receptor; antigen presentation; cell differentiation; cytokine; cytokine receptors; genetically modified animals; helper T lymphocyte; laboratory mouse; molecular cloning; ovalbumin; respiratory hypersensitivity

DESCRIPTION: (Adapted from applicant's abstract) This proposal is to establish the role of glycosylation-inhibiting factor (GIF), a 13-kDa cytokine in the generation and function of Th effector cells. GIF inhibits IgG1 and IgE antibody formation when administered around the time of immunization with DNP-Ova. GIF secreted from T cells is cysteinylated, i.e., modified by binding of free cysteine to Cys-60 in the sequence. Cys-modified recombinant human GIF(rhGIF) inhibited IgG1 and IgE antibody formation in vivo, whereas unmodified rhGIF did not. The modification is required not only for the bioactivity but also for the capability to bind to receptors on target cells. The receptor for this cytokine is detected on activated T and B cells and fresh NKT cells, whereas it is undetectable on small resting T and B cells, fresh conventional NKT cells, macrophage lines and dendritic cells. GIF is chemically cross-linked to a 50-52 kDa protein on target cells. This cytokine inhibited B cell Ig switch to IgG1 and IgE in vitro. It also inhibited antigen presentation mediated by B cell antigen receptor (BCR), whereas it showed little effect on that independent of BCR. GIF-/- T cells secreted more IL-2 and proliferated more than GIF+/+ T cells following stimulation with anti-CD3 and anti-CD28. GIF -/- T cells differentiated in vitro into Th effectors which secreted more IL-4 than GIF +/+ T cells, whereas the capability to secrete IFNg was similar between GIF -/- and +/+ Th effectors. It is hypothesized that this cytokine regulates the generation and function of Th effector cells by acting on cognate T-B interaction and directly on T cells. The effect of GIF on activation events on T and B cells will be analyzed by using antigen-specific T and B cells derived from transgenic mice that express antigen-specific TCR and BCR, respectively. The effect of GIF on antigen internalization and processing will be determined. Since BCR-mediated signaling regulates antigen presentation, the effect of GIF on signaling events induced by antigen will be investigated. The mechanism by which GIF regulates expansion and differentiation of T cells will be examined in vitro by using GIF -/- and +/+ T cells. To determine the in vivo function of this cytokine, GIF-/- and +/+ mice on BALB/c genetic background will be immunized and challenged with aerosolized Ova and allergic airway inflammation will be analyzed. Finally, in order to define the significance of this cytokine in the immune response, cDNA for GIF receptor will be cloned. For the receptor cloning, monoclonal antibodies (mAb) that specifically block the binding of GIF to target cells will be raised. Receptor protein will be purified by using the mAb and its partial amino acid sequences will be determined. cDNA libraries will be screened based on the amino acid sequences, or by expression cloning of the receptor through direct binding of anti-receptor mAb or 125I-GIF The experimental system proposed here will clarify the role of this cytokine and fill a critical gap in our knowledge regarding cytokine-mediated regulation of allergy.

描述：（改编自申请人摘要）本提案旨在建立 糖基化抑制因子（GIF），一种13-kDa的细胞因子， Th效应细胞的产生和功能。GIF抑制IgG1和IgE 当在免疫接种前后施用时的抗体形成 DNP-Ova从T细胞分泌的GIF是半胱氨酸化的，即，通过绑定修改 游离半胱氨酸与Cys-60的结合。半胱氨酸修饰的重组人 GIF（rhGIF）抑制体内IgG 1和IgE抗体的形成，而 未修饰的rhGIF则没有。修改不仅是为了 生物活性，而且结合靶细胞上的受体的能力。 这种细胞因子的受体在活化的T和B细胞以及新鲜的 NKT细胞，而在小的静息T和B细胞上检测不到，新鲜 常规NKT细胞、巨噬细胞系和树突细胞。GIF在化学上 与靶细胞上的50 - 52 kDa蛋白交联。这种细胞因子抑制B 体外细胞IG转变为IgG 1和IgE。它还抑制抗原呈递 它通过B细胞抗原受体（BCR）介导，而对 独立于BCR。GIF-/-T细胞分泌更多的IL-2并增殖 在用抗-CD3和抗-CD28刺激后比GIF +/+ T细胞更多。gif -/-T细胞在体外分化为分泌更多IL-4的Th效应细胞 而分泌IFNg的能力与GIF +/+ T细胞相似 在GIF-/-和+/+ Th效应子之间。假设这种细胞因子 调节Th效应细胞的产生和功能， T-B相互作用和直接作用于T细胞。GIF对激活事件的影响 将通过使用抗原特异性T和B细胞分析T和B细胞上的 来源于表达抗原特异性TCR和BCR的转基因小鼠， 分别GIF对抗原内化和加工的影响将 被确定。由于BCR介导的信号调节抗原呈递， 将研究GIF对抗原诱导的信号传导事件的影响。的 GIF调节T细胞扩增和分化的机制将 通过使用GIF-/-和+/+ T细胞进行体外检测。为了确定体内 这种细胞因子的功能，GIF-/-和+/+小鼠对BALB/c遗传背景 将进行免疫接种，并使用雾化Ova和过敏气道进行攻击 将分析炎症。最后，为了明确 这种细胞因子在免疫应答中的作用，将GIF受体的cDNA克隆出来。为 受体克隆，特异性阻断受体的单克隆抗体（mAb）， 将提高GIF与靶细胞的结合。受体蛋白将是 通过使用mAb纯化，其部分氨基酸序列将被 测定将基于氨基酸序列筛选cDNA文库， 或通过直接结合受体的表达克隆， 抗受体mAb或125I-GIF本文提出的实验系统将 阐明这种细胞因子的作用，填补了我们知识中的一个关键空白， 关于奎宁介导的过敏调节。