Functional characterization of IL-40, a novel cytokine

 描述（由申请人提供）：我们已经鉴定了一种由未表征基因（C17 Orf 99）编码的新型分泌蛋白，其在胎儿肝脏、骨髓和活化B细胞中特异性表达。该基因序列预测约27 KDa的分泌蛋白，具有信号肽，并且与任何已知的细胞因子家族没有同源性。然而，它在几种B细胞系或用刺激物激活的正常B细胞中被强烈诱导，所述刺激物例如 CD 40 L、IL-4或脂多糖（LPS）。这些特征（小分泌蛋白，表达限于活化的淋巴细胞）表明，该基因编码一种新的细胞因子，我们将其命名为白细胞介素-40。在这项提案中，我们的目标是功能特性IL-40。我们已经克隆并表达了小鼠和人IL-40，并将利用这些重组蛋白来探索其生物学功能。我们已经生产了针对IL-40肽的单克隆抗体，目前正在用重组蛋白进行筛选。我们还获得了IL-40-/-小鼠，其表现出派伊尔集合淋巴结异常和乳腺和粪便中伊加水平低。在具体目标1中，我们将通过鉴定IL-40的细胞来源来进行IL-40的体外生物学表征。我们假设IL-40是由某些B细胞亚群产生的。接下来我们将确定胎儿肝脏和骨髓中产生IL-40的细胞。这些结果将有助于我们了解它在这些器官中的生物学功能。我们假设IL-40的产生与IL-4的产生有关。因此，我们将探索其他强IL-4产生细胞（如iNKT和Th 2 CD 4+细胞）是否也产生IL-40。IL 4是与各种B细胞有丝分裂原组合诱导增殖的B细胞共刺激因子，并且这些是诱导B细胞产生IL-40的相同条件。因此，我们假设IL-40可能参与IL-4的某些已知生物学活性。我们将通过使用IL-40-/-或野生型（WT）小鼠B细胞进行IL-4驱动的增殖和分化测定来对此进行测试。在具体目标2中，我们将使用IL-40-/-小鼠探索IL-40的生理学，这些小鼠是活的和可繁殖的。我们将探索这些小鼠的派尔集合淋巴结，以探索由于缺乏IL-40而发生的细胞变化，并且我们将量化IL-40-/-小鼠的派尔集合淋巴结中IgA产生细胞的数量。我们将测量已被证明影响伊加表达的其他基因的表达，如CCL 28及其受体CCR 10，以及TGFβ。伊加水平与肠道微生物组有关，因此我们将分析IL-40-/-小鼠的肠道微生物组是否异常。最后，我们将寻找分子缺陷， 包括IL-40-/-小鼠的B细胞中的类别转换重组的免疫球蛋白产生。通过这些具体的目标，我们将开辟一个新的研究领域，即IL-40的生物学研究。

 DESCRIPTION (provided by applicant): We have identified a novel secreted protein encoded by an uncharacterized gene (C17Orf99) that is specifically expressed in the fetal liver, bone marrow and activated B cells. The gene sequence predicts a ~27KDa secreted protein with a signal peptide and exhibits no homology to any known cytokine family. However, it is strongly induced upon activation in several B cell lines or in normal B cells activated with stimuli such as CD40L, IL-4 or Lipopolysaccharide (LPS). These characteristics (small secreted protein, expression restricted to activated lymphoid cells) suggest that this gene encodes a novel cytokine which we have named Interleukin-40. In this proposal, we aim to functionally characterize IL-40. We have cloned and expressed mouse and human IL-40, and will use these recombinant proteins to explore its biological functions. We have produced monoclonal antibodies against IL-40 peptides which we are currently screening with recombinant protein. We have also obtained an IL-40-/- mouse that exhibits abnormalities in the Peyer's patches and low levels of IgA in the mammary gland and feces. In Specific Aim 1, we will undertake the biological characterization of IL-40 in vitro by identifying the cellular sources of IL-40. We hypothesize that IL-40 is produced by certain B cell subsets. We will next determine the cells that produce IL-40 in the fetal liver and bone marrow. These results will help us understand its biological function in these organs. We hypothesize that IL-40 production is linked to the production of IL-4. We will therefore explore whether other strong IL-4 producing cells (like iNKT and Th2 CD4+ cells) also produce IL-40. IL4 is a B cell costimulatory factor that induces proliferation in combination with various B cell mitogens, and these are the same conditions that induce IL-40 production by B cells. We therefore hypothesize that IL-40 may be involved in some of the known biological activities of IL-4. We will test this by performing IL-4 driven proliferation and differentiation assays using either IL-40-/- or wild type (WT) mouse B cells. In Specific Aim 2, we will explore the physiology of IL-40 using an IL-40-/- mouse, which are viable and fertile. We will explore the Peyer's patches of these mice to explore the cellular changes that have taken place due to the lack of IL-40, and we will quantify the number of IgA-producing cells in the Peyer's patches of IL-40-/- mice. We will measure the expression of other genes that have been shown to influence IgA expression like CCL28 and its receptor CCR10, as well as TGFβ. IgA levels are linked to the gut microbiome, so we will analyze the gut microbiome of the IL-40-/- mouse for abnormalities. Finally, we will search for molecular defects that may affect the production of Immunoglobulin production including class switch recombination in the B cells of the IL-40-/- mouse. Through these specific aims we will open a new field of research, namely, the study of the biology of IL-40.