Functional characterization of IL-40, a novel cytokine

 描述(申请人提供)：我们已经鉴定出一种新的分泌型蛋白，由一个未知基因(C17Orf99)编码，该基因在胎肝、骨髓和活化的B细胞中特异表达。该基因序列预测了一个~27 KDa的分泌性蛋白和一个信号肽，与任何已知的细胞因子家族都没有同源性。然而，在几个B细胞系或在刺激下激活的正常B细胞中，它被强烈诱导 CD40L、IL-4或脂多糖(LPS)。这些特征(小的分泌蛋白，仅限于激活的淋巴细胞表达)表明该基因编码一种新的细胞因子，我们将其命名为IL-40。在这项提案中，我们的目标是从功能上表征IL-40。我们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。我们已经制备了抗IL-40多肽的单抗，目前正在用重组蛋白进行筛选。我们还获得了一只IL-40-/-小鼠，该小鼠在Peyer氏结中显示异常，在乳腺和粪便中显示低水平的IgA。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的体外生物学特性研究。我们假设IL-40是由某些B细胞亚群产生的。接下来，我们将确定胎肝和骨髓中产生IL-40的细胞。这些结果将有助于我们了解其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，我们将探索其他产生IL-4的强大细胞(如iNKT和Th2 CD4+细胞)是否也产生IL-40。IL-4是一种B细胞共刺激因子，与多种B细胞有丝分裂原共同诱导增殖，与诱导B细胞产生IL-40的条件相同。因此，我们推测IL-40可能参与了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驱动的增殖和分化试验来测试这一点。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生育的。我们将探索这些小鼠的Peyer‘s斑块，以探索由于缺乏IL-40而发生的细胞变化，我们将量化IL-40-/-小鼠的Peyer’s斑块中产生IgA的细胞的数量。我们将测量其他已被证明影响免疫球蛋白A表达的基因的表达，如CCL28及其受体CCR10，以及转化生长因子β。免疫球蛋白水平与肠道微生物群有关，因此我们将分析IL-40-/-小鼠的肠道微生物群是否异常。最后，我们将寻找可能影响 IL-40-/-小鼠B细胞产生免疫球蛋白，包括类开关重组。通过这些具体目标，我们将开辟一个新的研究领域，即对IL-40生物学的研究。

 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.