RUI: Approaches to Somatic Immunoglobulin Diversification in Vitro

9405257 Goldsby多样性是抗体库的标志，在小鼠和人类的二次免疫反应中，体细胞突变产生显著的多样性。B细胞中编码免疫球蛋白的基因V区每一代体细胞突变率高达千分之一。这是一个非同寻常的突变率，对应于每两次细胞分裂引入一个新的免疫球蛋白突变。超突变的机制仍然是免疫学尚未解决的主要问题之一。培养系统和细胞系的可用性将允许在体外观察和研究这一现象，这将大大扩展我们分析这一过程的能力。由于牛的回肠Peyer’s patch （IPP）含有超过1亿个B细胞，并且是Ig基因大量、发育调节、广泛的体细胞突变的位点，因此它被选为建立免疫球蛋白V基因体细胞多样化的体外系统的靶组织。我们计划了多种方法来优化IPP B细胞的体外培养，包括添加有丝分裂原和细胞因子、IPP滤泡“基质”细胞的培养和单个IPP滤泡的培养。牛λ V基因体细胞多样化的检测将采用RT-PCR和单链构象多态性（SSCP）作为检测方法。支持体细胞免疫球蛋白V基因从组织向组织培养转移这一过程的系统的建立，很好地促进了免疫球蛋白基因多样化的dell和分子生物学分析。哺乳动物免疫反应的一个主要组成部分是产生大量抗体的能力，这些抗体能够特异性地结合各种外来物质（抗原）并促进它们从血液中清除。个体抗体所能识别的特异性范围称为个体抗体库。生物体抗体库的巨大多样性产生的分子机制仍然是现代免疫学的核心问题之一。产生抗体多样性的主要机制之一是编码抗体分子中外源抗原结合部分的基因的快速突变。该项目的总体目标是开发细胞培养系统，以支持抗体基因的持续多样化。这项研究将利用羊和牛免疫系统发育的某些独特特性，这表明该项目应该是可行的。如果这项研究成功，该培养系统的可用性将极大地促进免疫球蛋白基因多样化的细胞和分子机制的分析。这项研究将在一所本科院校进行，并将直接涉及本科生这个前沿问题。* * *

9405257 Goldsby Diversity is the hallmark of the antibody repertoire, and somatic mutation generates significant diversity during secondary immune responses in mice and humans. The V regions of genes encoding immunoglobulin in B cells undergo rates of somatic mutation as high as one base per thousand per generation. This is an extraordinary mutation rate and corresponds to the introduction of one new immunoglobulin mutation for every two cell divisions. The mechanism underlying hypermutation remains one of the major unsolved problems of immunology. The availability of culture systems and cell lines that would permit the observation and study of this phenomenon in vitro would provide a significant expansion of our capacity to analyze this process. Because the ileal Peyer's patch (IPP) of cattle contains more than one hundred million B cells and is the site of massive, developmentally regulated, extensive, somatic mutation of Ig genes, it has been chosen as a target tissue from which to establish in vitro systems that somatically diversify immunoglobulin V genes. A variety of approaches are planned to optimize the in vitro culture of IPP B cells, including the addition of mitogens and cytokines, culture on outgrowths of IPP follicular "stromal" cells and the culture of individual IPP follicles. The assay for occurrence of somatic diversification of bovine lambda V genes will employ RT-PCR and single strand conformational polymorphism (SSCP) as an assay. The development of a system that supports the transfer of this process of somatic immunoglobulin V gene diversification from tissue to tissue culture well facilitate the analysis of the dell and molecular biology of immunoglobulin gene diversification. %%% A major component of the mammalian immune response is the ability to produce a vast array of antibodies that are capable of specifically binding a wide range of foreign substances (antigens) and facilitating their removal from the blood. The range of s pecificities that the antibodies of an individual can recognize is termed the individual's antibody repertoire. The molecular mechanism(s) by which the vast diversity of an organism's antibody repertoire is generated remains one of the central questions of modern immunology. One of the major mechanisms that generates antibody diversity is by a rapid rate of mutation in the genes that encode the foreign antigen-binding portions of antibody molecules. The overall objective of this project is to develop cell culture systems that will support the continued diversification of antibody genes. This research will take advantage of certain unique properties of the development of the immune system in sheep and cattle which suggest that this project should be feasible. If this research is successful, the availability of this culture system will greatly facilitate the analysis of the cellular and molecular mechanisms of immunoglobulin gene diversification. The research will be done at an undergraduate institution and will directly involve undergraduate students in this cutting edge problem. ***