SOMATIC MUTATION AND SELECTION OF MEMORY B CELLS

描述(申请人提供)：表达高亲和力的记忆B细胞 体细胞突变免疫球蛋白编码的表面免疫球蛋白受体 可变(V)区基因在预防感染中发挥重要作用 代理和自身免疫也有贡献。尽管如此，三年多来 经过几十年的深入调查，我们对这一问题的认识存在许多差距。 记忆B细胞的细胞起源和亲和力依赖的选择，以及 产生体细胞突变的分子机制。这两种模式都 累积的突变和双链DNA断裂是一种 体细胞突变的中间阶段，导致了容易出错的假设 DNA修复可能是在V区引入碱基替换的原因 DNA因为它的已知作用是一种容易出错的聚合酶，DNA聚合酶 泽塔(Pol Zeta)被认为是这一过程中的关键调解人。老鼠 表达针对PolZeta RNA展示的转基因编码的反义RNA 低水平的polZeta RNA。尽管在免疫后，这些小鼠会产生 GC反应强烈，同种类型转换抗体效价正常， 记忆B细胞的数量，它们的抗体亲和力成熟缓慢，它们的 抗体V区积累的体细胞突变比正常少，有明显的 亲和力增强突变的减少。这是一份学习申请书。 进一步明确这些反义转基因小鼠对Low的影响 Pol Zeta水平对体细胞突变和亲和力随时间成熟的影响 用多种抗原进行免疫。因为在体内选择可能 极大地偏离累积突变的数量和质量，细胞来自 这些小鼠也将在体外进行克隆水平的免疫评估。 体细胞的反应性、耐受性及其积累 突变。最后，前体细胞混合物的过继转移研究 从常规、绿色荧光蛋白转基因和反义P01 将进行Zeta转基因小鼠以评估其细胞来源 Memory B细胞以及正常突变与 亚正常突变细胞中选择进入记忆B细胞的细胞 游泳池。如果成功，这些实验将在以下方面提供重要进展 了解与B细胞有关的分子和细胞现象 记忆。

DESCRIPTION (provided by applicant): Memory B cells that express high affinity surface immunoglobulin (sIg) receptors encoded by somatically mutated Ig variable (V) region genes play a major role in protection against infectious agents and contribute to autoimmunity as well. In spite of this, and over three decades of intensive investigation, many gaps exist in our understanding of the cellular origins and affinity dependent selection of memory B cells, and the molecular mechanisms that generate somatic mutations. Both the pattern of accumulated mutations and the possibility that double strand DNA breaks are an intermediate in somatic mutation, have led to the hypothesis that error-prone DNA repair may be responsible for introducing base substitutions in V region DNA. Because of its known role as an error-prone polymerase, DNA polymerase zeta (pol zeta) has been suggested as a key mediator in this process. Mice that express transgene encoded antisense RNA specific for pol zeta RNA display very low levels of pol zeta RNA. Although upon immunization these mice generate vigorous GC reactions, normal titers of isotype switched antibodies, and normal numbers of memory B cells, their antibodies affinity-mature slowly and their antibody V regions accumulate fewer than normal somatic mutations with a marked reduction in affinity enhancing mutations. This is a application to study further these antisense transgenic mice to define precisely the impact of low pol zeta levels on somatic mutation and affinity maturation with time after immunization with a variety of antigens. Because in vivo selection could greatly bias the quantity and quality of accumulated mutations, cells from these mice will also be evaluated at the clonal level in vitro for their immune responsiveness, tolerance susceptibility and their accumulation of somatic mutations. Finally, adoptive transfer studies of mixtures of precursor cells from conventional, green fluorescence protein transgenic, and antisense p01 zeta transgenic mice will be carried out to evaluate the cellular origins of memory B cells and any competitive advantage of normally mutating vs. sub-normally mutating cells in the selection of cells into the memory B cell pool. If successful, these experiments will provide important advances in understanding the molecular and cellular phenomena responsible for B cell memory.