B Cell Repertoire and B cell Neoplasms in Old Mice

描述（由申请人提供）：该项目的长期目标是探索中年小鼠短暂性B谱系克隆扩增（BLCE）发展到持续性B细胞扩增（BLCE）以及持续性BLCE发展到晚期B细胞肿瘤的机制。我们假设遗传改变的有序进展是这种与年龄相关的进展的基础。我们的第一个目标是从dnp -人血清白蛋白（DNP-HSA）或鸡蛋溶菌酶（HEL）免疫的C57/BL/6小鼠中分离抗原特异性血清单克隆免疫球蛋白（mlg）出现前的抗原特异性BLCE和抗原特异性mlg或弥漫性大细胞淋巴瘤（dcl）出现后的抗原特异性克隆BM浆细胞。短暂或稳定的抗原特异性BLCE将从4-6月龄和14-18月龄小鼠部分脾切除术获得的冷冻保存的脾脏细胞中分离出来。从19月龄用抗原特异性mIg处死的小鼠骨髓中分离出分泌抗原特异性mIg的BM浆细胞。抗原特异性细胞用荧光标记抗原染色。在免疫小鼠中获得的结果将通过分离自发的、稳定的BLCE、骨髓浆细胞分泌mIg或dcl来证实。自发BLCE将从mIg或dcl发展之前获得的冷冻保存的脾脏细胞中分离出来，通过其表面或细胞内结合荧光标记的抗克隆型抗体。通过单细胞RT-PCR鉴定肿瘤细胞及其稳定BLCE前体的标志性IgH/IgL CDR3序列，将建立抗原诱导或自发BLCE与肿瘤B细胞之间的联系。我们的第二个目标是制定一个遗传路线图，定义BLCE向晚期b细胞肿瘤的进展。我们将定义瞬时BLCE到稳定BLCE和稳定BLCE到分泌mig的骨髓浆细胞和/或dcl转化过程中积累的遗传异常。从B细胞肿瘤发生前的小鼠中分离的BLCE将通过单细胞RT-PCR分析原癌基因的体细胞超突变，FISH分析染色体易位和非整倍体，以及定量RT-PCR分析原癌基因的表达。

DESCRIPTION (provided by applicant): The long-term goal of this project is to explore the mechanisms underlying the progression of transient to persistent B Lineage Clonal Expansions (BLCE) in middle-aged mice and the progression of persistent BLCE to late-life B cell neoplasms. We hypothesize that an ordered progression of genetic alterations underlies this age-associated progression. Our first aim is to isolate antigen-specific BLCE prior to the appearance of antigen-specific serum monoclonal immunoglobulin (mlg) and antigen-specific clonal BM plasma cells after the appearance of antigen-specific mlg or diffuse large cell lymphoma (DLCL) from DNP-human serum albumin (DNP-HSA)- or hen egg lysozyme (HEL)-immunized C57/BL/6 mice. The transient or stable antigen-specific BLCE will be isolated from cryopreserved, spleen cells obtained by partial splenectomy of 4-6- and 14-18-month-old mice. BM plasma cells secreting antigen-specific mIg will be isolated from bone marrow obtained from mice sacrificed at 19-months of age with antigen-specific mIg. The antigen-specific cells will be stained with fluorescent-labeled antigen. Results obtained in immunized mice will be confirmed by isolating spontaneous, stable BLCE, bone marrow plasma cell-secreting mIg, or DLCL. Spontaneous BLCE will be isolated from cryopreserved spleen cells obtained prior to the development of mIg or DLCL by their surface or intracellular binding of fluorescent-labeled anti-clonotypic antibody. The link between antigen-induced or spontaneous BLCE and neoplastic B cells will be established by identifying signature IgH/IgL CDR3 sequences of the neoplastic cells and their stable BLCE precursors by single-cell RT-PCR. Our second aim is to develop a genetic roadmap defining the progression of BLCE into late-life B-cell neoplasms. We shall define the genetic abnormalities that accumulate during the transformation of transient to stable BLCE and stable BLCE to mIg-secreting bone marrow plasma cells and/or to DLCL. BLCE isolated from mice prior to the development of B cell neoplasms will be analyzed for the presence somatic hypermutation of proto-oncogenes by single cell RT-PCR, for chromosomal translocations and aneuploidy by FISH and for proto-oncogene expression by quantitative RT-PCR.