B Cell Repertoire and B cell Neoplasms in Old Mice

描述（由申请方提供）：本项目的长期目标是探索中年小鼠中一过性至持续性B谱系克隆扩增（BLCE）进展以及持续性BLCE进展至晚期B细胞肿瘤的潜在机制。我们推测，这种与年龄相关的进展是遗传变异有序进展的基础。我们的第一个目的是分离抗原特异性BLCE之前出现抗原特异性血清单克隆免疫球蛋白（mlg）和抗原特异性克隆BM浆细胞后出现抗原特异性mlg或弥漫性大细胞淋巴瘤（DLCL）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。将从4-6月龄和14-18月龄小鼠的部分脾切除术获得的冻存脾细胞中分离瞬时或稳定的抗原特异性BLCE。将从19月龄时用抗原特异性mIg处死的小鼠获得的骨髓中分离分泌抗原特异性mIg的BM浆细胞。抗原特异性细胞将用荧光标记抗原染色。将通过分离自发、稳定的BLCE、骨髓浆细胞分泌mIg或DLCL确认免疫小鼠中获得的结果。自发BLCE将从mIg或DLCL形成前获得的冻存脾细胞中分离，方法是通过其表面或细胞内结合荧光标记的抗克隆型抗体。抗原诱导或自发BLCE与肿瘤性B细胞之间的联系将通过单细胞RT-PCR鉴定肿瘤性细胞及其稳定BLCE前体的标志性IgH/IgL CDR 3序列来建立。我们的第二个目标是制定一个遗传路线图，确定BLCE进展为晚期B细胞肿瘤。我们将定义在从一过性BLCE转化为稳定BLCE和稳定BLCE转化为分泌mIg的骨髓浆细胞和/或DLCL的过程中积累的遗传异常。将通过单细胞RT-PCR分析在发生B细胞肿瘤之前从小鼠中分离的BLCE是否存在原癌基因的体细胞超突变，通过FISH分析染色体易位和非整倍性，并通过定量RT-PCR分析原癌基因表达。

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.