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暂时性向永久性B细胞克隆性扩张(BLCE)进展的机制，以及持续性BLCE向晚年B细胞肿瘤的进展。我们假设，这种与年龄相关的进展是由一系列有序的基因改变引起的。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的BLCE，在抗原特异性的血清单抗免疫球蛋白(MLG)出现之前，以及在抗原特异性MLG或弥漫性大细胞淋巴瘤(DLCL)出现后，从C57/BL/6小鼠中分离抗原特异性的BLCE。从4-6月龄和14-18月龄小鼠脾部分切除后获得的冷冻保存的脾细胞中分离出暂时或稳定的抗原特异性BLCE。从19个月龄使用抗原特异性MiG的小鼠的骨髓中分离出分泌抗原特异性MiG的骨髓浆细胞。抗原特异性细胞将被荧光标记的抗原染色。在免疫小鼠中获得的结果将通过分离自发的、稳定的BLCE、骨髓浆细胞分泌的MiG或DLCL来证实。自发性BLCE将从MIG或DLCL发生前获得的冷冻保存的脾细胞表面或细胞内与荧光标记的抗克隆型抗体结合而分离。通过单细胞RT-PCR鉴定肿瘤细胞及其稳定的BLCE前体的特征IgH/IGL CDR3序列，将建立抗原诱导或自发性BLCE与肿瘤B细胞之间的联系。我们的第二个目标是开发一个基因路线图，定义BLCE向晚年B细胞肿瘤的进展。我们将定义在从暂时性BLCE到稳定型BLCE和稳定型BLCE到分泌MiG的骨髓浆细胞和/或DLCL转变过程中积累的遗传异常。从B细胞肿瘤发生前分离的小鼠BLCE用单细胞RT-PCR分析原癌基因的体细胞超突变，用FISH分析染色体易位和异倍体，用定量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.