Immunoglobulin Genetics-Ontogeny Cell Differentiation

兔阑尾和鸡法氏囊是鸡的初级淋巴器官，B细胞抗体库主要通过类似基因转换的过程在生发中心发育。在人和小鼠中，次级淋巴器官生发中心的体细胞超突变导致V基因多样化，导致亲和力成熟。我们询问在特定的免疫反应中，在兔的脾生发中心(GC)中是否发生基因转换、体细胞超突变或两者兼而有之。免疫兔产生经典的抗DNP抗体反应，以研究在已知亲和力成熟的抗体反应期间克隆性VH和VL区域的变化。用显微操作法采集脾生发中心单个细胞。对单细胞抗体重链和轻链重排基因进行了扩增和测序。DNA水平的变化可能导致亲和力成熟，通过基因转换和体细胞超突变发生(2)。通过基因转换形成不同的潜在重链和轻链对，从而影响互补决定区(CDR)中的氨基酸，这可能是兔子产生异种高亲和力抗DNP抗体的已知能力。我们推测，如果基因转换或受体修改后不再与免疫抗原反应的序列改变的细胞是GC反应的副产品，兔脾或人扁桃体GC可能会产生新的B细胞库成员，从而在成人中发挥类似于幼兔肠道相关淋巴组织的作用[1]。虽然CD5在B细胞上的功能尚不清楚，但我们在兔体内的研究表明，CD5与表面免疫球蛋白的VH骨架区域的相互作用可能有助于B细胞的存活和扩增。我们利用B-慢性淋巴细胞白血病(B-CLL)细胞和转化的B-CLL患者的B细胞系，将CD5-Ig相互作用的研究扩展到人B细胞。CD5+B细胞在个体发育早期发育，并通过自我更新维持终生。通过流式细胞仪检测，人免疫球蛋白与CD5+CD19+B细胞结合，这种相互作用可被抗CD5抗体抑制。固定化免疫球蛋白从表达CD5的细胞系的裂解物中分离CD5分子。人免疫球蛋白与纯化的重组CD5结合。结合映射到CD5-D2区域，而被单抗识别的CD5表位定位在D1域。不同VH家族的免疫球蛋白对CLL细胞及阑尾和扁桃体组织切片抗CD5染色的抑制效果不同。我们认为，VH骨架区域与CD5的相互作用可能维持、选择或扩大自身免疫或转化的B细胞，也有助于正常人类VH谱系的倾斜(1)。

Rabbit appendix and chicken bursa of Fabricius are primary lymphoid organs where the B cell antibody repertoire develops in germinal centers mainly by a gene conversion-like process. In man and mouse, V-gene diversification by somatic hypermutation in germinal centers of secondary lymphoid organs leads to affinity maturation. We asked whether gene conversion, somatic hypermutation or both occur in rabbit splenic germinal centers (GC) during specific immune responses. Rabbits were immunized to make classical anti-DNP antibody responses in order to study clonal VH and VL region diversification during antibody responses known to exhibit affinity maturation. Individual cells from splenic germinal centers were collected by micromanipulation. The rearranged genes for antibody heavy and light chains in single cells were PCR-amplified and sequenced. The changes at the DNA level that may lead to affinity maturation occur by both gene conversion and somatic hypermutation (2). The development of different potential heavy and light chain pairs through gene conversion that affects amino acids in complementarity determining regions (CDRs) may account for the rabbit's known ability to produce heterogeneous high affinity anti-DNP antibodies. We speculate that if cells with sequences altered by gene conversion or receptor revision that no longer react with the immunizing antigen are a by-product of the GC reaction, the rabbit splenic or human tonsillar GC could produce new members of the B-cell repertoire and thus play a role in adults similar to that of the gut associated lymphoid tissues of young rabbits (1). Although the function of CD5 on B cells is unknown, our studies in the rabbit, suggested that CD5 interaction with VH framework regions of surface immunoglobulins may contribute to survival and expansion of B cells. We extended our investigations of CD5-Ig interaction to human B cells using B-chronic lymphocytic leukemia (B-CLL) cells and transformed B-cell lines from B-CLL patients. CD5+ B cells develop early in ontogeny and are maintained throughout life by self-renewal. By flow cytometry, human IgG binds CD5+CD19+ B cells and this interaction can be inhibited by anti-CD5 antibodies. Immobilized immunoglobulin isolates CD5 molecules from lysates of CD5-expressing cell lines. Human immunoglobulin binds to purified recombinant CD5. The binding maps to the CD5-D2 domain whereas CD5 epitopes recognized by monoclonal antibodies are localized in the D1 domain. Immunoglobulins of different VH families demonstrated different effectiveness as inhibitors of anti-CD5 staining of CLL cells and appendix and tonsil tissue sections. We propose that interactions of VH framework regions with CD5 may maintain, select or expand autoimmune or transformed B cells and also contribute to skewing of the normal human VH repertoire (1)