Immunoglobulin Genetics-Ontogeny Cell Differentiation

兔阑尾和鸡法氏囊是原发性淋巴器官，其B细胞抗体库主要通过基因转换样过程在生发中心形成。在人和小鼠中，通过在次级淋巴器官生发中心的体细胞超突变，v -基因多样化导致亲和成熟。我们询问兔脾生发中心（GC）在特异性免疫应答中是否发生基因转换、体细胞超突变或两者兼有。兔免疫产生经典的抗dnp抗体反应，以研究在已知表现亲和成熟的抗体反应中克隆VH和VL区域的多样化。通过显微操作收集脾生发中心的单个细胞。对单细胞抗体重链和轻链重排基因进行pcr扩增和测序。DNA水平上可能导致亲和成熟的变化发生在基因转化和体细胞超突变两种情况下(2)。通过影响互补决定区（cdr）氨基酸的基因转换，产生不同的潜在重链和轻链对，这可能解释了兔产生异质高亲和力抗dnp抗体的能力。我们推测，如果基因转换或受体修饰改变了细胞序列，不再与免疫抗原反应的细胞是GC反应的副产物，兔脾或人扁桃体GC可以产生新的b细胞库成员，从而在成人中发挥类似幼兔肠道相关淋巴组织的作用(1)。虽然CD5对B细胞的作用尚不清楚，但我们在兔身上的研究表明，CD5与表面免疫球蛋白的VH框架区相互作用可能有助于B细胞的存活和扩增。我们将CD5-Ig相互作用的研究扩展到人类B细胞，使用B-慢性淋巴细胞白血病（B- cll）细胞和从B- cll患者转化的B细胞系。CD5+ B细胞在个体发育早期发育，并通过自我更新维持一生。通过流式细胞术，人IgG结合CD5+CD19+ B细胞，这种相互作用可以被抗CD5抗体抑制。固定化免疫球蛋白从表达CD5的细胞系裂解物中分离CD5分子。人免疫球蛋白与纯化的重组CD5结合。这种结合映射到CD5- d2结构域，而单克隆抗体识别的CD5表位定位在D1结构域。不同VH家族的免疫球蛋白作为CLL细胞、阑尾和扁桃体组织切片抗cd5染色抑制剂的效果不同。我们认为VH框架区与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，也可能导致正常人类VH库的扭曲(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)