Ig Genetics--ontogeny And Differentiation Of Cells Of Th

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。在先前的研究中，我们发现在兔脾生发中心（GC）特异性免疫应答过程中，基因转换和体细胞超突变都发生。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。为了确定抗体亲和力是否在经过基因转换的克隆相关序列中增加，我们使用噬菌体展示来分析来自个体生发中心的Fab。免疫组化法鉴定抗原特异性脾生发中心，Leica LMD显微解剖法从免疫兔脾脏连续组织切片中恢复单个生发中心(2)。来自各个生发中心的噬菌体展示文库的生成和分析正在进行中。幼兔阑尾是一个动态的原发性淋巴器官，其中广泛的B细胞免疫前库主要通过基因转换样过程产生。从3周龄到5周龄的单个克隆中产生的组合位点的多样性(3)表明，一些已知需要肠道菌群的克隆扩增和选择可能是由微生物成分（超抗原）的间接作用驱动的，而不仅仅是由它们被识别为特定的外来抗原驱动的。这也可能包括B细胞受体框架区域与内源性超抗原（如CD5）之间的相互作用。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。目前正在对兔阑尾B淋巴细胞的发育和选择进行详细的分析。正常家兔出生3天后，B细胞开始形成小卵泡。通过流式细胞术和免疫组织化学检测，它们是CD79a阳性，表达IgM，但缺乏CD5抗原。到第8天，阑尾B细胞开始表达CD5，到2周时，大多数阑尾B细胞CD5阳性。这些发现与脾脏和PBL形成对比，其中大多数B细胞（IgM+ CD79a+）在出生后3天就表达CD5。正常ali/ali兔与突变ali/ali兔的B细胞发育动力学存在差异。由于突变ali/ali家兔缺乏与缺失的VH1a2基因相关的正常VH框架区域，它们的B细胞发育被延迟。B细胞扩增似乎与经历基因转换的B细胞的发育相关，随后VH框架区域的表达更类似于缺失的VH1a2基因编码的区域。ali/ali家兔阑尾卵泡B细胞发育不均匀。VHa2+ B细胞在一些卵泡中开始发育，但在其他卵泡中没有。一旦VHa2阳性卵泡发育，它们的B细胞也开始上调CD5。B细胞在这些卵泡中的生长和扩增也伴随着凋亡死亡，这表明正选择和负选择过程都影响B细胞库的形成。糖基化的发育开关区分了鸡法氏囊中三个B细胞群体（新移民、基因转化和法氏囊退出后）。我们正在研究是否可以通过研究细胞表面糖基化来确定可比较的发育阶段。特别是CD15 （Lewis-x）、CD15s （Sialyl Lewis-x）、CD15u （sulphated Lewis-x）和CD5在发育中的B细胞表面的出现在阑尾和脾脏的发育过程中。正常1周龄家兔，10%的B细胞上发现CD15s。这一比例在4周龄时逐渐下降至2%。这些可能代表阑尾发育早期新迁移的B细胞。与正常和ali/ali家兔比较，B细胞发育延迟，显示CD15s+ B细胞水平低于年龄匹配的正常a2/a2家兔，CD15+ B细胞出现延迟（ali/ali家兔1周龄和4周龄分别为2.6%和43%，而a2/a2家兔为36.3%和67.7%）。我们一直在进一步研究被称为脂筏的膜微结构域是否可能在阑尾卵泡B细胞发育过程中参与BCR信号传导。最早的信号转导级联事件涉及到脂筏中src家族激酶Lyn对Ig α和Ig β免疫受体酪氨酸激活基序的磷酸化。有人提出，静息细胞中的BCR被排除在筏外，抗原结合和寡聚化后，BCR易位到筏中，在筏中被Lyn磷酸化，启动信号级联。我们早在出生后3天就从脾细胞中分离出了CD79a、VHa2免疫球蛋白分子和Lyn。BCR在阑尾的转运延迟，大约在1周龄开始，与此同时B细胞开始上调CD5的表达。

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. In previous studies we showed that gene conversion and somatic hypermutation 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. 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. In order to determine whether antibody affinity increases in clonally related sequences that undergo gene conversion, we are employing phage display to analyze Fab from individual germinal centers. Antigen-specific splenic germinal centers were identified by immunohistochemistry and Leica LMD microdissection was used to recover individual germinal centers from serial tissue sections of spleens of immunized rabbits (2). Generation and analyses of phage display libraries from individual germinal centers are in progress. The young rabbit appendix is a dynamic primary lymphoid organ where a broad B cell preimmune repertoire is produced mainly by a gene conversion-like process. The variety of combining sites generated within individual clones from 3 to 5 weeks of age (3) suggest that some clonal expansion and selection, known to require gut flora, may be driven through indirect effects of microbial components (superantigens) rather that solely by their recognition as specific foreign antigens. This may also include interactions between B cell receptor framework regions and endogenous superantigens such as CD5. 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. A detailed analysis of the development and selection of rabbit appendix B lymphocytes is in progress. In normal rabbits 3 days after birth, B cells start to form small follicles. As detected by flow cytometry and immunohistochemistry they are CD79a positive and express IgM but lack the CD5 antigen. By day 8, appendix B cells start to express CD5 and by 2 weeks, the majority of appendix B cells are CD5 positive. These findings contrast to the spleen and PBL where most B cells (IgM+ CD79a+) already express CD5 3 days after birth. There are differences between the kinetics of B cell development in normal compared to mutant ali/ali rabbits. Since mutant ali/ali rabbits lack normal VH framework regions associated with the deleted VH1a2 gene, their B cell development is delayed. B-cell expansion seems to correlate with development of B cells that have undergone gene conversion and consequent expression of VH framework regions more similar to those encoded by the missing VH1a2 gene. B cell development is not uniform in all appendix follicles of ali/ali rabbits. VHa2+ B cells start to develop in some follicles but not in the others. Once VHa2 positive follicles develop, their B cells also start to up-regulate CD5. B cell growth and expansion in such follicles is also accompanied by apoptotic death suggesting that both positive and negative selection processes are affecting B cell repertoire formation. Developmental switches in glycosylation distinguish three populations of B cells in the chicken bursa of Fabricius (new immigrants, gene converted, and post bursal exiting). We are investigating whether comparable defined stages of development can be identified by studies of cell-surface glycosylation. In particular the appearance of CD15 (Lewis-x), CD15s (Sialyl Lewis-x ), CD15u (Sulfated Lewis-x) and of CD5 on surfaces of developing B cells is being followed during development in appendix and spleen. In normal 1 week-old rabbits CD15s was found on 10% of B cells. The percentage declined gradually to 2% by 4 weeks of age. These may represent newly immigrating B cells in the early phase of appendix development. Comparisons of normal with ali/ali rabbits, in which B cell development is delayed, showed low levels of CD15s+ B cells compared to age matched normal a2/a2 rabbits and a delay in appearance of CD15+ B cells (2.6% and 43% at 1 and 4 week of age in ali/ali compared to 36.3% and 67.7% in a2/a2 rabbits). We have been further studying whether the membrane microdomains, termed lipid rafts, might play a role in BCR signalling during B cell development in appendix follicles. The earliest event of a signal transduction cascade involves the phosphorylation of the immunoreceptor tyrosine based activation motifs of Ig alpha and Ig beta by the src family kinase Lyn in lipid rafts. It has been proposed that the BCR in resting cells is excluded from rafts and that after antigen binding and oligomerization, the BCR translocates into rafts, where it is phosphorylated by Lyn, initiating a signaling cascade. We have detected CD79a, VHa2 immunoglobulin molecules and Lyn in rafts isolated from splenocytes as early as 3 days after birth. BCR translocation into rafts is delayed in the appendix and starts around 1 week of age, at about the same time as B cells start to up regulate the expression of CD5.