Ig Genetics--ontogeny And Differentiation Of Cells Of Th

免疫球蛋白遗传学--Th细胞的个体发育和分化

• 批准号: 6668789
• 负责人: rose G. mage
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6668789
• 关键词: B cell receptor; B lymphocyte; CD antigens; CD5 molecule; cell differentiation; developmental genetics; enzyme linked immunosorbent assay; flow cytometry; gene conversion; gene expression; gene rearrangement; gut associated lymphoid tissue; human tissue; immunocytochemistry; immunogenetics; immunoglobulin genes; laboratory rabbit; polymerase chain reaction; protein structure function

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.

Fabricius的Rabbit附录和鸡肉囊是原发性淋巴器官，在该器官中，B细胞抗体库主要通过基因转换过程在生发中心发展。在人和小鼠中，v-Gene多样化在继发性淋巴机器人的生发中心中通过体细胞超数导致亲和力成熟。在先前的研究中，我们表明在特定免疫反应期间，基因转化率和体细胞超名都发生在兔脾发芽中心（GC）中。对兔子进行免疫以进行经典的抗DNP抗体反应，以研究在已知表现出亲和力成熟的抗体反应期间的克隆VH和VL区域的多样化。通过基因转化的基因转化的不同潜在的重链和轻型链的发展，这些转化率在互补性确定区域（CDR）中影响氨基酸，这可能是兔子产生异质高亲和力抗DNP抗体的已知能力。为了确定抗体亲和力是否增加了经历基因转化的克隆相关序列，我们正在使用噬菌体显示来分析各个生发中心的FAB。通过免疫组织化学鉴定了抗原特异性的脾发芽中心，并使用Leica LMD显微解剖来从免疫兔子的脾脏的串行组织切片中恢复单个生发中心（2）。从各个生发中心的噬菌体显示库的生成和分析正在进行中。年轻的兔附录是一种动态的原发性淋巴器官，其中主要由基因转换过程产生宽B细胞免疫库。在3至5周龄的单个克隆中产生的组合位点的种类（3）表明，某些克隆膨胀和选择（已知需要肠道菌群）可以通过微生物成分（超抗原）的间接影响来驱动，而仅通过其识别为特定的外国抗原。这也可能包括B细胞受体框架区域与内源性超抗原（例如CD5）之间的相互作用。尽管CD5对B细胞的功能尚不清楚，但我们在兔子中的研究表明，CD5与表面免疫球蛋白表面免疫球蛋白的VH框架区域的相互作用可能有助于B细胞的存活和扩张。对兔附录B淋巴细胞的发展和选择进行了详细分析。在出生后3天的正常兔子中，B细胞开始形成小卵泡。如流式细胞术和免疫组织化学所检测到的，它们是CD79A阳性和表达IgM，但缺乏CD5抗原。到第8天，附录B细胞开始表达CD5，到2周，大多数附录B细胞为CD5阳性。这些发现与大多数B细胞（IGM+ CD79A+）出生后3天已经表达CD5的脾脏和PBL形成鲜明对比。与突变的Ali/Ali兔子相比，正常的B细胞发育动力学之间存在差异。由于突变的Ali/Ali兔子缺乏与已删除的VH1A2基因相关的正常VH框架区域，因此它们的B细胞发育延迟。 B细胞的扩展似乎与经历基因转化的B细胞的发育相关，因此VH框架区域的表达与缺失的VH1A2基因所编码的区域更相似。在Ali/Ali兔子的所有附录卵泡中，B细胞发育均不均匀。 VHA2+ B细胞在某些卵泡中开始发育，而在其他卵泡中则不在。一旦VHA2阳性卵泡发展，其B细胞也会开始上调CD5。 B细胞的生长和此类卵泡中的扩张还伴有凋亡死亡，这表明正选择过程和负面选择过程都会影响B细胞库的形成。糖基化的发育开关区分了Fabricius鸡肉囊中的三个B细胞（新移民，基因转换和后囊后）。我们正在研究是否可以通过细胞表面糖基化的研究来鉴定出可比的开发阶段。特别是在附录和脾脏的发育过程中，在发育过程中遵循CD15（Lewis-X），CD15S（Siallyl Lewis-X），CD15U（硫化Lewis-X）和CD5的出现。在正常的1周大兔子中，CD15在10％的B细胞上发现。到4周龄时，该百分比逐渐下降至2％。这些可能代表附录开发的早期阶段新移民的B细胞。与年龄匹配的正常A2/A2兔子相比，B细胞发育延迟的ALI/ALI兔的正常比较表现出低水平的CD15S+ B细胞，并且CD15+ B细胞的外观延迟（ALI/ALI的1和4周，ALI/ALI的1和43％和43％和43％，与36.3％和67.3％和67.7％和67.7％的A2 rabit In2/A2 rabb相比）。我们一直在研究附录卵泡中B细胞发育过程中BCR信号传导中是否在BCR信号中起作用。信号转导级联的最早事件涉及脂质筏中SRC家族激酶lyn的Igα和Igβ的免疫受体酪氨酸的活化基序的磷酸化。已经提出，静息细胞中的BCR被排除在筏子之外，并且在抗原结合和寡聚后，BCR转移到筏中，在其中lyn磷酸化，引发了信号级联。我们已经在出生后3天后检测到从脾细胞中分离出的CD79A，VHA2免疫球蛋白分子和Lyn。在附录中，BCR易位延迟到1周大约开始，大约在B细胞开始调节CD5表达的同时。