Ig Genetics, Ontogeny and Differentiation of Cells of th

Ig 遗传学、个体发育和细胞分化

• 批准号: 6807769
• 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/6807769
• 关键词: B cell receptor; B lymphocyte; CD antigens; CD5 molecule; cell differentiation; enzyme linked immunosorbent assay; flow cytometry; gene conversion; gene expression; gut associated lymphoid tissue; human tissue; immunocytochemistry; immunogenetics; immunoglobulin genes; laboratory rabbit; polymerase chain reaction; protein structure function

Recruitment of Blood-Borne B-cells to Neonatal Rabbit Appendix Young rabbit appendix is a homologue of chicken bursa of Fabricius; both are crucial sites for preimmune B-cell repertoire development. These are primary lymphoid organs where the B cell antibody repertoire develops in germinal centers mainly by a gene conversion-like process. Although B cell Ig-gene rearrangements occur in sites such as bone marrow of young rabbits, immature IgM+ B cells undergo further Ig-repertoire diversification in appendix and other gut associated lymphoid tissues. We have now characterized some of the molecules involved in the multi-step recruitment of blood-borne B cells into neonatal rabbit appendix. Expression of peripheral lymph node addressin (PNAd) on appendix high endothelial venules (HEV), and of its counter-receptor, CD62L, on B cells in peripheral blood and in close association with PNAd-positive HEV suggests their role in tethering. Sialyl-Lewis-x, known to be involved in tethering of pre-bursal cells on chicken bursal vasculature, was also found on appendix B cells. The interaction of chemokine receptor CCR7 on peripheral blood B cells and one of its ligands, CCL21, on appendix HEV, could cause activation of integrins such as LFA-1 and alpha 4/beta-1 and lead to firm adhesion of B cells to HEV. We found these integrins expressed on peripheral blood B cells. Simultaneous down-regulation of CCR7 and up-regulation of CXCR5 on appendix B cells compared to peripheral blood suggests a role for CXCR5 in B-cell recruitment into follicles during appendix development. The proportions of appendix B cells expressing CD62L, sialyl-Lewis-x and alpha-4/beta-1 declined between day 3 and 4 weeks after birth while percentages of Lewis-x+ appendix B cells increased. These changes correlate with the stage of repertoire diversification by gene conversion in both rabbits and chickens. Previous work from our laboratory showed that in young ali/ali appendix, fewer B cells were proliferating compared to wild-type rabbits. Therefore we compared the expression of Lewis-x on wild-type and mutant ali/ali appendix B cells. The percentages of Lewis-x+ B cells in ali/ali appendix were significantly lower at one and two weeks compared to wild-type controls. In contrast to the Lewis-x expression profile, the proportions B cells expressing adhesion molecules (CD62L and sialyl-Lewis-x) that are involved in tethering and rolling on HEV were comparable between the wild-type and mutant rabbits. We conclude that migration and recruitment of B cells to appendix was normal in ali/ali rabbits but that their intrinsic defect in rearrangement, due to deletion of the VH1 gene leads to delayed development. The Kit activating Mutation in Mast Cells, and Oligovlonal B-Lymphocytes of Mastocytosis Patients Mastocytosis is characterized by focal heterotypic clusters of mast cells and lymphocytes in the bone marrow and by a somatically acquired activating Kit mutation, D816V. The relationship of the occurrence of this mutation to the heterotypic clusters of mast cells and lymphocytes in bone marrow is unknown. We collaborated with the laboratory of Dr. Dean Metcalfe to investigate whether these two unique features of mastocytosis were related. To explore this hypothesis, laser capture microdissected mast cells, B cells and T cells, from both lesional and non lesional areas of bone marrow biopsy tissues from patients with mastocytosis, were examined for the D816V mutation in their DNA, employing HinfI restriction digestion of nested PCR products amplified from extracts of dissected cells. The D816V mutation was detected in mast cells, B cells and T cells from lesional but not non lesional areas of bone marrow tissues. We found that mast cells and lymphocytes within focal aggregates in the bone marrow of those with mastocytosis are more frequently positive for the codon 816 activating mutation. Control B cells were obtained from normal human appendix tissue. We found that the B cell population in lesional areas of mastocytosis as well as B cells in normal appendix represented oligoclonal populations. Clonal proliferation is unlikely to be the basis of clustering of B cells in lesional areas of patient bone marrow. Studies of Rabbit CD5 The variety of combining sites generated within individual clones from appendix follicles of young rabbits suggests that clonal expansion and selection, known to require gut flora, may not be driven by specific antigens but rather through indirect effects of microbial components (superantigens). In addition, there may be survival signals supplied by 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. For further studies of the interaction of CD5 and VH-regions, a fragment of CD5 protein containing the three extracellular domains has been cloned and expressed as recombinant protein. In addition, rabbit VH1a2 has been expressed as a recombinant protein. In a collaboration with Drs. David Garboczi and Brandt Burgess, crystallization of CD5 has been accomplished. We hope to also generate co-crystals of CD5 and VH. Purified CD5 protein was also used to produce polyclonal goat anti-rabbit CD5. In addition we have generated anti-peptide mAbs specific for each extracellular domain of CD5. These reagents are being used to continue a detailed analysis of the development and selection of rabbit appendix B lymphocytes. 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. Studies of Rabbit Activation Induced Deaminase Cloned cDNAs encoding the rabbit homolog of Activation Induced Deaminase have been produced, the encoded sequence has been analyzed and peptides chosen for use as immunizing antigens. MAP-4 conjugates of the peptides were used as immunogens and polyclonal antibodies were raised in rabbits. These antibodies have been affinity purified and used for immunohistochemical , immunofluoresence and confocal microscopy studies.

招募血液传播B细胞到新生儿兔附录 年轻的兔子附录是Fabricius鸡肉囊的同源物；两者都是免疫前B细胞曲目发展的关键部位。这些是原发性淋巴机器人，其中B细胞抗体库主要通过基因转化样过程在生发中心发展。尽管B细胞Ig-Gene重排发生在诸如年轻兔子的骨髓等部位，但未成熟的IgM+ B细胞在附录和其他肠道相关的淋巴组织中会进一步经历Ig-Repertoire多样化。现在，我们已经表征了一些参与血液传播B细胞募集到新生儿兔附录中的分子。在附录高内皮静脉（HEV）上的外周淋巴结地址（PNAD）及其反受体CD62L在外周血中的B细胞上的表达，并与PNAD阳性HEV密切相关，这表明它们在绑带中的作用。在附录B细胞上也发现了siAlyl-Lewis-X，已知与鸡囊脉管脉络膜上的绑扎前细胞绑扎。趋化因子受体CCR7在外周血B细胞及其配体CCL21在附录HEV上的相互作用可能导致整联蛋白的激活，例如LFA-1和Alpha 4/beta-1，并导致B细胞对HEV的粘附。我们发现这些整合素在外周血B细胞上表达。与外周血相比，CCR7同时下调CCR7和CXCR5上的上调表明CXCR5在附录发育过程中B细胞募集到卵泡中的作用。表达CD62L，SiAllyl-Lewis-X和Alpha-4/beta-1的附录B细胞的比例在出生后第3至4周之间下降，而Lewis-X+附录B细胞的百分比增加。这些变化与兔子和鸡的基因转化的曲目多样化阶段相关。我们实验室的先前工作表明，在年轻的Ali/Ali附录中，与野生型兔子相比，B细胞的增殖较少。因此，我们比较了Lewis-X在野生型和突变体Ali/Ali附录B细胞上的表达。与野生型对照相比，ALI/ALI附录中Lewis-X+ B细胞的百分比显着降低。与Lewis-X表达曲线相反，与野生型和突变型兔之间的链接和滚动有关的比例B细胞（表达粘附分子（CD62L和siAllyl-Lewis-X）与链球上和滚动的比例相当。我们得出的结论是，在Ali/Ali兔子中，B细胞向附录的迁移和募集是正常的，但是由于VH1基因的缺失导致其重排的内在缺陷导致发育延迟。 肥大患者的肥大细胞和寡聚型B淋巴细胞中的套件激活突变 肥大细胞增多症的特征是骨髓中肥大细胞和淋巴细胞的局灶性异型簇，以及由体外获得的激活套件突变D816V。骨髓中肥大细胞和淋巴细胞的异型簇的发生与骨髓中的异型簇的关系是未知的。我们与Dean Metcalfe博士的实验室合作，研究了这两个独特的肥大性特征是否相关。为了探讨这一假设，检查了从植物核细胞增多症患者的骨髓活检组织的病变和非病变区域中捕获的微解析的肥大细胞，B细胞和T细胞，在其DNA中检查了d816v突变的DNA中的d816v突变，均采用了hinfi限制了从嵌套的PCR产物中的hInfi限制了从筑巢的PCR产物中挖掘出嵌套的细胞。在骨髓组织的病变但非病变区域的肥大细胞，B细胞和T细胞中检测到D816V突变。我们发现，肥大细胞增多症患者骨髓中焦点骨料内的肥大细胞和淋巴细胞对于密码子816激活突变的阳性更常见。对照B细胞是从正常的人类阑尾组织获得的。我们发现，肥大细胞增多症的病变区域的B细胞群以及正常附录中的B细胞代表寡群群体。克隆增殖不太可能成为患者骨髓病变区域B细胞聚类的基础。 兔子CD5的研究 幼兔附录卵泡中各个克隆中产生的组合位点的种类表明，克隆膨胀和选择（已知需要肠道菌群）可能不会由特定的抗原而驱动，而是通过微生物成分（超抗原）的间接影响来驱动。此外，B细胞受体框架区域与内源性超抗原（例如CD5）之间的相互作用可能会提供生存信号。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。为了进一步研究CD5和VH区域的相互作用，已克隆了包含三个细胞外域的CD5蛋白的片段，并表示为重组蛋白。另外，兔VH1A2已被表示为重组蛋白。与Drs合作。 David Garboczi和Brandt Burgess，CD5的结晶已完成。我们希望也能产生CD5和VH的共结晶。纯化的CD5蛋白也用于产生多克隆山羊抗兔CD5。此外，我们已经产生了针对CD5的每个细胞外结构域特异的抗肽mAB。这些试剂用于继续对兔附录B淋巴细胞的发育和选择进行详细分析。在出生后3天的正常兔子中，B细胞开始形成小卵泡。如流式细胞仪和免疫组织化学所检测到的，它们为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细胞发育并不均匀。 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。 兔活化诱导脱氨酶的研究 已经产生了编码激活型脱氨酶的兔同源物的克隆cDNA，已经分析了编码的序列，并选择肽作为免疫抗原。将肽的MAP-4共轭物用作免疫原子，并在兔子中饲养多克隆抗体。这些抗体已被纯化并用于免疫组织化学，免疫荧光和共聚焦显微镜研究。