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: 7299886
• 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/7299886
• 关键词: Ig; Genetics; Ontogeny; Differentiation; Cells

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. Purified CD5 protein was also used to produce polyclonal goat anti-rabbit CD5. In addition we generated anti-peptide mAbs specific for each extracellular domain of CD5. These reagents were used to continue a detailed analysis of the development and selection of rabbit appendix B lymphocytes (1). Although only a small proportion of mouse and human B cells are CD5+, most adult rabbit B cells express CD5. However, CD5 was not detectable on the majority of B cells in neonatal appendix 1 and 3 days after birth. Cell trafficking studies demonstrated that CD5+ and CD5- CD62L+ B cells from bone marrow migrated into appendix. There, CD5+ B cells were preferentially expanded and predominated by ~2 weeks of age. In mutant ali/ali rabbits, VHa2+ B cells develop through gene conversion-like alteration of rearranged VH genes upstream of deleted VH1a2. Correlated appearance of individual CD5+ germinal centers and VHa2+ B-cells in mutants' appendix suggest that CD5 binding positively selects cells with a2+ framework regions. Following negative and positive selection, cells with diversified rearranged heavy- and light-chain sequences exit appendix, migrate to peripheral tissues and constitute the preimmune repertoire of CD5+ B cells that encounter foreign antigens. (2). Studies of Rabbit Activation Induced Deaminase Studies in mouse, human and chicken suggest that activation-induced deaminase (AID) is involved in the three known processes leading to antibody diversification: somatic hypermutation, gene conversion, and class switch recombination. Developing rabbit appendix provides a particularly good site for studying all three of these B-cell maturation events. We extended knowledge about AID to a mammalian species that uses gene conversion to diversify rearranged immunoglobulin genes by cloning and sequencing rabbit AID, isolating AID protein from rabbit appendix-cell nuclear and cytoplasmic extracts (identity confirmed by mass spectrometry). We produced anti-AID antibody that identified AID protein in cells by immuno-histochemical and -fluorescent staining techniques and described co-localization of AID and other molecules important for Ab diversification. Although much work remains to understand fully the mechanism of action of AID and its association with other cellular components, the rabbit system now offers a particularly useful model for future studies of these dynamics (3) Recruitment of and trafficking of B-cells in young 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 previously 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. We found that appendix regulates precursor lymphocyte recruitment for further development by modulating the sites of extravasation. The total area of peripheral node addressin-positive (PNAd+) high endothelial venules (HEVs) increased from 1-day to 1-week after birth, remained constant up to 2-weeks and declined to a low and persistent amount by 3-weeks. In normal 1-week and manipulated 5-week-appendix where growth of follicles was retarded, PNAd+ HEVs were present in the basolateral sides of B-cell follicles whereas, in normal 5-wk-appendix these were restricted to T-cell areas. The PNAd was expressed on the lumenal surface of HEVs. The proportions of CD62L+ B cells in appendix declined from ~40% at 3-days to 2-3% at 4-weeks. In lymphocyte transfer experiments, CD62L+ B cells were preferentially recruited compared with CD62L- B cells, anti-PNAd antibody blocked migration of B cells by ~50%, and 100 times more B cells were recruited in 1-week compared to 6-week appendix. Thus, a unique spatiotemporal expression pattern of PNAd+ HEVs is associated with development of B-cell follicles. This regulates migration of blood-borne B-lymphocytes into developing appendix by interacting with L-selectin (CD62L) (4).Having previously shown that peripheral lymph node addressin detected by mAb MECA-79 played a role in recruitment of immature blood-borne B cells into neonatal rabbit appendix, we recently found expression of an ~127 kD O-linked sulphated proteoglycan on developing B cells in appendix and Peyer's patches recognized by the mAb MECA-79. Binding of the mAb to B lymphocytes was sensitive to enzyme treatment with O-sialoglycoprotease and expression was partially inhibited by sodium chlorate, a metabolic inhibitor of sulfation. The proportions of MECA-79+ B lymphocytes gradually increased from <0.5% at three days to >70 % at six weeks in appendix and Peyer?s patches. The proportions of MECA-79+ B lymphocytes in spleen and peripheral blood were very low (<0.5-2 %). However, the MECA-79 determinant was detected on B cells in splenic germinal centers after immunization. In situ labelling of appendix cells showed that the MECA-79 determinant was expressed on fluorescein-labelled B lymphocytes that migrated from appendix into mesenteric lymph nodes. B-cell MECA-79 may be involved in interactions with T cells and/or dendritic cells. Alternatively, because we found that lymphatic endothelium in the thymus-dependent area of appendix, a site for lymphocyte exit, expressed P-selectin (CD62P), interaction of the MECA-79 determinant on B cells with CD62P may have a role in the exit of B lymphocytes from rabbit appendix (5).

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。纯化的CD5蛋白也用于产生多克隆山羊抗兔CD5。另外，我们生成了针对CD5的每个细胞外域特异的抗肽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（1）。尽管只有一小部分小鼠和人类B细胞是CD5+，但大多数成年兔B细胞表达CD5。然而，在出生后1和3天，在新生儿附录中的大多数B细胞中无法检测到CD5。细胞运输研究表明，来自骨髓的CD5+和CD5-CD62L+ B细胞迁移到附录中。在那里，CD5+ B细胞优先扩展并占主导地位约2周。在突变的Ali/Ali兔子中，VHA2+ B细胞通过删除VH1A2上游的重新排列的VH基因的基因转换样变化而发展。突变体附录中单个CD5+生发中心和VHA2+ B细胞的相关外观表明，CD5结合积极地选择具有A2+框架区域的细胞。在负面选择和阳性选择之后，具有多样化的重排重和轻链序列的细胞退出附录，迁移到外围组织，构成遇到异物抗原的CD5+ B细胞的免疫前曲目。 （2）。 兔活化诱导脱氨酶的研究 对小鼠，人和鸡肉的研究表明，激活诱导的脱氨酶（AID）参与了导致抗体多样化的三个已知过程：体外超突变，基因转化和类转​​换重组。开发兔子附录为研究所有这三个B细胞成熟事件提供了一个特别好的站点。我们将有关辅助的知识扩展到了使用基因转化率通过克隆和测序兔辅助，从兔附录 - 附录 - 细胞核和细胞质提取物中分离出辅助蛋白的哺乳动物物种，从而使重排的免疫球蛋白基因多样化（通过质谱法证实了兔子附录核和细胞质提取物（通过质谱证实）。我们通过免疫 - 归化和荧光染色技术鉴定出细胞中的AID蛋白，并描述了对AB多样化重要的AID和其他分子的共定位。尽管还有很多工作要充分了解援助的作用机理及其与其他细胞组件的关联，但兔子系统现在为未来的这些动力学提供了一个特别有用的模型（3） 在年轻兔子附录中招募和贩运B细胞 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。我们以前表征了一些参与血液传播B细胞多步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。我们发现，附录可以调节前体淋巴细胞募集，以通过调节渗出部位来进一步发育。外围节点地址阳性（PNAD+）高内皮静脉（HEVS）的总面积从1天到出生后的1天增加到1周，最多持续2周，并以3周的速度下降至低且持续的数量。在正常的1周且操纵5周的卵泡生长中，B细胞卵泡的基底外侧有PNAD+ HEV，而PNAD+ HEV存在于B细胞卵泡的基底外侧，而在正常的5-WK-Appendix中，这些均受T-Cell区域仅限于T细胞区域。 PNAD在HEV的流体表面表达。附录中CD62L+ B细胞的比例从3天的40％下降到4周的2-3％。在淋巴细胞转移实验中，与CD62L-B细胞相比，优先募集CD62L+ B细胞，抗PNAD抗体阻断B细胞的迁移量降低了约50％，与6周的附录相比，在1周内募集了1周的B细胞。因此，PNAD+ HEV的独特时空表达模式与B细胞卵泡的发展有关。这可以调节血液传播的B淋巴细胞通过与L-选择素相互作用（CD62L）通过相互作用（4）。先前的表明，MAB MECA-79检测到的外周淋巴结寻址素在招募无效的Bl-Brol-By-by supareix ropercrance in ne neonate ropercrance中起作用MAB MECA-79识别的附录和Peyer斑块中的B细胞的蛋白聚糖。 MAB与B淋巴细胞的结合对用O-乳糖蛋白酶治疗的酶处理敏感，并且表达被氯酸钠（一种代谢抑制剂的硫酸盐抑制剂）部分抑制。 MECA-79+ B淋巴细胞的比例从附录和Peyer的斑块中的六周逐渐增加到> 70％。脾和外周血中MECA-79+ B淋巴细胞的比例非常低（<0.5-2％）。但是，免疫后，在脾生发中心的B细胞上检测到MECA-79的决定因素。附录细胞的原位标记表明，MECA-79的决定因素是在荧光素标记的B淋巴细胞上表达的，该B淋巴细胞从附录迁移到肠系膜淋巴结。 B细胞MECA-79可能与T细胞和/或树突状细胞相互作用。另外，因为我们发现附录的乳腺依赖性区域的淋巴内皮是淋巴细胞退出的位点，表达了P-链霉素（CD62P），因此B细胞上B细胞与CD62P的MECA-79在B lymphocytes（5）中可能在B lymphocytes（5）中起作用。