Ig Genetics, Ontogeny and Differentiation of Cells of the Rabbit Immune System

兔免疫系统细胞的 Ig 遗传学、个体发育和分化

• 批准号: 7592110
• 负责人: rose G. mage
• 金额: $ 37.96万
• 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/7592110
• 关键词: Animal Model; Animals; Antibodies; Antibody Repertoire; Antigens; Appendix; Archives; Area; B cell differentiation; B cell repertoire; B-Cell Development; B-Lymphocytes; Binding; Binding Sites; Biotechnology; Blocking Antibodies; Blood; Bone Marrow; Breeding; Bursa of Fabricius; CSNK1A1 gene; Cell Differentiation process; Cell Maturation; Cells; Chickens; Clonal Expansion; Cloning; Collaborations; Communicable Diseases; Computer Systems Development; Databases; Dendritic Cells; Depth; Development; Educational workshop; Enzymes; Event; Extracellular Domain; Extramural Activities; Extravasation; Fluorescein; Fluoresceins; Framework Regions; Future; Gene Conversion; Gene Rearrangement; Genes; Genetic; Genetic Polymorphism; Genome; Growth; Gut associated lymphoid tissue; High Endothelial Venule; Homologous Gene; Human; Humoral Immunities; IGH@ gene cluster; Immune system; Immunization; Immunoglobulin Allotypes; Immunoglobulin Class Switching; Immunoglobulin Genes; Immunoglobulin M; Immunoglobulin Somatic Hypermutation; Immunoglobulin Switch Recombination; Immunoglobulins; Immunologic Memory; Immunology; In Situ; Individual; Infectious Disease Immunology; Inorganic Sulfates; Institutes; Knowledge; L-Selectin; Label; Laboratories; Light; Link; Lymphatic Endothelium; Lymphocyte; Lymphoid; Mesentery; Metabolic; Modeling; Mus; National Institute of Allergy and Infectious Disease; Neonatal; Nuclear; O-sialoglycoprotein endopeptidase; Organ; Oryctolagus cuniculus; P-Selectin; PNAd; Paper; Pattern; Play; Process; Proteins; Proteoglycan; RNA Splicing; Receptors, Antigen, B-Cell; Recombinant Proteins; Recombinants; Recommendation; Records; Recruitment Activity; Reporting; Research Personnel; Resources; Role; SELL gene; SELP gene; Schedule; Signal Transduction; Site; Spleen; Staining method; Stains; Structure of aggregated lymphoid follicle of small intestine; Structure of germinal center of lymph node; Study models; Superantigens; Support Contracts; Surface Immunoglobulins; System; T-Lymphocyte; Techniques; Thymus Gland; Time; Transfer Agreement; United States National Institutes of Health; Unspecified or Sulfate Ion Sulfates; Week; Work; Yang; activation-induced cytidine deaminase; animal breeding; day; design; gene cloning; genetic resource; human disease; inhibitor/antagonist; interest; intracellular protein transport; lymph nodes; microbial; migration; mutant; peripheral blood; programs; protein localization location; protein structure function; receptor; research study; sialyl Lewis x; sodium chlorate; spatiotemporal; sulfation; trafficking; variable region gene

Rabbits are the only or best animal model for several infectious diseases. The sequence of the rabbit genome is currently unfinished at 2x, but deeper 7x coverage is starting soon. A 2006 NHGRI recommendation that rabbit be sequenced more deeply, describes the NIAID allotype-defined rabbits as a valuable resource for future SNP discovery. They have polymorphisms of a variety of immune system genes including allelic allotypes of the VH, CH, and CL regions of antibody molecules. The colony also contains descendants of rabbits formerly at the Basel Institute for Immunology, including mutant and wild-type parental of VH1a2-deleted Alicia, CK1 splicing defective Basilea, and several VH-CH recombinant heavy chain types. These rabbits are now available to interested individuals, particularly to sites where breeding colonies can be established. A 4D relational database contains more than 45 years of breeding records and other information about animals in the colony. The mutant ali animals have a deletion of a key variable region gene in the immunoglobulin heavy chain locus that is present in related 2R1 wild type rabbits. The mutants may be more susceptibe to infectious disease because they have abnormal delayed development of humoral immunity. In part as the result of a White Paper I coauthored in 2005 proposing deep sequencing of the rabbit genome, 7x coverage and assembly is scheduled to start in September 2007 at Broad Institute MIT and Harvard. The sequences we have been able to find and use in the incomplete 2x trace archive, have already proven useful for various studies in progress. As a result of my efforts, two websites are now available one maintained by the National Center for Biotechnology Information (NCBI) http://www.ncbi.nlm.nih.gov/projects/genome/guide/rabbit/ -- and one by NIAID on Rabbit in Immunology & Infectious Disease (http://www3.niaid.nih.gov/research/resources/ri/. The latter site offers a summary of an NIAID workshop on Rabbit Models of Human Infectious diseases held in 2005 that I helped the extramural NIAID DMID to program and chair.. Studies of 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. Modified versions of the initially expressed construct were designed and expressed in order to obtain recombinant protein(s) that may crystallize more readily 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 and producing anti-AID antibody that identified AID protein in cells by immuno-histochemical and -fluorescent staining techniques. Although much work remains to understand fully the mechanism of action of AID and its association with other cellular components,co-localization of AID and other molecules important for Ab diversification was observed. The rabbit system now offers a particularly useful model for future studies of these dynamics (Yang G, Obiakor H, Sinha RK, Newman BA, Hood BL, Conrads TP, Veenstra TD, Mage RG, Activation-induced deaminase cloning, localization, and protein extraction from young VH-mutant rabbit appendix. Proc Natl Acad Sci U S A 102:17083-8, 2005). The antibodies characterized in this report are being used in collaboration with other investigators. 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. We found that appendix regulates precursor lymphocyte recruitment for further development by modulating the sites of extravasation. 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). 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 then found expression of an 127 kD O-linked sulphated proteoglycan on developing B cells in appendix and Peyers 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 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 (1).

兔是几种传染病的唯一或最好的动物模型。目前，兔基因组的2倍序列尚未完成，但更深入的7倍覆盖很快就会开始。2006年NHGRI建议对兔子进行更深入的测序，将NIAID同种型定义的兔子描述为未来发现SNP的宝贵资源。它们具有多种免疫系统基因的多态性，包括抗体分子的VH、CH和CL区域的等位基因异型。该群体还包含以前在巴塞尔免疫研究所的兔子后代，包括vh1a2缺失的突变型和野生型亲本Alicia， CK1剪接缺陷的Basilea，以及几种VH-CH重组重链型。这些兔子现在可供感兴趣的个人使用，特别是可以建立繁殖殖民地的地点。一个4D关系数据库包含了超过45年的繁殖记录和其他关于该群体动物的信息。突变的ali动物在相关的2R1野生型兔子中存在免疫球蛋白重链位点的关键可变区基因缺失。突变体可能更容易感染传染病，因为它们的体液免疫发育异常延迟。2005年，我与人合著了一份白皮书，提议对兔子基因组进行深度测序，这部分是由于这份白皮书的结果，7倍的覆盖范围和组装计划于2007年9月在麻省理工学院和哈佛大学的布罗德研究所开始。我们已经能够在不完整的2x跟踪档案中找到并使用的序列，已经被证明对正在进行的各种研究有用。由于我的努力，现在有两个网站可用，一个是由国家生物技术信息中心（NCBI） http://www.ncbi.nlm.nih.gov/projects/genome/guide/rabbit/维护的，另一个是由NIAID维护的兔免疫学和传染病网站（http://www3.niaid.nih.gov/research/resources/ri/）。后一个站点提供了2005年我帮助NIAID DMID组织和主持的关于人类传染病兔子模型的NIAID研讨会的摘要。