An Advanced Model to Analyze CD4T Cel Reactions to Antibody and BCR V Regions

分析 CD4T 细胞对抗体和 BCR V 区反应的高级模型

• 批准号: 8311662
• 负责人: Lawrence J Wysocki
• 金额: $ 7.93万
• 依托单位: NATIONAL JEWISH HEALTH
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-04 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8311662
• 关键词: 4-hydroxy-5-nitrophenyl acetic acid; A/J Mouse; Abbreviations; Ablation; Adoptive Transfer; Affect; Affinity; Animals; Antibodies; Antibody Formation; Antigen Presentation; Antigen Receptors; Antigen-Antibody Complex; Antigens; Autoantigens; Autoimmune Diseases; Autoimmune Process; Autoimmunity; B-Cell Development; B-Lymphocytes; BAC (bacterial artificial chromosome); Back; Binding; Bone Marrow; Breeding; C57BL/6 Mouse; CD4 Positive T Lymphocytes; Chromosomes, Human, 4-5; Clinical; Complementary DNA; Complex; Cross Reactions; Cyan Fluorescent Protein; Data; Deposition; Development; Dissection; Engineering; Epitopes; Event; Fc Receptor; Fluorescence; Future; Gene Targeting; Genes; Genetic; Genetic Recombination; Genomics; Green Fluorescent Proteins; Haptens; Health Hazards; Helper-Inducer T-Lymphocyte; Immune system; Immunity; Immunoglobulin G; Immunoglobulin M; Immunoglobulin Somatic Hypermutation; Immunoglobulins; Inflammation; Inflammatory; Knock-in Mouse; Laboratories; Lectin; Libraries; Life; Light; Link; Location; Lymphopoiesis; Mediating; Memory B-Lymphocyte; Modeling; Mouse Strains; Mus; Mutation; Nuclear; Nuclear Antigens; Organ; Ovalbumin; Ovum; Patients; Peptide antibodies; Peptides; Physiological; Play; Point Mutation; Polysaccharides; Process; Production; Publishing; Reaction; Reagent; Receptors, Antigen, B-Cell; Refractory; Reporter; Role; Self Tolerance; Site; Source; Specificity; Staining method; Stains; Structure of germinal center of lymph node; Surface; Systemic Lupus Erythematosus; T cell differentiation; T cell response; T-Lymphocyte; Testing; Therapeutic; Therapeutic Monoclonal Antibodies; Thymus Gland; Tissues; Transgenic Mice; Transgenic Organisms; Work; acquired immunity; adverse outcome; antigen processing; autoreactive B cell; base; chemokine receptor; genetic analysis; immunogenic; immunogenicity; immunoregulation; mutant; neutralizing antibody; novel; prevent; receptor; response; sound; systemic autoimmune disease; therapy design

DESCRIPTION (provided by applicant): An advanced model to analyze CD4 T cell reactions to antibody and BCR V regions Project Summary This is an R03 proposal to generate an antibody construct and a corresponding transgenic mouse for genetic analyses of T cell reactions to V region peptides derived from antibodies and B cell antigen receptors (BCR). CD4 T cells make a living by interacting with B cells that present antigenic peptides in MHC II. While some of these peptides come from foreign antigen, others are neoantigens generated in the BCR through the processes of V/D/J gene recombination in the bone marrow and somatic hypermutation in the periphery. Neoantigenic BCR peptides in MHC II open a potential avenue of T cell help to B cells, in violation of the principle of linked recognition. We refer to this as the receptor presentation avenue of help. Neoantigenic Ab peptides may also activate CD4 T cells at sites of Ab deposition where APC gather Ab through Fc receptors or surface lectins. At present, we have only a rudimentary understanding of how the immune system regulates potentially dangerous interactions between CD4 T cells and B cells or other APC that are mediated by BCR and Ab peptides. As an example, IgG is ostensibly tolerogenic; yet therapeutic mAb sometimes elicit an endogenous neutralizing antibody response with a potential clinical health hazard. We are pursuing adoptive transfer studies to reveal the consequences of T cell reactions to BCR- and Ab- derived peptides using a model based in mice of the strain A genetic background. Our most recent results indicate that the "receptor presentation" avenue of T help to B cells is subject to immunoregulation. T-B interactions in the GC that are mediated by BCR-derived peptides impede GC participation and memory B cell development and ultimately induce a refractory state in the T cells. Efforts to dissect this novel form of immunoregulation and to explore the issue of Ab immunogenicity versus tolerogenicity, would be greatly enhanced if we could utilize the available library of genetically altered C57BL/6 (B6) mice. Accordingly, we would like to develop a corresponding model in the B6 strain so that genetic alterations can be introduced into adoptively transferred B cells, T cells or recipient mice. A B6 model would also enable us to genetically analyze CD4 T cell reactions to an antibody V region peptide in adoptive recipients of T cells that are injected with mAb carrying the cognate peptide. To this end, we propose to engineer an Ig; light chain construct that contains a sequence encoding a peptide called 3K, which is immunogenic for CD4 T cells in the context of by I-Ab (Aim 1). This will enable production of hapten-specific (NP) mAb of various isotypes for planned future studies of T cell responses to 3K;Ig, immune complexes thereof and glycan modified forms of 3K;Ig. For these studies, we would use a currently available 12TCR Tg mouse as a source of 3K-specific CD4 T cells. In Aim 2, we propose to engineer a modified 3K; genomic construct with a GFP reporter and generate a corresponding B6 transgenic mouse. This mouse will be crossed with a knock-in mouse (B1-8) that carries a complementary targeted heavy chain from an Ig; NP-specific mAb to produce a mouse with NP-specific 3K; B cells. These B cells can then be used with 3K-specific T cells in future co-adoptive transfer genetic studies of receptor presentation. The products of this work will enable a genetic dissection of mechanisms that regulate T cell responses to Ig V region peptides in the BCR or secreted antibody. The anticipated results from this model will advance our understanding of immunoregulation in the face of neoantigens that frequently arise in BCR and Ab and are presented to CD4 T cells on a regular basis during immunity. Abbreviations ANA: Anti-nuclear antibody BAC: Bacterial artificial chromosome BCR: B cell antigen receptor CFP: Cyan fluorescent protein GFP: Green fluorescent protein GC: Germinal Center NP: (4-hydroxy-3-nitro-phenyl)acetyl SHM: Somatic hypermutation

描述（由申请人提供）：用于分析 CD4 T 细胞对抗体和 BCR V 区反应的高级模型 项目摘要 这是一项 R03 提案，旨在生成抗体构建体和相应的转基因小鼠，用于对 T 细胞对 V 区肽的反应进行遗传分析源自抗体和 B 细胞抗原受体 (BCR)。 CD4 T 细胞通过与在 MHC II 中呈递抗原肽的 B 细胞相互作用而生存。虽然这些肽中的一些来自外来抗原，但其他肽是通过骨髓中的 V/D/J 基因重组和外周体细胞超突变过程在 BCR 中产生的新抗原。 MHC II 中的新抗原 BCR 肽开辟了 T 细胞帮助 B 细胞的潜在途径，违反了连锁识别原理。我们将此称为受体呈递帮助途径。新抗原 Ab 肽还可以激活 APC 通过 Fc 受体或表面凝集素聚集 Ab 的 Ab 沉积位点的 CD4 T 细胞。目前，我们对免疫系统如何调节 CD4 T 细胞与 B 细胞或其他由 BCR 和 Ab 肽介导的 APC 之间潜在危险的相互作用只有初步了解。例如，IgG 表面上具有耐受性；然而，治疗性单克隆抗体有时会引起内源性中和抗体反应，从而对临床健康造成潜在危害。我们正在进行过继转移研究，以使用基于 A 品系遗传背景的小鼠模型来揭示 T 细胞对 BCR 和 Ab 衍生肽反应的后果。我们最新的结果表明，T 细胞对 B 细胞的帮助的“受体呈递”途径受到免疫调节的影响。 GC 中由 BCR 衍生肽介导的 T-B 相互作用会阻碍 GC 参与和记忆 B 细胞发育，并最终诱导 T 细胞进入难治状态。如果我们能够利用现有的基因改造 C57BL/6 (B6) 小鼠文库，那么剖析这种新型免疫调节形式并探索抗体免疫原性与耐受原性问题的努力将会大大加强。因此，我们希望在 B6 品系中开发相应的模型，以便将基因改变引入过继转移的 B 细胞、T 细胞或受体小鼠中。 B6 模型还使我们能够对注射了携带同源肽的 mAb 的 T 细胞收养受体中的 CD4 T 细胞对抗体 V 区肽的反应进行基因分析。为此，我们建议设计一种 Ig；轻链构建体包含编码 3K 肽的序列，该肽在 I-Ab 的背景下对 CD4 T 细胞具有免疫原性（目标 1）。这将能够生产各种同种型的半抗原特异性 (NP) mAb，用于计划的未来研究 T 细胞对 3K;Ig、其免疫复合物和 3K;Ig 聚糖修饰形式的反应。对于这些研究，我们将使用目前可用的 12TCR Tg 小鼠作为 3K 特异性 CD4 T 细胞的来源。在目标 2 中，我们建议设计一个改进的 3K；带有 GFP 报告基因的基因组构建体并生成相应的 B6 转基因小鼠。该小鼠将与携带 Ig 互补靶向重链的敲入小鼠 (B1-8) 杂交； NP 特异性单克隆抗体，用于产生具有 NP 特异性 3K 的小鼠； B细胞。然后，这些 B 细胞可以与 3K 特异性 T 细胞一起用于未来受体呈递的共收转移遗传学研究。这项工作的产品将能够对调节 T 细胞对 BCR 或分泌抗体中 Ig V 区肽的反应的机制进行基因剖析。该模型的预期结果将增进我们对新抗原的免疫调节的理解，这些新抗原经常出现在 BCR 和 Ab 中，并在免疫过程中定期呈递给 CD4 T 细胞。缩写 ANA：抗核抗体 BAC：细菌人工染色体 BCR：B 细胞抗原受体 CFP：青色荧光蛋白 GFP：绿色荧光蛋白 GC：生发中心 NP：(4-羟基-3-硝基-苯基)乙酰基 SHM：体细胞超突变