Somatic Diversification of Immunoglobulin Genes in Galt

描述(申请人提供)：肠道共生微生物区系是脊椎动物淋巴组织和粘膜免疫发育所必需的。然而，人们对这些过程发生的机制知之甚少。以前在兔身上的工作已经证明，在免疫前抗体库的发展过程中，肠道共生微生物区系是B细胞扩张和Ig基因体细胞多样化所必需的，这两者都存在于肠道相关淋巴组织(GALT)中。这些过程在出生后几天内以不依赖于抗原和T细胞的方式发生，令人惊讶的是，它们需要激活补体。我们假设了一个高尔特发育模型，在这个模型中，B细胞在出生后不久就从骨髓进入GalT，并迁移到滤泡相关上皮(FAE)，并从管腔与C‘包被的细菌相互作用。然后将这些复合体传递给FDC，FDC为B细胞激活和上调AID表达提供刺激信号；在出生后几周内，基本上所有的Ig基因都是体细胞多样化的，从而为兔子提供了不同的抗体库。这笔赠款的目标是测试高尔特开发的这种模式。在目标1中，我们将使用可溶性趋化因子受体来抑制趋化因子驱动的迁移，并确定B细胞的迁移是如何改变的；在目标2中，我们将使用无菌GALT外植体以及TLR和NLR激动剂和拮抗剂来确定TLR和NLR配体以及与IgM结合的细菌超抗原样分子对B细胞增殖和Ig基因多样化的贡献；我们还将使用GALT无菌外植体以及体外FDC：B细胞培养系统来确定补体激活如何促进GALT的发育。在目标3中，我们将在诺生菌CD4-/-小鼠中寻找以抗原和T细胞无关的方式增殖并经历Ig基因体细胞多样化的GalT样B细胞。这些实验很重要，因为它们将开始确定共生细菌和补体对粘膜淋巴组织的发育并最终对人类健康和过敏和炎症性肠病等疾病做出贡献的机制。

DESCRIPTION (provided by applicant): The intestinal commensal microbiota is required for development of lymphoid tissues and mucosal immunity in vertebrates. However, little is known of the mechanism by which these processes occur. Previous work in rabbits has demonstrated that the intestinal commensal microbiota is required for expansion of B cells and somatic diversification of Ig genes during development of the preimmune antibody repertoire, both of which occur in gut-associated lymphoid tissues (GALT). These processes occur within a few days after birth in an antigen- and T cell-independent manner, and surprisingly, they require activation of complement. We hypothesize a model for GALT development in which B cells enter GALT from the bone marrow soon after birth and migrate to the follicle-associated epithelium (FAE) and interact with C'-coated bacteria from the lumen. These complexes are then delivered to FDCs which provide stimulatory signals for B cell activation and upregulation of AID expression; within a few weeks after birth, essentially all Ig genes are somatically diversified, thereby providing the rabbits with a diverse antibody repertoire. The goal of this grant is to test this model of GALT development. In Aim 1 we will use soluble chemokine receptors to inhibit chemokine-driven migration and determine how the migration of B cells is altered; in Aim 2, we will use sterile GALT explants and TLR and NLR agonists and antagonists to determine the contribution of TLR and NLR ligands and IgM-binding bacterial superantigen-like molecules to B cell proliferation and Ig gene diversification; we will also use sterile explants of GALT as well as an in vitro FDC:B cell culture system to determine how complement activation contributes to GALT development. In Aim 3, we will search in gnotobiotic CD4-/- mice for GALT-like B cells that proliferate in an antigen and T-cell independent manner and undergo somatic diversification of the Ig genes. These experiments are important because they will begin to determine the mechanism by which commensal bacteria and complement contribute to development of mucosal lymphoid tissues and ultimately to human health and disease such as allergy and inflammatory bowel disease.