Human B1 Cell Immunoglobulin

描述（由申请人提供）：该项目涉及人类B1细胞，以及该B细胞亚群产生的免疫球蛋白（Ig）。近年来，B细胞耗竭疗法的成功提高了B细胞作为自身免疫的关键贡献者的形象。然而，B细胞促进自身免疫的手段，特别是致病自身抗体的确切来源，还没有完全弄清楚。多年来，人们一直认为B1细胞可能与狼疮和相关疾病有关，但由于缺乏人类B1细胞的识别标记，因此无法对其进行评估。我们最近的发现填补了这一空白，即人类B1细胞共表达CD20、CD27和CD43，缺乏CD70的表达。有了B1细胞的表型在手，就有可能检查B1细胞产生的Ig的性质。人类B1细胞产生的Ig具有许多与自身免疫有关的特征。人B1细胞产生抗dna自身抗体；人B1细胞表达体细胞突变的抗体；并且，人B1细胞源性抗体可以进行同型转换。在狼疮中，不仅B1细胞在所有B细胞中的比例增加了2倍，而且表达IgG的B1细胞的比例也显著增加了16倍（占所有B1细胞的35%）。因此，产生自身反应性Ig、体细胞突变Ig以及更大程度（狼疮）或更小程度（正常）的同型转换Ig的人B1细胞表达了成为被认为是致病的自身反应性/突变/转换自身抗体来源的必要属性。我们假设狼疮的B1细胞，特别是IgG+ B1细胞，确实是致病自身抗体的来源。为了验证这一假设，我们将研究由B1、幼稚B细胞和记忆B细胞产生的Ig，比较狼疮患者的B细胞与正常对照，并比较IgG+ B1细胞与未转换的B1细胞。具体而言，1)我们将通过单细胞分选、PCR扩增和测序来评估B1细胞Ig的结构，重点关注VH/VD/VJ/VL的使用、体细胞突变、n区添加和CDR3的长度和电荷；2)我们将通过表达克隆、抗体纯化、HEp-2免疫荧光、ELISA测定和自身抗原阵列来评估B1细胞Ig的库，重点关注多反应性、自身反应性抗体的频率；3)我们将通过小鼠体内自身抗体给药后的肾小球结合/免疫荧光和肾脏组织学检查来评估B1细胞Ig的致病性，重点研究抗dna自身反应性/多反应性抗体对肾脏的损害能力。我们假设，在狼疮中，B1细胞产生致病的肾源性抗体，这些抗体是体细胞突变和同型转换的，因此至少代表了一些“经典”型自身反应性Ig的起源

DESCRIPTION (provided by applicant): This project concerns human B1 cells, and the immunoglobulin (Ig) produced by this B cell subpopulation. In recent years the success of B cell depletion therapies has raised the profile of B cells as key contributors to autoimmunity. However, the means by which B cells promote autoimmunity, and especially the precise origin of pathogenic autoantibodies, have not been fully worked out. For many years it has been suggested that B1 cells might be involved in lupus and related illnesses, but this has been impossible to evaluate because identifying markers for human B1 cells have been lacking. This gap has now been filled with our recent finding that human B1 cells co-express CD20, CD27 and CD43, and lack expression of CD70. With the phenotype of B1 cells in hand it is possible to examine the nature of B1 cell-generated Ig. Human B1 cells produce Ig with many features that suggest a relationship to autoimmunity. Human B1 cells produce anti-DNA autoantibody; human B1 cells express antibody that is somatically mutated; and, human B1 cell- derived antibody can be isotype-switched. And in lupus, not only is the proportion of B1 cells among all B cells increased 2-fold, but the fraction of B1 cells that express IgG is markedly increased 16-fold (to 35% of all B1 cells). Thus, human B1 cells that produce autoreactive Ig, somatically mutated Ig, and to a greater (lupus) or lesser (normal) extent, isotype-switched Ig, express the necessary attributes to be a source of autoreactive/mutated/switched autoantibody that is recognized as pathogenic. We hypothesize then that in lupus B1 cells, particularly IgG+ B1 cells, do, in fact, represent a source of pathogenic autoantibody. To address this hypothesis, we will study Ig produced by B1, naive, and memory B cells, comparing B cells from lupus patients with normal controls, and comparing IgG+ B1 cells with non-switched B1 cells. Specifically, 1) We will evaluate the structure of B1 cell Ig by single cell sorting, PCR amplification, and sequencing, focusing on VH/VD/VJ/VL usage, somatic mutation, N-region addition, and CDR3 length and charge; 2) We will evaluate the repertoire of B1 cell Ig by expression cloning, antibody purification, HEp-2 immunofluorescence, ELISA assay, and autoantigen array, focusing on the frequency of polyreactive, autoreactive antibodies; and, 3) We will evaluate the pathogenicity of B1 cell Ig by glomerular binding/immunofluorescence and histological examination of kidneys after murine in vivo autoantibody administration, focusing on the capacity of anti-DNA autoreactive/polyreactive antibodies to produce renal damage. We hypothesize that in lupus, B1 cells generate pathogenic, nephritogenic antibodies that are somatically mutated and isotype-switched, and thus represent the origin of at least some of the "classical" type of autoreactive Ig that has been thought to be purview of post-germinal center conventional B cells. The results of this work will lay the foundation for a new and discrete therapeutic target in lupus that may eliminate the need for wholesale destruction of all mature B cells. PUBLIC HEALTH RELEVANCE: The recent successful use of B cell depletion therapy shows that B cells play an important role in autoimmune diseases. Newly discovered human B1 cells are a unique subpopulation of B cells capable of making autoantibodies and thus could be directly involved in initiating autoimmunity; further, we have found that human B1 cells are expanded in patients with lupus autoimmunity. The goal of the present proposal is to gather evidence indicating that B1 cells are a source of autoantibodies in lupus and thereby contribute to lupus disease, which will provide the foundation for new targeted therapies designed to eliminate the "bad" pathogenic B1 cells without affecting the majority population of B cells needed for immunity against infectious pathogens.