Human B1 Cell Immunoglobulin

描述(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地位。然而，B细胞促进自身免疫的方式，尤其是致病性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，这一点一直无法评估。我们最近发现人类B1细胞共表达CD20、CD27和CD43，而不表达CD70，填补了这一空白。有了B1细胞的表型，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具有许多特征，表明与自身免疫有关。人类B1细胞产生抗DNA自身抗体；人类B1细胞表达体细胞突变的抗体；人类B1细胞来源的抗体可以同型转换。在狼疮中，不仅B1细胞在所有B细胞中的比例增加了2倍，而且表达免疫球蛋白的B1细胞的比例显著增加了16倍(达到所有B1细胞的35%)。因此，产生自身反应性免疫球蛋白、体细胞突变免疫球蛋白以及在较大(狼疮)或较小程度(正常)程度上产生同型转换免疫球蛋白的人B1细胞，表达了作为自身反应/突变/转换自身抗体来源的必要属性，该抗体被认为是致病的。我们假设在狼疮B1细胞中，特别是在免疫球蛋白+B1细胞中，实际上确实是致病自身抗体的来源。为了解决这一假设，我们将研究由B1、幼稚和记忆B细胞产生的Ig，将狼疮患者的B细胞与正常对照进行比较，并将免疫球蛋白+B1细胞与非转换的B1细胞进行比较。具体地说，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，重点是VH/VD/VJ/VL的使用、体细胞突变、N区添加以及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法和自身抗原阵列来评估B1细胞Ig的谱系，重点是多反应性抗体和自身反应性抗体的频率；3)我们将通过肾小球结合/免疫荧光和体内注射小鼠自身抗体后肾脏的组织学检查来评估B1细胞Ig的致病性，重点是抗DNA自身反应/多反应抗体对肾脏造成损害的能力。我们假设，在狼疮中，B1细胞产生致病的、致肾病的抗体，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”类型的自身反应性Ig的起源。 这一直被认为是生发中心后常规B细胞的范围。这项工作的结果将为狼疮的新的和离散的治疗靶点奠定基础，该靶点可能消除对所有成熟B细胞的大规模破坏的需要。

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.