Development of novel reagents to identify xenograft reactive B cells

项目3：开发鉴定异种移植反应性B细胞的新试剂 项目摘要 供体器官短缺是当今器官移植面临的最严峻挑战。猪是 被认为是一种有希望的替代组织来源。不幸的是，异种移植，共享 由于人类产生了很强的体液免疫反应，所以不能用于临床。 朝向猪的组织。我们的长期目标是使猪的组织适合于人类使用，方法是防止 人类抗体的结合。这个项目的中心假设是猪的主要组织相容性 复合(MHC)蛋白在猪中被称为猪白细胞抗原(SLA)，有助于异种抗原性。 我们已经证明，具有人类白细胞抗原抗体的患者通常会发生交叉反应。 与相应的二语习得。我们还发现，大约25%的缺乏人类白细胞抗原的人口 抗体还具有结合SLA的免疫球蛋白G和免疫球蛋白M。停用SLA以消除其对 人类抗猪免疫是有问题的，因为SLA是帮助保护器官免受感染的关键 和癌症。这笔赠款的目的是：(1)增进我们对以下频率的了解 患者有抗SLA抗体；(Ii)确定产生这些抗体的B细胞的来源，以及(Iii) 确定他们对B细胞耗竭疗法(利妥昔单抗)的敏感性。我们的方法是创新的，因为它将 开发以类似方法为基础的新工具，这些方法在 同种异体移植。我们的理由是，这种知识将使我们更接近使用猪作为一种 通过帮助更好地匹配捐赠者和接受者，器官来源。在特定的目标1中，我们将创建重组 SLA，产生这些蛋白质的珠状阵列，并使用该阵列来筛选500份人类血清中存在的 SLA抗体。具体目标2.1的重点是在利妥昔单抗治疗前后筛查25名患者 用SLA珠阵列来确定这些异种反应性抗体是否减少。具体目标2.2将确定 并对SLA特异性B细胞进行表征，以确定天然抗体是否与SLA的某些反应性有关。 这项技术开发将极大地促进将猪捐赠者与人类配对的能力 通过避免或消除SLA特异性抗体。该项目将依靠以下几个组件 这个奖项是为了实现它的目标。Core C将提供用于血清筛查和SLA四聚体的珠子阵列 来分析核心B提供的B细胞。项目2调查人员和核心D将帮助分类和 四聚体染色B细胞的分析。项目1将提供Rituximab样本和数据，并协助 与他们确定B细胞耗尽是否会影响SLA特异性抗体的工作进行比较 出现类似的抗多糖抗体。

Project 3: Development of Novel Reagents to Identify Xenograft Reactive B Cells Project Summary A shortage of available donor organs is the most critical challenge facing organ transplantation today. Pigs are considered a promising source of replacement tissues. Unfortunately, xenotransplantation, the sharing of organs across species, is not clinically applied because humans exert a strong humoral immune response toward pig tissues. Our long-term goal has been to make pig tissues suitable for use in humans by preventing the binding of human antibodies. The central hypothesis of this project is that pig Major Histocompatibility Complex (MHC) proteins, known as Swine Leukocyte Antigens (SLA) in the pig, contribute to xenoantigenicity. We have shown that patients having antibodies against Human Leukocyte Antigens (HLA) often cross-react with the homologous SLA. We have also found that approximately 25% of the population who lack HLA antibodies also have IgG and IgM capable of binding SLA. Inactivating SLA to eliminate their contribution to human anti-pig immunity is problematic because the SLA are key to helping protect the organ from infection and cancer. The objectives of this grant are to: (i) improve our understanding of the frequency with which patients have anti-SLA antibodies; (ii) determine the origin of B cells which produce those antibodies, and (iii) define their susceptibility to a B-cell depleting therapy (Rituximab). Our approach is innovative because it will develop novel tools that build on similar approaches that have been successful in the setting of allotransplantation. Our rationale is that this knowledge will bring us bring us closer to the use of pigs as an organ source by helping to better match donors with recipients. In Specific Aim 1 we will create recombinant SLA, produce a bead array of these proteins, and use the array to screen 500 human sera for the presence of SLA antibodies. The focus of Specific Aim 2.1 is to screen 25 patients before and after Rituximab treatment with the SLA bead array to determine if those xenoreactive antibodies diminish. Specific Aim 2.2 will identify and characterize SLA-specific B cells to determine if natural antibodies account for some of the SLA reactivity. This technology development will contribute significantly to the ability to match pig donors with human recipients by avoiding or eliminating SLA-specific antibodies. This project will rely on several components of this award to achieve its goals. Core C will provide bead arrays for serum screening and SLA tetramers with which to analyze B cells provided by Core B. Project 2 investigators and Core D will help with the sorting and analyses of tetramer-stained B cells. Project 1 will provide Rituximab samples and data and assist in comparisons to their work to determine if B cell depletion affects SLA-specific antibodies and naturally occurring anti-glycan antibodies similarly.