Role of CD47 in xenograft rejection by macrophages

描述(申请人提供)：猪的异种移植有可能解决人类器官捐赠者日益短缺的问题。由于供者和宿主之间广泛的分子不相容，先天免疫在异种移植排斥反应中的作用远大于在同种异体移植排斥反应中的作用。CD47广泛表达，是巨噬细胞和DC的抑制性受体SIRP？的配基。在目前的资助期间，我们证明了CD47-SIRP中缺乏跨物种的相互作用？巨噬细胞介导的异基因造血和非造血细胞移植排斥反应主要是通过巨噬细胞途径引起的。CD47缺陷细胞移植可诱导同基因野生型(WT)小鼠快速的先天免疫激活。此外，CD47-SIRP？供者特异性输血(DST)后，需要信号抑制受体CD11hiCD8？-DC的活化，诱导耐受。基于申请书中提供的这些和其他数据，我们假设供体CD47和宿主SIRP之间的相互作用？对于控制SIRP？+巨噬细胞和DC的激活至关重要，这种相互作用的缺失会激活宿主巨噬细胞和DC，从而刺激抗供体T细胞反应。在这里，我们提出了三个具体的目标来检验我们的假设。目的1阐明CD47-SIPR的作用。异种肝细胞移植后SIRP？+天然免疫细胞激活的调节信号。我们将CD47KO小鼠肝细胞移植到同基因WT小鼠体内，以分析CD47差异单独诱导的先天免疫细胞活化和移植物排斥反应。我们还将人CD47转基因猪肝细胞移植到人源化小鼠，以确定人CD47表达是否可能抑制人类对异种猪移植的先天免疫反应。目的2探讨“缺失CD47”诱导的天然免疫细胞活化在肝细胞移植后T细胞异种反应中的作用。我们将首先在同种移植模型中解决这个问题，在该模型中，CD47 KO小鼠将被用来模拟CD47血型不合的异种供者。我们将通过体外和过继T细胞转移试验来检测抗供体T细胞反应。然后，我们将研究人类CD47在猪肝细胞上表达的可能性，以减弱具有人类免疫系统功能的人源化小鼠的人类T细胞异种免疫反应。目的3是确定供体细胞上的CD47促进DST加共刺激阻断治疗受者耐受诱导的机制。我们将确定在CD47缺陷的DST后迅速激活的DC和巨噬细胞群体，以及它们在T细胞激活和耐受诱导中的作用。我们将在CD47缺陷的同种异体心脏移植的小鼠模型和猪到小鼠的异种移植模型中解决这些问题，该模型涉及从人CD47转基因猪到人源化小鼠的DST。这些研究有望为CD47血型不合激活先天和获得性异种免疫反应的机制，以及使用人CD47转基因猪作为供者促进异种耐受诱导和异种移植存活的可能性提供重要的见解。

DESCRIPTION (provided by applicant): Xenotransplantation from pigs has the potential to resolve the growing shortage of human organ donors. Because of the extensive molecular incompatibilities between the donor and host, innate immunity plays a much greater role in xenograft rejection than in allograft rejection. CD47 is ubiquitously expressed and serves as a ligand of SIRP?, an inhibitory receptor on macrophages and DCs. During the current funding period, we demonstrated that the lack of cross-species interaction in CD47-SIRP? pathway largely accounts for macrophage-mediated rejection of hematopoietic and non-hematopoietic cellular xenografts. Transplantation of CD47-deficient cells induces rapid innate immune activation in syngeneic wild-type (WT) mice. Furthermore, CD47-SIRP? signal is required to repress recipient CD11hiCD8?- DC activation and induce tolerance after donor-specific transfusion (DST). Based on these and other data presented in the application, we hypothesize that the interaction between donor CD47 and host SIRP? is essential for controlling activation of SIRP?+ macrophages and DCs, and that the absence of this interaction activates host macrophages and DCs, hence stimulating anti-donor T cell responses. Here, we propose 3 specific aims to test our hypothesis. Aim 1 is to elucidate the role of CD47-SIPR? signaling in the regulation of SIRP?+ innate immune cell activation after hepatocyte xenotransplantation. We will transplant CD47 KO mouse hepatocytes into syngeneic WT mice to analyze innate immune cell activation and graft rejection induced solely by CD47 disparity. We will also transplant human CD47 transgenic vs. control pig hepatocytes into humanized mice to determine whether human CD47 expression may inhibit human innate immune responses to pig xenografts. Aim 2 is to determine the role of "missing CD47"-induced innate immune cell activation in T cell xenoresponses after hepatocyte transplantation. We will first address this question in allotransplant models, in which CD47 KO mice will be used to mimic CD47-incompatible xenogeneic donors. We will examine anti-donor T cell responses by in vitro and adoptive T cell transfer assays. We will then investigate the potential of human CD47 expression on pig hepatocytes to attenuate human T cell xenoimmune responses using humanized mice with a functional human immune system. Aim 3 is to determine the mechanisms by which CD47 on donor cells facilitates tolerance induction in DST plus costimulatory blockade-treated recipients. We will identify the DC and macrophage populations that are rapidly activated after CD47-deficient DST and their roles in T cell activation and tolerance induction. We will address these questions in a mouse model of CD47-deficient cardiac allotransplantation, and a pig-to-mouse xenotransplantation model that involves DST from human CD47-transgenic pigs into humanized mice. These studies are expected to provide significant insights into the mechanisms by which CD47 incompatibility activates innate and adaptive xenoimmune responses, and the potential of using human CD47 transgenic pigs as donors to facilitate xenotolerance induction and xenograft survival.