Fas ligand+ blood cells to reduce transplant rejection

描述(由申请人提供)： 稳定地转导淋巴造血祖细胞(HSCs)和子代细胞的能力使我们能够对HSCs和子代进行基因工程，特别是树突状细胞(DC)，以作为细胞工具来改进移植。异基因(异基因)血液和骨髓移植(BMT)将继续是许多遗传性和获得性疾病患者的重要治疗选择。我们最近的初步结果表明，转导FasL+同种异体小鼠DC或HSCs的体外或体内治疗抑制了对该同种异体抗原的反应，增加了同种异体细胞的植入，而对受体小鼠没有免疫消融或明显的毒性。在这项建议的目标1-2中，我们建议进行研究，以测试这一概念并阐明转导的树突状细胞或表达Fas配体(FasL)的HSCs似乎减少了介导移植排斥反应的T(和NK)细胞，而没有明显的器官毒性或一般免疫损害的主要机制。AIMS 1-2试图在同种异体免疫应答过程中加速和增强FasL的自然稳态作用，潜在的结果是同种免疫T细胞在激活/扩增过程中可能被更早地杀死，或者在激活诱导细胞死亡(AICD)期间被更有效地杀死，分别被转导的FasL+DC(Aim 1)或HSCs/子代(Aim 2)所杀死。在我们研究这一点时，我们将检查同种异体免疫细胞中的凋亡途径。此外，由于我们预计转导的FasL+HSCs可能在体内具有潜在的毒性，我们将同时研究限制潜在的FasL毒性的技术，例如，仅转导一小部分高质量的HSCs，通过使用无法切割的FasL缺失突变体来释放可溶性FasL(SFasL)，通过在对HSCs产生耐受后消除转导细胞本身或其FasL表达，或者通过使用谱系/阶段特异性启动子来限制FasL的表达。一种新的减少移植物排斥反应的FasL+细胞治疗方法可能最终用于临床异基因骨髓移植。此外，稳定的淋巴造血嵌合体的实现也将被预测为对移植的同种器官或多能干细胞产生耐受性。因此，在目标3中，我们将评估在有或没有其他免疫抑制方法(如共刺激阻断)的情况下，由这种FasL策略产生的造血巨嵌合体中的同种异体心脏移植的耐受性。 特定目标1：评估FasL+DC是否特异性地减少同种异体免疫反应并增强异基因造血干细胞的植入 特异性目的2：确定FasL+HSCs在allo BMT中是否产生特异性耐受。 具体目的3：评估用FasL+DC/HSCs产生的造血嵌合体对心脏移植的耐受性。

DESCRIPTION (provided by applicant): The ability to stably transduce lymphohematopoietic stem-progenitor cells (HSCs) and progeny cells allows us to genetically engineer HSCs and progeny, especially dendritic cells (DCs), to serve as cellular tools to improve transplantation. Allogeneic (allo) blood and marrow transplantation (BMT) will continue to be an important treatment option in patients with many inherited and acquired diseases. Our recent Preliminary Results indicate that ex vivo or in vivo treatment with transduced FasL+ allog mouse DCs or HSCs suppressed the response to that alloantigen and increased engraftment of allo cells, without immunoablation or evident toxicity to the recipient mice. In Aims 1-2 of this proposal, we propose studies to test the concept and elucidate the principal mechanisms by which transduced DCs or HSCs constitutively expressing Fas ligand (FasL) appear to reduce the T (and NK) cells that mediate graft rejection without overwhelming organ toxicity or general immunologic impairment. Aims 1-2 attempt to accelerate and enhance the natural homeostatic role of FasL during the course of the alloimmune response, with the potential outcome that alloimmune T cells may be killed earlier in their activation/expansion, or killed more potently later during activation-induced cell death (AICD), by transduced FasL+ DCs (Aim 1) or HSCs/progeny (Aim 2), respectively. As we investigate this, we will examine the apoptotic pathways in alloimmune cells. In addition, since we expect that transduced FasL+ HSCs might potentially be toxic in vivo, we will in parallel investigate technologies to limit potential FasL toxicity, eg by transducing only a small percent of high quality HSCs, by employing a FasL deletion mutant that cannot be cleaved to release soluble FasL (sFasL), by eliminating the transduced cells themselves or their FasL expression after tolerance to HSCs has been generated, or by using lineage/stage-specific promoters to restrict FasL expression. A novel FasL+ cell therapy approach to reduce graft rejection may eventually be used in clinical allo BMT. Furthermore, achievement of stable lymphohematopoietic chimerism would also be predicted to generate tolerance for transplanted allo organs or pluripotent stem cells. Therefore, in Aim 3, we will assess tolerance to cardiac allografts in hematopoietic macro-chimeras, generated by this FasL strategy, with or without other immunosuppressive methodologies such as co-stimulatory blockade. SPECIFIC AIM 1: Assess whether FasL+ DCs specifically reduce an alloimmune response and enhance engraftment of allo HSCs SPECIFIC AIM 2: Determine whether FasL+ HSCs generate specific tolerance in allo BMT. SPECIFIC AIM 3: Evaluate tolerance to cardiac allografts in hematopoietic chimeras generated using FasL+ DCs/HSCs.