Fas ligand+ blood cells to reduce transplant rejection

描述（由申请人提供）： 稳定地扩增淋巴造血干祖细胞（HSC）和后代细胞的能力允许我们对HSC和后代，特别是树突状细胞（DC）进行基因工程改造，以用作改善移植的细胞工具。异基因（allo）血液和骨髓移植（BMT）将继续成为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，用转导的FasL+同种异体小鼠DC或HSC的离体或体内治疗抑制了对该同种异体抗原的应答，并增加了同种异体细胞的植入，而没有免疫消融或对受体小鼠的明显毒性。在本提案的目标1-2中，我们提出研究来测试概念并阐明组成性表达Fas配体（FasL）的转导的DC或HSC似乎减少介导移植物排斥的T（和NK）细胞而没有压倒性器官毒性或一般免疫损伤的主要机制。目的1-2试图在同种免疫应答过程中加速和增强FasL的天然稳态作用，其潜在结果是同种免疫T细胞可以在其活化/扩增中较早被杀死，或者在活化诱导的细胞死亡（AICD）期间较晚被转导的FasL+ DC（Aim 1）或HSC/子代（Aim 2）更有效地杀死。当我们研究这一点时，我们将研究同种免疫细胞中的凋亡途径。此外，由于我们预期转导的FasL+ HSC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，例如通过仅转导小百分比的高质量HSC，通过采用不能被切割以释放可溶性FasL（sFasL）的FasL缺失突变体，通过在对HSC的耐受性已经产生后消除转导的细胞本身或其FasL表达，或通过使用谱系/阶段特异性启动子来限制FasL表达。一种新的FasL+细胞治疗方法，以减少移植排斥反应，最终可能会用于临床同种异体BMT。此外，还预测实现稳定的淋巴造血嵌合体会产生对移植的同种异体器官或多能干细胞的耐受性。因此，在目标3中，我们将评估由这种FasL策略产生的造血巨嵌合体对心脏同种异体移植物的耐受性，无论是否使用其他免疫抑制方法，例如共刺激阻断。 特异性目的1：评估FasL+ DC是否特异性降低同种异体免疫应答并增强同种异体HSC的植入 特异性目的2：确定FasL+ HSC是否在同种异体BMT中产生特异性耐受。 具体目的3：评价使用FasL+ DC/HSC产生的造血嵌合体对心脏同种异体移植物的耐受性。

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.