Using donor dendritic cells to optimize GvHD and GvL in allogeneic stem cell transplantation

摘要：首要目标是通过识别同种异体干细胞移植中的 以及丰富捐赠者树突状细胞(DC)群体，以减少移植物抗宿主疾病并提高存活率。 目前的建议是基于发表的研究结果，即供体DC在同种异体移植物中和在 异基因移植受者的血液对临床结果有重大影响。小鼠的临床前数据 骨髓移植模型系统表明，供者的血浆细胞样树突状细胞(PDC)来自骨髓，但不是G-CSF 动员的移植物可保护胸腺的家园GvHD，并限制CCR9-、IL12-和IDO- 依赖的态度。作为患者，优化移植物中的免疫细胞的问题非常重要 移植了更多的骨髓供者的PDC存活率提高了20%，慢性移植物抗宿主病较少，而没有 与供者较少的PDC受者相比，复发风险增加。悬而未决的问题限制了 这些令人兴奋的观察结果在广泛的临床实践中的应用包括解决DC的异质性 在骨髓和细胞因子动员的移植物中识别允许有利移植的关键亚群(S) 成果；确定捐助者DC管理GvHD的机制；以及确定具有成本效益的程序 用于动员和采集需要最小体外操作的最佳造血祖细胞移植物。 为了解决这些问题，并朝着我们的总体目标取得进展，我们提出了三个具体目标： 目的1.明确供体DC限制移植物抗宿主病的机制和分子水平。假设：小鼠 人骨髓含有免疫抑制DC亚群(S)，频率较高 与G-CSF动员的移植物相比，G-CSF动员的移植物抑制了T细胞对同种抗原的激活，并限制了GvHD。 目的2.提高树突状细胞在同种异体移植中的免疫调节能力。假设：治疗 短程Flt3供者-L将增加幼稚粒细胞的数量和免疫效力 异基因受者移植后限制移植物抗宿主病的供者DC。 目的3.研究供体来源DC的归巢和持久性对移植物抗宿主病和移植物抗宿主病的影响。 假设：移植受者血淋巴组织所在的供者PDC通过 胸腺中同种反应性T细胞的阴性选择和/或Treg的产生。 这项拟议的研究将使用单细胞RNAseq和质量细胞术来定义小鼠树突状细胞簇 和具有良好免疫学特性的人骨髓和细胞因子动员的移植物，制定时间表 增加免疫调节性树突状细胞的频率和效力，并使用生物... 荧光和荧光成像示踪供体DC在小鼠体内的归巢、扩增和功能 BMT模型。这项研究将对供者DC亚群(S)如何调节免疫产生机制的理解 在异基因干细胞移植后，这将为实体器官移植和过继T细胞的研究提供信息 像CART这样的疗法。阳性结果可能导致Flt3-L治疗干细胞捐赠者的临床试验。

ABSTRACT: The overarching goal is to improve outcomes in allogeneic stem cell transplantation by identifying and enriching donor dendritic cell (DC) populations that reduce graft-versus-host disease and improve survival. The current proposal is grounded on published findings that the content of donor DC in the allograft and in the blood of allo-transplant recipients have a significant impact on clinical outcomes. Preclinical data from murine BMT model systems demonstrate that donor plasmacytoid dendritic cells (pDC) from marrow but not G-CSF mobilized grafts protect against GvHD, home to the thymus, and limit GvHD in an CCR9-, IL12-, and IDO- dependent manner. The question of optimizing immune cells in the graft is highly significant, as patients transplanted with more marrow donor pDC have 20% better survival, with less chronic GvHD, and without increased risk of relapse compared with recipients of fewer donor pDC. Unanswered questions that limit application of these exciting observations to widespread clinical practice include resolving heterogeneity of DC in marrow and cytokine-mobilized grafts to identify the critical subset(s) that confer favorable transplant outcomes; defining the mechanism by which donor DC regulate GvHD; and identifying cost-effective procedures for mobilization and harvesting optimal hematopoietic progenitor grafts requiring minimal ex vivo manipulation. To address these questions, and make progress towards our overall goal, we propose three Specific Aims: Aim 1. To define the mechanisms and molecular profile of donor DCs that limit GvHD. Hypothesis: Murine and human bone marrow contain immunosuppressive DC subset(s) that are present at higher frequencies than in G-CSF-mobilized grafts, that inhibit T cell activation in response to allo-antigen, and that limit GvHD. Aim 2. To enhance the immuno-regulatory potency of DCs in allotransplantation. Hypothesis: Treatment of donors with a short-course of Flt3-L will increase the numbers and immunological potency of immature donor DC that limit GvHD following transplantation to allogeneic recipients. Aim 3. To determine how homing and persistence of donor-derived DC affects GvHD and GvL. Hypothesis: donor pDC that home to hemato-lymphoid tissue in transplant recipients support tolerance via negative selection of allo-reactive T cells in the thymus and/or the generation of Treg. The proposed research will use single cell RNAseq and mass cytometry to define clusters of DC from mouse and human marrow and cytokine-mobilized grafts with favorable immunological properties, develop schedules of Flt3-L administration that increase the frequency and potency of immune-regulatory DC, and use bio- luminescent and fluorescent imaging to track in vivo homing, expansion, and function of donor DC in mouse BMT models. This research will yield a mechanistic understanding of how donor DC subset(s) regulate immunity after allogeneic stem cell transplant that will inform studies in solid organ transplantation and adoptive T cell therapies such as CART. Positive results can lead to a clinical trial of Flt3-L treatment of stem cell donors.