Clonal analysis of in vivo hematopoiesis

我们利用分子生物学技术对造血干细胞和祖细胞（HSPCs）在体内的行为有了新的认识。 我们继续积极开发和利用慢病毒“条形码化”，将高多样性的31-35个碱基对遗传条形码引入靶细胞中，以研究非人灵长类动物模型中的体内造血。 我们的合作者Rong Lu首先设计了这种非常强大的方法，并将其应用于研究小鼠造血。我们现在已经用条形码标记的自体CD 34+细胞移植了27只猕猴，并且已经能够以定量和高度可重复的方式跟踪数千个个体HSPC随时间（长达8年）和多个谱系的造血输出。 我们目前正在修改我们的条形码策略，以允许同时检索条形码和单细胞RNASeq，以便开始能够将我们的细胞命运发现（条形码定义的个体发生）与基因表达方面的“状态”表征联系起来，我们希望在单细胞水平上进行表观遗传标记。 新载体将条形码放置在允许通过10 X和其他标准单细胞RNASeq平台进行高表达和检索的位置。在CMV感染之前和之后对NK细胞进行的初步单细胞RNASeq实验记录了信息性聚类和对NK亚群之间关系的新见解，如RNA速度方法所示。 我们已经取得了许多重要和新颖的发现，包括成熟NK细胞的令人惊讶的生命史，表明循环成熟细胞毒性NK细胞（CD 16 + CD 56-）的主要部分不与B、T或骨髓细胞或其推定的前体CD 56 brightCD 16 neg NK细胞共享条形码，甚至在移植后80个月。体外和鼠模型以前不能阐明NK细胞谱系关系。这些循环NK细胞由快速扩增和寡克隆群体组成，以一种模式出现和消失，表明对特定环境线索（如病毒感染或病毒再活化）的反应。我们的数据提供了克隆NK反应的第一个直接证明，为NK记忆的可能机制提供了见解。我们使用差异表达的KIR表面分子，以前与NK病毒和肿瘤反应，排序NK细胞表达不同的KIR，并记录这些特定的KIR表达NK细胞群内的克隆分离。这是第一次直接证明具有特异性受体特征的NK细胞克隆群体的产生和持续存在，推测是表观遗传维持的。在基于CD 16表达的体内NK耗竭的情况下，再次出现相同的扩增克隆，没有从高度多克隆HSPC募集，但从残留的高度增殖性CD 16 dim NK亚群募集。这项工作最近发表。我们假设这些克隆可能是在对CMV的反应的背景下产生的，基于人类移植和献血者研究中的相关数据，并且正在通过CMV阴性猕猴中的条形码移植来测试这一点，显示特定的克隆变化发生在成熟的NK细胞群体中，发表于本财年。我们现在正在努力了解NK对疫苗接种和其他挑战的反应，以及猕猴HLA-E等同物在特定记忆样NK反应中的参与。 我们最近完成了一个项目，比较了年轻与老年HSPC的克隆行为，证明了克隆模式的显著差异，特别是在髓系、B和T细胞系中，多系克隆的贡献非常延迟，而单系贡献持续存在，与小鼠模型相反，小鼠模型表明仅存在髓系偏向性克隆的蓄积。在年龄最大的动物中，我们观察到克隆脱落和克隆扩增，可能提供与衰老相关的人类“CHIP”模型（不确定预后的克隆造血）。这一发现最近被认为与人类衰老中发现的同一组体细胞突变有关，即“年龄相关的克隆造血”，而小鼠模型中缺乏这些突变。 我们继续分析了体外扩增的HSPC、CAR-T细胞和NK细胞植入后的克隆模式，这些主题具有翻译和临床重要性。我们还开始比较HSPC植入后特定预处理方案对克隆模式的影响，注意TBI和白消安之间的差异，并将研究扩展到抗体介导的预处理。

We have utilized molecular techniques to gain new insights into the behavior of hematopoietic stem and progenitor cells (HSPCs) in vivo. We have continued active development and utilization of lentiviral "barcoding" with high-diversity 31-35 base pair genetic barcodes introduced into target cells in order to study in vivo hematopoiesis in the non-human primate model. Our collaborator Rong Lu first devised this very powerful approach and applied it to study murine hematopoiesis. We have now transplanted 27 macaques with barcoded autologous CD34+ cells, and have been able to track hematopoietic output from thousands of individual HSPCs over time (up to 8 years) and in multiple lineages in a quantitative and highly reproducible manner. We are currently modifying our barcoding strategy to allow simultaneous retrieval of the barcode and single cell RNASeq in order to begin to be able to connect our cell fate findings (barcode-defined ontogeny) together with "state" characterization in terms of gene expression and we hope epigenetic marks at a single cell level. The new vector has the barcode placed in a position that allows high expression and retrieval via 10X and other standard single cell RNASeq platforms. Preliminary single cell RNASeq experiments on NK cells before and after CMV infection document informative clustering and new insights into the relationship between NK subsets, as indicated by RNA velocity approaches. We have already made a number of important and novel discoveries, including a surprising life history for mature NK cells, showing that the major fraction of circulating mature cytotoxic NK cells(CD16+CD56-) do not share barcodes with B, T or myeloid cells or their putative precursor CD56brightCD16neg NK cells, even 80 months post-transplant. In vitro and murine models have not previously been able to shed light on NK cell lineage relationships. These circulating NK cells consist of massively-expanded and oligoclonal populations, waxing and waning in a pattern suggesting responses to specific environmental cues such as viral infection or viral reactivation. Our data provides the first direct demonstration of clonal NK responses, providing insights into possible mechanisms for NK memory. We used differentially-expressed KIR surface molecules, previously linked to NK viral and tumor responses, to sort NK cells expressing different KIR, and documented clonal segregation within these specific KIR-expressing NK populations. This is the first direct demonstration of the generation and persistence of clonal populations of NK cells with specific receptor characteristics, presumably epigenetically-maintained. With in vivo NK depletion based on CD16 expression, the same expanded clones arise again, without recruitment from highly polyclonal HSPC but with recruitment from a residual highly proliferative CD16dim NK subset. This work was published recently. We hypothesize that these clones might be generated in the context of a response to CMV, based on correlative data in human transplantation and blood donor studies, and are testing this via barcoded transplantation in CMV negative macaques, showing specific clonal changes occur in the mature NK cell populations, published this FY. We are now working to understand NK response to vaccinations and other challenges, and the involvement of the macaque equivalent of HLA-E in specific memory-like NK responses. We have recently completed a project comparing the clonal behavior of young versus aged HSPC, demonstrating marked differences in clonal patterns, specifically very delayed contributions from multilineage clones with persistence of unilineage contributions, in both myeloid, B and T cell lineages, in contrast to murine models suggesting accumulation of only myeloid-biased clones. In the oldest animal, we observed clonal dropout and clonal expansions, potentially providing a model of human "CHIP" associated with aging (clonal hematopoiesis of indeterminate prognosis). This finding has recently been linked to the presence of the same panel of somatic mutations found in humans with aging, ie "age-related clonal hematopoiesis", in contrast to the lack of these mutations in murine models. We have continued to analyze clonal patterns following engraftment of ex vivo expanded HSPC, CAR-T cells and NK cells, topics of translational and clinical importance. We have also begun to compare the impact of specific conditioning regimens on clonal patterns following HSPC engraftment, noting differences between TBI and busulfan, and extending the studies to antibody-mediated conditioning.