Clonal analysis of in vivo hematopoiesis

我们利用分子技术对体内造血干细胞和祖细胞(HSPC)的行为有了新的了解。我们继续积极开发和利用慢病毒条形码，利用高多样性的31-35个碱基对的遗传条形码来标记单个造血干细胞和祖细胞，以研究非人类灵长类动物模型的体内造血。我们的合作者荣路首先设计了这一非常强大的方法，并将其应用于研究小鼠的造血。我们现在已经移植了15只带有条形码自体CD34细胞的猕猴，并能够以定量和高度重复性的方式跟踪数千个个体HSPC随着时间的推移(长达4.5年)和多个谱系的造血输出。我们已经有了许多重要和新的发现，包括缺乏证据表明灵长类动物中有共同的淋巴祖细胞产生T和B细胞，直到移植后期才有B和T细胞的共同克隆衍生，以及更早的髓系和B细胞的共同克隆衍生。我们还首次发现了自然杀伤(NK)细胞的主要部分的独特谱系来源和生活史。即使在移植后48个月，CD16-CD56-细胞毒性NK细胞也没有与B、T或髓系细胞共享条形码。在体外和小鼠模型之前还不能阐明NK细胞的谱系关系。我们继续使用这些猕猴的条形码细胞来进一步剖析体内NK细胞的个体发育和NK细胞的体外扩增过程，与NHLBI的Rick Childs博士小组、Tulane非人灵长类设施的Miti Kaur博士小组以及瑞典斯德哥尔摩卡罗林斯卡研究所的Yenan Bryceson博士小组合作，NK细胞的体外扩增与采用细胞疗法的开发高度相关。我们已经将克隆唯一的条形码映射到具有“适应性”表型的NK细胞，在小鼠研究中与NK记忆有关，在人类中与假定的对CMV等病毒的NK适应性反应有关。在具有适应性表型的NK细胞中，我们发现了寡克隆和大量扩增的个体克隆，在其他谱系中没有发现贡献，并且具有暗示对特定环境线索的反应的消长模式，如病毒感染或重新激活。我们的数据首次提供了克隆性NK反应的直接证据，可能解释了NK记忆。我们继续寻找这些细胞的前体，追踪来自血液、骨髓、淋巴结、肝脏以及阴道和肠道淋巴集合体的表型纯化样本中的显性克隆。随着体内NK的严重耗尽，相同的克隆再次出现，而不是从高度多克隆的HSPC重新招募。 我们延长了对特定骨髓部位单个HSPC地理隔离的长期分析，证实了我们之前在小鼠身上使用乐高成像技术的发现，现在使用条形码在猕猴模型中使用。我们可以直接证明B细胞、CD56 NK细胞和髓系细胞在局部的骨髓壁龛中原位产生，令人惊讶的是，我们现在有强有力的证据表明T细胞在骨髓中原位产生。 我们还在几种动物身上跟踪了数千个单个HSPC克隆的输出长达4年，并证明了长期重新繁殖产生髓系、B细胞和T细胞系的克隆以及CD56brightCD16-NK细胞的输出具有显著的克隆稳定性。我们已经证明，髓系和淋巴系的偏见是常见的，并且随着时间的推移，这种偏见在个体克隆中保持稳定。 我们最近在许多其他方向扩展了条形码模型，包括：1)比较年轻和老年HSPC的克隆行为，通过移植两只带有条形码细胞的老年猕猴的初步数据，显示出与年轻动物非常不同的动力学和克隆模式。2)分析干细胞扩增的新方法，对体内扩增的细胞和未扩增的细胞进行定量和谱系分析。3)红系和血小板系克隆个体发育的研究。4)与纽约大学的Rahul Sajita合作，将单细胞RNAseq应用于条形码种群，以进一步定义个体发育和识别罕见的前体细胞种群。我们现在已经在NHLBI与安德烈·拉罗切尔合作建立了DropSeq单细胞方法学，并有证据表明我们可以从单细胞中检索条形码和转录组。这种方法提供了许多调查机会。

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 utilized to label individual hematopoietic stem and progenitor cells 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 15 macaques with barcoded autologous CD34+ cells, and have been able to track hematopoietic output from thousands of individual HSPCs over time (up to 4.5 years) and in multiple lineages in a quantitative and highly reproducible manner. We have already made a number of important and novel discoveries, including the lack of evidence for a common lymphoid progenitor producing T and B cells in primates, with no shared clonal derivation of B and T cells until late after transplant, and much earlier shared clonal derivation of myeloid and B cells. We have also for the first time discovered the unique lineage derivation and life history of the major fraction of natural killer (NK) cells. CD16+CD56- cytotoxic NK cells did not share barcodes with B, T or myeloid cells even 48 months post-transplant. In vitro and murine models have not previously been able to shed light on NK cell lineage relationships. We have continued to use barcoded cells from these macaques to further dissect in vivo NK cell ontogeny, and the process of ex vivo expansion of NK cells, highly relevant for adoptive cell therapy development, in collaboration with Dr. Rick Childs' group in NHLBI, Dr Miti Kaur's group at the Tulane Non-human Primate facility, and Dr Yenan Bryceson's group at the Karolinska Institut in Stockholm Sweden. We have mapped the clonally-unique barcodes to NK cells with an "adaptive" phenotype, linked in murine studies to NK memory, and in humans to putative NK adaptive responses to viruses such as CMV. Within NK cells with an adaptive phenotype, we find oligoclonal and massively expanded individual clones, with no contributions found in other lineages, and with waxing and waning patterns suggesting responses to specific environmental cues such as viral infection or reactivation. Our data provides the first direct demonstration of clonal NK responses, possibly explaining NK memory. We continue to search for the precursor to these cells, tracking dominant clones in phenotypically purified samples from blood, bone marrow, lymph nodes, liver, and vaginal and intestinal lymphoid aggregates. With profound in vivo NK depletion, the same clones arise again, without recruitment from highly polyclonal HSPC. We have extended our analysis of the geographic segregation of individual HSPCs long term in specific marrow sites, confirming our prior findings in mice using LEGO imaging techniques now in the macaque model utilizing barcoding. We can directly demonstrate in situ production of B cells, CD56+ NK cells and myeloid cells in localized marrow niches, and surprisingly, we now have strong evidence for in situ marrow production of T cells. We have also followed the output of thousands of individual HSPC clones in several animals for up to 4 years, and demonstrate marked clonal stability of output from long-term repopulating clones producing myeloid, B cell and T cell lineages, along with CD56brightCD16- NK cells. We have shown that myeloid vs lymphoid bias is common, and that this bias remains stable in individual clones over time. We have recently extended the barcoding model in a number of additional directions, including: 1) Comparison of the clonal behavior of young versus aged HSPC, with preliminary data from transplants of two aged macaques with barcoded cells demonstrating a very different kinetic and clonal pattern compared to young animals. 2) Analysis of novel methodologies for stem cell expansion, with quantitative and lineage analytics performed on expanded versus non-expanded cells in vivo. 3) Investigation of the clonal ontogeny of erythroid and platelet lineages. 4) Collaboration with Rahul Sajita at NYU to apply single cell RNAseq to barcoded populations in order to further define ontogeny as well as identify rare precursor cell populations. We now have set up the DropSeq single cell methodology at NHLBI in collaboration with Andre Larochelle, and have evidence that we can retrieve both the barcode and the transcriptome from single cells. This approach offers many investigative opportunities.