Clonal and imaging analyses of in vivo hematopoiesis, immune cell ontogeny and adoptive cell therapies

我们利用分子技术对体内造血干细胞和祖细胞(HSPC)和免疫细胞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，将高多样性的31-35个碱基对的遗传条形码引入靶细胞，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30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长达8年的时间和多个谱系的造血输出。 我们最近修改了我们的条形码策略，允许同时检索条形码和单细胞RNAseq/ATACSeq，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育)与基因表达方面的“状态”表征联系在一起，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10X和其他标准单细胞RNAseq平台进行高表达和检索的位置。第一只猕猴现在已经被移植了这种新的载体，对扩增的NK细胞克隆出现的分析正在进行中。 我们已经取得了许多重要和新的发现，包括令人惊讶的成熟NK细胞的生活史，表明循环中的成熟细胞毒性NK细胞(CD16+CD56-)的主要部分不与B、T或髓系细胞或其假定的前体CD56brightCD16neg NK细胞共享条形码，即使在移植后80个月。在体外和小鼠模型之前还不能阐明NK细胞的谱系关系。这些循环中的NK细胞由大量扩增的寡克隆群体组成，以某种模式消长，暗示着对特定环境线索的反应，如病毒感染或病毒重新激活。我们的数据首次提供了克隆性NK反应的直接证据，为NK记忆的可能机制提供了洞察力。我们使用以前与NK病毒和肿瘤反应相关的差异表达的KIR表面分子，对表达不同KIR的NK细胞进行分类，并记录了这些表达KIR的特定NK群体中的克隆性分离。这是第一次直接证明具有特定受体特征的NK细胞克隆群体的产生和持续存在，推测这种克隆群体是由表观遗传学维持的。随着体内基于CD16表达的NK耗尽，同样的扩增克隆再次出现，没有从高度多克隆的HSPC中招募，但从残留的高度增殖的CD16dim NK亚群中招募。 我们已经分析了组织驻留的NK细胞，在小鼠和猴子的过继转移研究中显示了对NK记忆功能的重要作用。我们在脾、肺、肠道和肝脏等组织中发现了明显扩增的NK克隆，扩增克隆在这些组织中共享，这表明扩增后血液中的所有组织都有特定的归巢。我们还在血液中鉴定了包含这些扩增克隆的CD56enerCD16neg NK细胞的少量表型群体，表明这些细胞可能是这些组织驻留的NK细胞的前体，并记录了这些细胞上趋化因子应答归巢分子的表达。 基于人类移植和献血者研究中的相关数据，我们假设扩增的NK细胞克隆可能是在对CMV的应答的背景下产生的，并且我们通过条形码移植在CMV阴性猕猴中检验了这一假设，表明在实验性CMV感染后成熟的NK细胞群体发生了特定的克隆性变化。CMV感染前后NK细胞的单细胞RNAseq实验记录了NK亚群和NK细胞对CMV应答之间的关系的信息聚集和新的见解。 针对巨细胞病毒的适应性/记忆性NK反应与涉及人类白细胞抗原-E的相互作用有关。我们现在正在努力了解NK对基于恒河猴CMV平台的疫苗的反应，该平台之前被我们的合作者Louis Picker证明可以提供独特的有效保护，使其免受SIV的攻击。我们正在调查在条形码猕猴接种疫苗后是否会出现NK克隆性扩张，这取决于人类白细胞抗原-E，并至少部分解释了对这一有希望的疫苗平台的独特反应。 我们继续分析体外扩增的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。我们也已经开始比较特定的调节方案对HSPC植入后克隆模式的影响，注意到TBI和白花丹之间的差异，现在正在积极地将研究扩展到抗体介导的调节，实现了抗CD345调节后条形码细胞的高水平植入，更令人惊讶的是，至少对外来转基因的部分耐受。 我们还将跟踪方法应用于猕猴模型中自然杀伤细胞和CAR-T细胞的过继转移。我们已经分析了体外扩增的NK细胞的克隆组成，并与Richard Childs实验室合作，在体外维持和扩增了扩增的假定适应性克隆。我们计划研究自然杀伤细胞在过继移植后的克隆模式和持久性。我们还将钠碘转运体基因(NIS)克隆到慢病毒载体中，这将允许体内成像和跟踪过继转移的CAR-T细胞，并分析各种共刺激结构域的影响。这项工作正在小鼠异种移植模型和猕猴模型中进行。对CAR-NK细胞的类似研究正在进行中

We have utilized molecular techniques to gain new insights into the behavior of hematopoietic stem and progenitor cells (HSPCs) and immune cells 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 30 macaques with barcoded autologous CD34+ cells, and have been able to track hematopoietic output from thousands of individual HSPCs over time for up to 8 years and in multiple lineages in a quantitative and highly reproducible manner. We have recently modified our barcoding strategy to allow simultaneous retrieval of the barcode and single cell RNASeq/ATACSeq 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. The first macaque has now been transplanted with this novel vector, and analyses focusing on emergence of expanded NK cell clones are in progress. 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. We have analyzed tissue-resident NK cells, shown to be important for function NK memory in murine and monkey adoptive transfer studies. We have discovered markedly expanded NK clones in tissues including spleen, lung, gut, and liver, with expanded clones shared across these tissues, suggesting specific homing to all tissues from the blood following expansion. We have also identified a minor phenotypic population in the blood of CD56negCD16neg NK cells that contain these expanded clones, suggesting these cells may be precursors for these tissue resident NK, and documented expression of chemokine-responsive homing molecules on these cells. We hypothesize that expanded NK cell clones might be generated in the context of a response to CMV, based on correlative data in human transplantation and blood donor studies, and we tested this hypothesis via barcoded transplantation in CMV negative macaques, showing specific clonal changes occur in the mature NK cell populations following experimental CMV infection. Single cell RNASeq experiments on NK cells before and after CMV infection document informative clustering and new insights into the relationship between NK subsets and the response of NK cells to CMV. Adaptive/memory NK responses to CMV have been linked to interactions involving HLA-E. We are now working to understand NK response to a vaccine based on a rhesus CMV platform previously shown to confer uniquely potent protection from SIV by our collaborator Louis Picker. We are investigating whether NK clonal expansion arise following vaccination in barcoded macaques, dependent on HLA-E and explaining at least in part the unique response to this promising vaccine platform. 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 compared the impact of specific conditioning regimens on clonal patterns following HSPC engraftment, noting differences between TBI and busulfan, and are now actively extending the studies to antibody-mediated conditioning, achieving high level engraftment of barcoded cells following anti-CD345 conditioning, and more surprisingly, at least partial tolerance to foreign transgenes. We are also applying tracking approaches to the adoptive transfer of natural killer cells and CAR-T cells in the macaque model. We have analyzed the clonal composition of ex vivo expanded NK cells and documented expanded putative adaptive clones are maintained and expanded in vitro, in collaboration with the Richard Childs laboratory. We plan to study the clonal patterns and persistence of NK populations following adoptive transfer. We have also cloned the sodium-iodide symporter gene (NIS) into a CAR lentiviral vector, which will allow in vivo imaging and tracking of adoptively-transferred CAR-T cells, and analysis of the impact of various co-stimulatory domains. This work is in progress in murine xenograft models and in the macaque model. Similar studies are ongoing with CAR-NK cells