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

体内造血、免疫细胞个体发育和过继细胞疗法的克隆和成像分析

• 批准号: 10929124
• 负责人: CYNTHIA E DUNBAR
• 金额: $ 182.94万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10929124
• 关键词: ATAC-seq; Adoptive Cell Transfers; Adoptive Transfer; Aging; Alzheimer' s Disease; Antibodies; Autologous; Bar Codes; Base Pairing; Behavior; Big Data Methods; Blood; Blood donor; Brain; Busulfan; CD34 gene; Cell Lineage; Cell Ontogeny; Cell Therapy; Cells; Central Nervous System Diseases; Characteristics; Clinical; Clonal Expansion; Clone Cells; Collaborations; Cues; Cytomegalovirus; Cytomegalovirus Infections; Data; Development; Disease; Donor person; Engraftment; Environment; Epigenetic Process; FCGR3B gene; Gene Expression; Generations; Genes; Genetic; Goals; Growth; Hematopoiesis; Hematopoietic; Hematopoietic Stem Cell Transplantation; Hematopoietic stem cells; Homing; Human; Image; Immune; Immunology; In Vitro; Individual; Infection; Knowledge; Laboratories; Lentivirus Vector; Link; Liver; Lung; Macaca; Macaca mulatta; Macrophage; Maintenance; Malignant Neoplasms; Mediating; Memory; Methodology; Methods; Minor; Modeling; Molecular; Monkeys; Mus; Myeloid Cells; NCAM1 gene; Natural Killer Cells; Organ; Output; Pattern; Phenotype; Population; Positioning Attribute; Primates; Process; Progenitor Cell Engraftment; Reaction; Regimen; Reproducibility; Residual state; Retrieval; Rhesus; SIV; SLC5A5 gene; Sorting; Spleen; Surface; Techniques; Testing; Time; Tissues; Transgenes; Transplantation; Vaccination; Vaccines; Viral; Virus Diseases; Waxes; Work; Xenograft Model; Xenograft procedure; chemokine; chimeric antigen receptor T cells; clinically relevant; conditioning; cytotoxic; differential expression; experimental study; fighting; gene therapy; genetic manipulation; hematopoietic engraftment; improved; in vivo; in vivo imaging; insight; life history; mouse model; nonhuman primate; novel; novel strategies; post-transplant; receptor; recruit; response; segregation; self-renewal; single-cell RNA sequencing; targeted treatment; trafficking; tumor; vaccine platform; vector

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

我们利用分子生物学技术对造血干细胞和祖细胞（HSPCs）以及免疫细胞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自体CD 34+细胞移植了30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随时间的造血输出长达8年，并且在多个谱系中。 我们最近修改了我们的条形码化策略，以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。第一只猕猴现在已经被移植了这种新型载体，并且正在进行针对扩增的NK细胞克隆出现的分析。 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。 我们已经分析了组织驻留NK细胞，在小鼠和猴过继转移研究中显示出对NK记忆功能的重要性。我们已经在包括脾、肺、肠和肝的组织中发现了显著扩增的NK克隆，这些组织中共有扩增的克隆，这表明扩增后从血液特异性归巢到所有组织。我们还鉴定了血液中含有这些扩增克隆的CD 56 + CD 16-NK细胞的次要表型群体，表明这些细胞可能是这些组织驻留NK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。 基于人类移植和血液供体研究中的相关数据，我们假设扩增的NK细胞克隆可能在对CMV的应答的背景下产生，并且我们通过CMV阴性猕猴中的条形码移植测试了这一假设，表明实验性CMV感染后成熟NK细胞群体中发生特异性克隆变化。在CMV感染之前和之后对NK细胞的单细胞RNASeq实验记录了信息聚类和对NK亚群与NK细胞对CMV的应答之间的关系的新见解。 对CMV的适应性/记忆NK应答与涉及HLA-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，该平台先前被我们的合作者Louis Picker证明具有独特的有效保护作用。我们正在研究在条形码猕猴中接种疫苗后是否会出现NK克隆扩增，这取决于HLA-E，并至少部分解释了对这种有前途的疫苗平台的独特反应。 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。我们还开始比较特定预处理方案对HSPC植入后克隆模式的影响，注意到TBI和白消安之间的差异，现在正在积极将研究扩展到抗体介导的预处理，实现抗CD 345预处理后条形码细胞的高水平植入，更令人惊讶的是，至少部分耐受外源转基因。 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。我们分析了体外扩增NK细胞的克隆组成，并与Richard查尔兹实验室合作，记录了体外扩增的假定适应性克隆的维持和扩增。我们计划研究过继转移后NK细胞群体的克隆模式和持久性。 我们还将钠-碘同向转运体基因（NIS）克隆到CAR慢病毒载体中，这将允许对过继转移的CAR-T细胞进行体内成像和跟踪，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。CAR-NK细胞的类似研究正在进行中

项目成果

Expanded NK cells used for adoptive cell therapy maintain diverse clonality and contain long-lived memory-like NK cell populations.

• DOI: 10.1016/j.omto.2022.12.006
• 发表时间: 2023-03-16
• 期刊: MOLECULAR THERAPY ONCOLYTICS
• 作者: Allan, David S. J.;Wu, Chuanfeng;Mortlock, Ryland D.;Chakraborty, Mala;Rezvani, Katayoun;Davidson-Moncada, Jan K.;Dunbar, Cynthia E.;Childs, Richard W.
• 通讯作者: Childs, Richard W.

Transient silencing of PTEN in human CD34(+) cells enhances their proliferative potential and ability to engraft immunodeficient mice.

• DOI: 10.1016/j.exphem.2011.10.001
• 发表时间: 2012-01
• 期刊: EXPERIMENTAL HEMATOLOGY
• 影响因子: 2.6
• 作者: Kim, Inho;Kim, Yoo-Jin;Metais, Jean-Yves;Dunbar, Cynthia E.;Larochelle, Andre
• 通讯作者: Larochelle, Andre

CD9 up-regulation on CD34+ cells with ingenol 3,20-dibenzoate does not improve homing in NSG mice.

巨大戟二萜醇 3,20-二苯甲酸酯对 CD34 细胞的 CD9 上调并不能改善 NSG 小鼠的归巢。

• DOI: 10.1182/blood-2011-01-332031
• 发表时间: 2011
• 期刊: Blood
• 影响因子: 20.3
• 作者: Desmond,Ronan;Dunfee,Ashley;Racke,Frederick;Dunbar,CynthiaE;Larochelle,Andre
• 通讯作者: Larochelle,Andre

Measurement of the absolute immature platelet number reflects marrow production and is not impacted by platelet transfusion.

• DOI: 10.1111/j.1537-2995.2012.03918.x
• 发表时间: 2013-06
• 期刊: Transfusion
• 影响因子: 2.9
• 作者: Bat T;Leitman SF;Calvo KR;Chauvet D;Dunbar CE
• 通讯作者: Dunbar CE

Barcode clonal tracking of tissue-resident immune cells in rhesus macaque highlights distinct clonal distribution pattern of tissue NK cells.

• DOI: 10.3389/fimmu.2022.994498
• 发表时间: 2022
• 期刊: FRONTIERS IN IMMUNOLOGY
• 影响因子: 7.3
• 作者: Wu, Chuanfeng;Liang, Jialiu A.;Brenchley, Jason M.;Shin, Taehoon;Fan, Xing;Mortlock, Ryland D.;Abraham, Diana M.;Allan, David S. J.;Thomas, Marvin L.;Hong, So Gun;Dunbar, Cynthia E.
• 通讯作者: Dunbar, Cynthia E.