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

基本信息

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
项目类别：
财政年份：
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&apos 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

项目摘要

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

我们利用分子技术对体内造血干细胞和祖细胞 (HSPC) 以及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碱基对遗传条形码引入靶细胞，以研究非人灵长类动物模型中的体内造血作用。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作用。我们现在已经为 30 只猕猴移植了带有条形码的自体 CD34+ 细胞，并且能够在长达 8 年的时间内以定量和高度可重复的方式追踪多个谱系中数千个 HSPC 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 10X 和其他标准单细胞 RNASeq 平台进行高表达和检索的位置。第一只猕猴现已被移植了这种新型载体，并且针对扩增的 NK 细胞克隆的出现的分析正在进行中。我们已经取得了许多重要而新颖的发现，包括成熟 NK 细胞令人惊讶的生命史，表明循环成熟细胞毒性 NK 细胞 (CD16+CD56-) 的主要部分不与 B、T 或骨髓细胞共享条形码或其假定的前体 CD56brightCD16neg NK 细胞，甚至在移植后 80 个月。体外和小鼠模型以前无法阐明 NK 细胞谱系关系。这些循环 NK 细胞由大规模扩增的寡克隆细胞群组成，其盛衰模式表明对病毒感染或病毒再激活等特定环境线索的反应。我们的数据首次直接展示了克隆性 NK 反应，为 NK 记忆的可能机制提供了见解。我们使用先前与 NK 病毒和肿瘤反应相关的差异表达的 KIR 表面分子来对表达不同 KIR 的 NK 细胞进行分类，并记录了这些特定的表达 KIR 的 NK 群体内的克隆分离。这是首次直接证明具有特定受体特征（可能是表观遗传维持的）NK 细胞克隆群的产生和持续存在。随着基于 CD16 表达的体内 NK 耗竭，相同的扩增克隆再次出现，没有从高度多克隆 HSPC 中招募，而是从残留的高度增殖性 CD16dim NK 亚群中招募。我们分析了组织驻留 NK 细胞，这些细胞在小鼠和猴子过继转移研究中被证明对 NK 记忆功能很重要。我们在脾、肺、肠和肝等组织中发现了显着扩增的 NK 克隆，并且扩增的克隆在这些组织中共享，这表明扩增后从血液中特异性归巢到所有组织。我们还在血液中鉴定出含有这些扩增克隆的 CD56negCD16neg 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响应性归巢分子的表达。根据人类移植和献血者研究的相关数据，我们假设扩大的 NK 细胞克隆可能是在对 CMV 反应的情况下产生的，并且我们通过在 CMV 阴性猕猴中进行条形码移植来测试了这一假设，显示特定的克隆变化发生在实验性 CMV 感染后的成熟 NK 细胞群。 CMV 感染前后 NK 细胞的单细胞 RNASeq 实验记录了有关 NK 亚群与 NK 细胞对 CMV 反应之间关系的信息聚类和新见解。 NK 对 CMV 的适应性/记忆反应与涉及 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，我们的合作者 Louis Picker 此前已证明该平台可提供针对 SIV 的独特有效保护。我们正在研究条形码猕猴接种疫苗后是否会出现依赖于 HLA-E 的 NK 克隆扩增，并至少部分解释对这一有前途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，这是具有转化和临床重要性的主题。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，并且现在正在积极将研究扩展到抗体介导的预处理，以在抗 CD345 后实现条形码细胞的高水平植入条件作用，更令人惊讶的是，至少对外来转基因具有部分耐受性。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。我们与 Richard Childs 实验室合作，分析了离体扩增的 NK 细胞的克隆组成，并记录了在体外维持和扩增的扩增假定适应性克隆。我们计划研究过继转移后 NK 种群的克隆模式和持久性。我们还将碘化钠同向转运蛋白基因 (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

期刊论文数量（9）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

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
影响因子：
0
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

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

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

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.