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2.
Nat Immunol ; 23(5): 791-801, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35393592

RESUMO

Clonal expansion is a core aspect of T cell immunity. However, little is known with respect to the relationship between replicative history and the formation of distinct CD8+ memory T cell subgroups. To address this issue, we developed a genetic-tracing approach, termed the DivisionRecorder, that reports the extent of past proliferation of cell pools in vivo. Using this system to genetically 'record' the replicative history of different CD8+ T cell populations throughout a pathogen-specific immune response, we demonstrate that the central memory T (TCM) cell pool is marked by a higher number of prior divisions than the effector memory T cell pool, owing to the combination of strong proliferative activity during the acute immune response and selective proliferative activity after pathogen clearance. Furthermore, by combining DivisionRecorder analysis with single-cell transcriptomics and functional experiments, we show that replicative history identifies distinct cell pools within the TCM compartment. Specifically, we demonstrate that lowly divided TCM cells display enriched expression of stem-cell-associated genes, exist in a relatively quiescent state, and are superior in eliciting a proliferative recall response upon activation. These data provide the first evidence that a stem-cell-like memory T cell pool that reconstitutes the CD8+ T cell effector pool upon reinfection is marked by prior quiescence.


Assuntos
Linfócitos T CD8-Positivos , Memória Imunológica
3.
Nature ; 601(7891): 125-131, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34880496

RESUMO

All cancers emerge after a period of clonal selection and subsequent clonal expansion. Although the evolutionary principles imparted by genetic intratumour heterogeneity are becoming increasingly clear1, little is known about the non-genetic mechanisms that contribute to intratumour heterogeneity and malignant clonal fitness2. Here, using single-cell profiling and lineage tracing (SPLINTR)-an expressed barcoding strategy-we trace isogenic clones in three clinically relevant mouse models of acute myeloid leukaemia. We find that malignant clonal dominance is a cell-intrinsic and heritable property that is facilitated by the repression of antigen presentation and increased expression of the secretory leukocyte peptidase inhibitor gene (Slpi), which we genetically validate as a regulator of acute myeloid leukaemia. Increased transcriptional heterogeneity is a feature that enables clonal fitness in diverse tissues and immune microenvironments and in the context of clonal competition between genetically distinct clones. Similar to haematopoietic stem cells3, leukaemia stem cells (LSCs) display heritable clone-intrinsic properties of high, and low clonal output that contribute to the overall tumour mass. We demonstrate that LSC clonal output dictates sensitivity to chemotherapy and, although high- and low-output clones adapt differently to therapeutic pressure, they coordinately emerge from minimal residual disease with increased expression of the LSC program. Together, these data provide fundamental insights into the non-genetic transcriptional processes that underpin malignant clonal fitness and may inform future therapeutic strategies.


Assuntos
Competição entre as Células , Células Clonais/patologia , Leucemia Mieloide Aguda/patologia , Análise de Célula Única , Animais , Competição entre as Células/efeitos dos fármacos , Linhagem Celular , Linhagem da Célula/efeitos dos fármacos , Células Clonais/efeitos dos fármacos , Células Clonais/metabolismo , Feminino , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Camundongos , Camundongos Endogâmicos C57BL , Inibidor Secretado de Peptidases Leucocitárias/metabolismo
4.
Immunity ; 54(6): 1338-1351.e9, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-33862015

RESUMO

Despite advances in single-cell multi-omics, a single stem or progenitor cell can only be tested once. We developed clonal multi-omics, in which daughters of a clone act as surrogates of the founder, thereby allowing multiple independent assays per clone. With SIS-seq, clonal siblings in parallel "sister" assays are examined either for gene expression by RNA sequencing (RNA-seq) or for fate in culture. We identified, and then validated using CRISPR, genes that controlled fate bias for different dendritic cell (DC) subtypes. This included Bcor as a suppressor of plasmacytoid DC (pDC) and conventional DC type 2 (cDC2) numbers during Flt3 ligand-mediated emergency DC development. We then developed SIS-skew to examine development of wild-type and Bcor-deficient siblings of the same clone in parallel. We found Bcor restricted clonal expansion, especially for cDC2s, and suppressed clonal fate potential, especially for pDCs. Therefore, SIS-seq and SIS-skew can reveal the molecular and cellular mechanisms governing clonal fate.


Assuntos
Células Dendríticas/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Animais , Diferenciação Celular/genética , Linhagem Celular , Linhagem da Célula/genética , Feminino , Expressão Gênica/genética , Células HEK293 , Humanos , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Células-Tronco/metabolismo
5.
Nat Cell Biol ; 23(3): 219-231, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33649477

RESUMO

Regulation of haematopoietic stem and progenitor cell (HSPC) fate is crucial during homeostasis and under stress conditions. Here we examine the aetiology of the Flt3 ligand (Flt3L)-mediated increase of type 1 conventional dendritic cells (cDC1s). Using cellular barcoding we demonstrate this occurs through selective clonal expansion of HSPCs that are primed to produce cDC1s and not through activation of cDC1 fate by other HSPCs. In particular, multi/oligo-potent clones selectively amplify their cDC1 output, without compromising the production of other lineages, via a process we term tuning. We then develop Divi-Seq to simultaneously profile the division history, surface phenotype and transcriptome of individual HSPCs. We discover that Flt3L-responsive HSPCs maintain a proliferative 'early progenitor'-like state, leading to the selective expansion of multiple transitional cDC1-primed progenitor stages that are marked by Irf8 expression. These findings define the mechanistic action of Flt3L through clonal tuning, which has important implications for other models of 'emergency' haematopoiesis.


Assuntos
Proliferação de Células/efeitos dos fármacos , Células Dendríticas/efeitos dos fármacos , Hematopoese/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Proteínas de Membrana/farmacologia , RNA-Seq , Análise de Célula Única , Transcriptoma/efeitos dos fármacos , Animais , Linhagem da Célula , Células Cultivadas , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/metabolismo , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fenótipo
6.
Nat Methods ; 16(6): 479-487, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31133762

RESUMO

Single cell RNA-sequencing (scRNA-seq) technology has undergone rapid development in recent years, leading to an explosion in the number of tailored data analysis methods. However, the current lack of gold-standard benchmark datasets makes it difficult for researchers to systematically compare the performance of the many methods available. Here, we generated a realistic benchmark experiment that included single cells and admixtures of cells or RNA to create 'pseudo cells' from up to five distinct cancer cell lines. In total, 14 datasets were generated using both droplet and plate-based scRNA-seq protocols. We compared 3,913 combinations of data analysis methods for tasks ranging from normalization and imputation to clustering, trajectory analysis and data integration. Evaluation revealed pipelines suited to different types of data for different tasks. Our data and analysis provide a comprehensive framework for benchmarking most common scRNA-seq analysis steps.


Assuntos
Adenocarcinoma/genética , Benchmarking , Biologia Computacional/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Neoplasias Pulmonares/genética , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Humanos , Software , Células Tumorais Cultivadas
7.
J Math Biol ; 79(2): 673-704, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31069504

RESUMO

Motivated by a recently proposed design for a DNA coded randomised algorithm that enables inference of the average generation of a collection of cells descendent from a common progenitor, here we establish strong convergence properties for the average generation of a super-critical Bellman-Harris process. We further extend those results to a two-type Bellman-Harris process where one type can give rise to the other, but not vice versa. These results further affirm the estimation method's potential utility by establishing its long run accuracy on individual sample-paths, and significantly expanding its remit to encompass cellular development that gives rise to differentiated offspring with distinct population dynamics.


Assuntos
Diferenciação Celular/genética , Replicação do DNA , Modelos Genéticos , Algoritmos , Técnicas de Cultura de Células , Morte Celular/genética , Proliferação de Células/genética , Simulação por Computador , Microscopia Intravital , Homeostase do Telômero/genética , Imagem com Lapso de Tempo
8.
Front Immunol ; 9: 1313, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29951060

RESUMO

Adaptation of antibody-mediated immunity occurs in germinal centers (GC). It is where affinity maturation, class switching, memory and plasma cell differentiation synergize to generate specific high-affinity antibodies that aid both to clear and protect against reinfection of invading pathogens. Within GCs, light and dark zone are two compartments instrumental in regulating this process, by segregating T cell-dependent selection and differentiation from generation of GC B cells bearing hypermutated antigen receptors. Spatial segregation of GC B cells into the two zones relies on the chemokine receptor CXCR4, with textbooks attributing high and low expression to a dark and light zone phenotype. Interestingly, this bipolarity is not reflected in the CXCR4 expression profile of GC B cells, which is highly variable and unimodal, indicating a continuum of intermediate CXCR4 levels rather than a binary dark or light zone phenotype. Here, analysis of published BrdU pulse-chase data reveals that throughout cell cycle, average CXCR4 expression in GC B cells steadily increases close to twofold, scaling with cell surface area. CXCR4 expression in recently divided GC B cells in G0/G1 or early S phase shows intermediate levels compared to cells in G2M phase, consistent with their smaller size. The lowest number of CXCR4 receptors are displayed by relatively aged GC B cells in G0/G1 or early S phase. The latter, upon progressing through S phase, however, ramp up relative CXCR4 expression twice as much as recently divided cells. Twelve hours after the BrdU pulse, labeled GC B cells, while initially in S phase, are desynchronized in terms of cell cycle and match the CXCR4 profile of unlabeled cells. A model is discussed in which CXCR4 expression in GC B cell increases with cell cycle and cell surface area, with highest levels in G2 and M phase, coinciding with GC B cell receptor signaling in G2 and immediately preceding activation-induced cytidine deaminase (AID) activity in early G1. In the model, GC B cells compete for CXCL12 expression on the basis of their CXCR4 expression, gaining a relative advantage as they progress in cell cycle, but loosing the advantage at the moment they divide.

9.
Cell Stem Cell ; 22(1): 64-77.e6, 2018 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-29276143

RESUMO

Bone marrow vascular niches sustain hematopoietic stem cells (HSCs) and are drastically remodeled in leukemia to support pathological functions. Acute myeloid leukemia (AML) cells produce angiogenic factors, which likely contribute to this remodeling, but anti-angiogenic therapies do not improve AML patient outcomes. Using intravital microscopy, we found that AML progression leads to differential remodeling of vasculature in central and endosteal bone marrow regions. Endosteal AML cells produce pro-inflammatory and anti-angiogenic cytokines and gradually degrade endosteal endothelium, stromal cells, and osteoblastic cells, whereas central marrow remains vascularized and splenic vascular niches expand. Remodeled endosteal regions have reduced capacity to support non-leukemic HSCs, correlating with loss of normal hematopoiesis. Preserving endosteal endothelium with the small molecule deferoxamine or a genetic approach rescues HSCs loss, promotes chemotherapeutic efficacy, and enhances survival. These findings suggest that preventing degradation of the endosteal vasculature may improve current paradigms for treating AML.


Assuntos
Células-Tronco Hematopoéticas/patologia , Leucemia Mieloide Aguda/patologia , Nicho de Células-Tronco , Animais , Medula Óssea/irrigação sanguínea , Medula Óssea/patologia , Contagem de Células , Hematopoese , Humanos , Microscopia Intravital , Camundongos Endogâmicos C57BL , Baço/patologia , Células Estromais/patologia , Fatores de Tempo , Microambiente Tumoral
10.
BMC Syst Biol ; 10(1): 43, 2016 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-27363727

RESUMO

BACKGROUND: Cellular barcoding is a recently developed biotechnology tool that enables the familial identification of progeny of individual cells in vivo. In immunology, it has been used to track the burst-sizes of multiple distinct responding T cells over several adaptive immune responses. In the study of hematopoiesis, it revealed fate heterogeneity amongst phenotypically identical multipotent cells. Most existing approaches rely on ex vivo viral transduction of cells with barcodes followed by adoptive transfer into an animal, which works well for some systems, but precludes barcoding cells in their native environment such as those inside solid tissues. RESULTS: With a view to overcoming this limitation, we propose a new design for a genetic barcoding construct based on the Cre Lox system that induces randomly created stable barcodes in cells in situ by exploiting inherent sequence distance constraints during site-specific recombination. We identify the cassette whose provably maximal code diversity is several orders of magnitude higher than what is attainable with previously considered Cre Lox barcoding approaches, exceeding the number of lymphocytes or hematopoietic progenitor cells in mice. CONCLUSIONS: Its high diversity and in situ applicability, make the proposed Cre Lox based tagging system suitable for whole tissue or even whole animal barcoding. Moreover, it can be built using established technology.


Assuntos
Engenharia Genética/métodos , Integrases/metabolismo , Recombinação Genética , Animais , Linfócitos T CD8-Positivos/metabolismo , Variação Genética , Sequências Repetidas Invertidas/genética , Camundongos
11.
J Math Biol ; 73(2): 491-523, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-26733310

RESUMO

For proliferating cells subject to both division and death, how can one estimate the average generation number of the living population without continuous observation or a division-diluting dye? In this paper we provide a method for cell systems such that at each division there is an unlikely, heritable one-way label change that has no impact other than to serve as a distinguishing marker. If the probability of label change per cell generation can be determined and the proportion of labeled cells at a given time point can be measured, we establish that the average generation number of living cells can be estimated. Crucially, the estimator does not depend on knowledge of the statistics of cell cycle, death rates or total cell numbers. We explore the estimator's features through comparison with physiologically parameterized stochastic simulations and extrapolations from published data, using it to suggest new experimental designs.


Assuntos
Divisão Celular , Células/citologia , Modelos Biológicos , Contagem de Células , Ciclo Celular , Células/metabolismo , Simulação por Computador , Probabilidade , Coloração e Rotulagem
12.
J Immunol ; 184(3): 1339-47, 2010 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-20053939

RESUMO

Immunization with a T cell-dependent Ag leads to the formation of several hundred germinal centers (GCs) within secondary lymphoid organs, a key process in the maturation of the immune response. Although prevailing perceptions about affinity maturation intuitively assume simultaneous seeding, growth, and decay of GCs, our previous mathematical simulations led us to hypothesize that their growth might be nonsynchronized. To investigate this, we performed computer-aided three-dimensional reconstructions of splenic GCs to measure size distributions at consecutive time points following immunization of BALB/c mice with a conjugate of 2-phenyl-oxazolone and chicken serum albumin. Our analysis reveals a broad volume distribution of GCs, indicating that individual GCs certainly do not obey the average time course of the GC volumes and that their growth is nonsynchronized. To address the cause and implications of this behavior, we compared our empirical data with simulations of a stochastic mathematical model that allows for frequent and sudden collapses of GCs. Strikingly, this model succeeds in reproducing the empirical average kinetics of GC volumes as well as the underlying broad size distributions. Possible causes of GC B cell population collapses are discussed in the context of the affinity-maturation process.


Assuntos
Subpopulações de Linfócitos B/citologia , Subpopulações de Linfócitos B/imunologia , Proliferação de Células , Citocinese/imunologia , Centro Germinativo/citologia , Centro Germinativo/imunologia , Modelos Imunológicos , Animais , Adesão Celular/imunologia , Agregação Celular/imunologia , Diferenciação Celular/imunologia , Estudos Transversais , Haptenos/administração & dosagem , Haptenos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Oxazolona/administração & dosagem , Oxazolona/análogos & derivados , Oxazolona/imunologia , Baço/citologia , Baço/imunologia , Processos Estocásticos
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