36 research outputs found
Perivascular niche cells sense thrombocytopenia and activate hematopoietic stem cells in an IL-1 dependent manner
No full textHematopoietic stem cells (HSCs) residing in specialized niches in the bone marrow are responsible for the balanced output of multiple short-lived blood cell lineages in steady-state and in response to different challenges. However, feedback mechanisms by which HSCs, through their niches, sense acute losses of specific blood cell lineages remain to be established. While all HSCs replenish platelets, previous studies have shown that a large fraction of HSCs are molecularly primed for the megakaryocyte-platelet lineage and are rapidly recruited into proliferation upon platelet depletion. Platelets normally turnover in an activation-dependent manner, herein mimicked by antibodies inducing platelet activation and depletion. Antibody-mediated platelet activation upregulates expression of Interleukin-1 (IL-1) in platelets, and in bone marrow extracellular fluid in vivo. Genetic experiments demonstrate that rather than IL-1 directly activating HSCs, activation of bone marrow Lepr+ perivascular niche cells expressing IL-1 receptor is critical for the optimal activation of quiescent HSCs upon platelet activation and depletion. These findings identify a feedback mechanism by which activation-induced depletion of a mature blood cell lineage leads to a niche-dependent activation of HSCs to reinstate its homeostasis
Deciphering the immune microenvironment in pediatric Acute Myeloid Leukaemia (AML) using single-cell multi-omics
No full textLAUREA MAGISTRALELa Leucemia Mieloide Acuta pediatrica (LMAp) è una neoplasia rara del midollo osseo, caratterizzata da un rapido accumulo di cellule mieloidi anomale e immature. I risultati terapeutici per i pazienti con LMAp sono poco soddisfacenti, in particolare in termini di sopravvivenza libera da eventi, a causa degli alti tassi di recidiva. La ricerca sta di recente investigando il ruolo prognostico e terapeutico del sistema immunitario nella LMAp, fondamentale nello sviluppo di terapie cellulari come le CAR-T.
Per caratterizzare il microambiente immunitario nella LMAp, in questo lavoro è stato analizzato un nuovo dataset con risoluzione a singola cellula, composto da 22 campioni longitudinali di midollo osseo da 13 pazienti LMA pediatrici ad alto rischio e 5 controlli sani. La tecnica di acquisizione CITE-seq, ha permesso una caratterizzazione multimodale delle cellule, fornendo una misura completa del loro trascritto e la quantificazione di 81 epitopi di superficie. Dopo il pre-processing, l’analisi si è concentrata su una selezione di circa 20,000 linfociti T/NK e 10,000 B, poi ri-annotati per identificare i sottotipi cellulari.
Analisi differenziali di abbondanza ed espressione genica sono state eseguite sulle popolazioni linfoidi, al fine di evidenziare eventuali differenze legate alla LMAp in sè o alla sua progressione. Queste, Insieme all’analisi di espressione delle proteine di superficie, hanno permesso di evidenziare alterazioni funzionali e fenotipiche nelle cellule linfoidi, legate alla LMA. Questo lavoro ha confermato alcuni risultati di studi precedenti, come la presenza delle Cellule B Atipiche nei campioni LMAp e lo stato di esaurimento delle cellule T in LMAp, associato alla progressiva perdita di espressione superficiale di CD26. Inoltre, sono emersi nuovi aspetti riguardo l’iper-attivazione dei linfociti NK in LMA. Questi risultati, da verificare in un maggior numero di pazienti, hanno una potenziale rilevanza nella progettazione di nuove terapie cellulari e strategie immunoterapeutiche, destinate a diventare la nuova frontiera nel trattamento della LMA.Paediatric Acute Myeloid Leukaemia (pAML) is a rare malignant disease of the bone marrow, characterized by the rapid accumulation of abnormal immature myeloid blood cells. Treatment outcomes for pAML patients are unsatisfactory, particularly in terms of event-free survival, due to the high relapse rates. Recently, increasing attention has been given to the role of the immune system in pAML, both as a prognostic index and as a therapeutic target, due to the advancement of cell-based approaches such as CAR-T cell therapies.
With the aim of characterising the pAML immune microenvironment, this work focuses on the analysis of a newly developed single-cell multi-omic dataset, comprising of 22 longitudinal bone marrow samples from 13 high-risk pAML patients and 5 healthy controls. The Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) allowed a multimodal characterisation of cells, providing the unbiased measure of their transcriptome and quantifying the levels of 81 surface proteins. After the pre-processing steps, the analysis focused on a selected subset of healthy lymphoid cells, consisting of ~20,000 T/NK and 10,000 B lymphocytes, which were carefully re-annotated to identify distinct cell subtypes. Differential abundance and gene expression analyses were performed on all identified lymphoid populations (AML vs Control cells) to evidence any AML or disease timepoint related differences. This analysis, combined with surface protein expression analysis, revealed functional and phenotypical alterations in the lymphoid compartment associated with AML.
This work confirmed previous findings such as the presence of Atypical B cells in pAML samples, and the AML-related exhaustion state of T cells, associated with the progressive loss of surface CD26. Moreover, some novel aspects have emerged regarding the hyper-activation of Natural Killer lymphocytes in AML. These results, still requiring validation in larger cohorts, have potential relevance in designing innovative cell-based and immune therapies, set to become the next frontier in AML treatment
miRNA-126 Orchestrates an Oncogenic Program in B Cell Precursor Acute Lymphoblastic Leukemia
No full textMicroRNA (miRNA)-126 is a known regulator of hematopoietic stem cell quiescence. We engineered murine hematopoiesis to express miRNA-126 across all differentiation stages. Thirty percent of mice developed monoclonal B cell leukemia, which was prevented or regressed when a tetracycline-repressible miRNA-126 cassette was switched off. Regression was accompanied by upregulation of cell-cycle regulators and B cell differentiation genes, and downregulation of oncogenic signaling pathways. Expression of dominant-negative p53 delayed blast clearance upon miRNA-126 switch-off, highlighting the relevance of p53 inhibition in miRNA-126 addiction. Forced miRNA-126 expression in mouse and human progenitors reduced p53 transcriptional activity through regulation of multiple p53-related targets. miRNA-126 is highly expressed in a subset of human B-ALL, and antagonizing miRNA-126 in ALL xenograft models triggered apoptosis and reduced disease burden
Driving CAR T Stem Cell Targeting in Acute Myeloid Leukemia: The Roads to Success
No full textCurrent treatment outcome for acute myeloid leukemia (AML) patients is unsatisfactory and characterized by high rates of relapse and poor overall survival. Increasing evidence points to a crucial role of leukemic stem cells (LSC) and the bone marrow (BM) leukemic niche, in which they reside, in AML evolution and chemoresistance. Thus, future strategies aiming at improving AML therapeutic protocols are likely to be directed against LSC and their niche. Chimeric antigen receptor (CAR) T-cells have been extremely successful in the treatment of relapsed/refractory acute lymphoblastic leukemia and B-cell non-Hodgkin lymphoma and comparable results in AML are highly desirable. At present, we are at the dawn of CAR T-cell application in AML, with several preclinical studies and few early phase clinical trials. However, the lack of leukemia-specific targets and the genetic and phenotypic heterogeneity of the disease combined with the leukemia-induced remodeling of the BM microenvironment are limiting CAR T-cell exploitation in AML. Here, we reviewed AML-LSC and AML-BM niche features in the context of their therapeutic targeting using CAR T-cells. We summarized recent progress in CAR T-cell application to the treatment of AML, and we discussed the remaining therapeutic challenges and promising novel strategies to overcome them
A Double-Switch Vector System Positively Regulates Transgene Expression by Endogenous microRNA Expression (miR-ON Vector)
No full textInternational audienceTo better understand and exploit microRNA (miR) regulation, a more precise characterization of miR expression patterns within a tissue or a lineage during development, differentiation, and homeostasis is needed. We previously showed that lentiviral vectors (LV) can be made responsive to miR to stringently control transgene expression as well as to report miR activity "live" and at the single-cell level. Although very useful, this approach reports miR activity by transgene suppression, hampering the direct identification and selection of miR-expressing cells. Here, we describe a strategy to couple transgene expression to the activity of the miR of interest. To this aim, we generated LV encoding two in-series OFF switches: a transcriptional repressor tagged with miR target sequences and a reporter cassette under the control of the repressor. Reporter expression is ON only when the miR is active and represses translation of the transcriptional repressor. We successfully applied this design to different types of repressors, multiple gene encoding vectors and delivered the system either by two separate or a self-contained vector. We demonstrated its performance by live monitoring of two miRs in different stages of human primary hematopoietic stem/progenitor cell differentiation in vivo. Further applications of this approach include imaging of rare miR-expressing cells and positive regulation of a therapeutic or selector gene in target cells identified by the expression of selected miRs
Systemic and targeted delivery of semaphorin 3A inhibits tumor angiogenesis and progression in mouse tumor models.
No full textOBJECTIVE:
The role of semaphorins in tumor progression is still poorly understood. In this study, we aimed at elucidating the regulatory role of semaphorin 3A (SEMA3A) in primary tumor growth and metastatic dissemination.
METHODS AND RESULTS:
We used 3 different experimental approaches in mouse tumor models: (1) overexpression of SEMA3A in tumor cells, (2) systemic expression of SEMA3A following liver gene transfer in mice, and (3) tumor-targeted release of SEMA3A using gene modified Tie2-expressing monocytes as delivery vehicles. In each of these experimental settings, SEMA3A efficiently inhibited tumor growth by inhibiting vessel function and increasing tumor hypoxia and necrosis, without promoting metastasis. We further show that the expression of the receptor neuropilin-1 in tumor cells is required for SEMA3A-dependent inhibition of tumor cell migration in vitro and metastatic spreading in vivo.
CONCLUSIONS:
In sum, both systemic and tumor-targeted delivery of SEMA3A inhibits tumor angiogenesis and tumor growth in multiple mouse models; moreover, SEMA3A inhibits the metastatic spreading from primary tumors. These data support the rationale for further investigation of SEMA3A as an anticancer molecule
Single-cell transcriptomics uncovers distinct molecular signatures of stem cells in chronic myeloid leukemia
No full textRecent advances in single-cell transcriptomics are ideally placed to unravel intratumoral heterogeneity and selective resistance of cancer stem cell (SC) subpopulations to molecularly targeted cancer therapies. However, current single-cell RNA-sequencing approaches lack the sensitivity required to reliably detect somatic mutations. We developed a method that combines high-sensitivity mutation detection with whole-transcriptome analysis of the same single cell. We applied this technique to analyze more than 2,000 SCs from patients with chronic myeloid leukemia (CML) throughout the disease course, revealing heterogeneity of CML-SCs, including the identification of a subgroup of CML-SCs with a distinct molecular signature that selectively persisted during prolonged therapy. Analysis of nonleukemic SCs from patients with CML also provided new insights into cell-extrinsic disruption of hematopoiesis in CML associated with clinical outcome. Furthermore, we used this single-cell approach to identify a blast-crisis-specific SC population, which was also present in a subclone of CML-SCs during the chronic phase in a patient who subsequently developed blast crisis. This approach, which might be broadly applied to any malignancy, illustrates how single-cell analysis can identify subpopulations of therapy-resistant SCs that are not apparent through cell-population analysis
High-dimensional functional phenotyping of preclinical human CAR T cells using mass cytometry
No full textHere, we present a comprehensive protocol for the generation and functional characterization of chimeric antigen receptor (CAR) T cells and their products by mass cytometry in a reproducible and scalable manner. We describe the production of CAR T cells from human peripheral blood mononuclear cells. We then detail a three-step staining protocol with metal-labeled antibodies and the subsequent mass cytometry analysis. This protocol allows simultaneous characterization of CAR T cell intracellular signaling, activation, proliferation, cytokine production, and phenotype in a single assay
