Single cell RNA-seq on human T cells isolated from humanized mice engraft with human PBMC and treated or not with PTCy post-transplantation
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ABSTRACT: In order to assess the impact of PTCy on T cells in our humanized mouse GVHD model, we performed single cell RNA-sequencing on human T cells isolated from spleen of control and PTCy-treated mice on day 6 after hPBMC injection
Project description:Single-cell transcriptome profiling using a 3' droplet-based platform (Chromium,10x Genomics) of human CD45+ leukocytes isolated from leukemic HuSGM3 mice infused with CD19.28z CAR-T cells, two days after cytokine release syndrome (CRS) onset and 5 days later.
Project description:The goal of the experiment is to describe the overall transcriptomic alterations and signaling pathways activated by T cells upon transplantation in NSG or NSG-HLA-A2/HHD mice. PBMCs and CD3/CD28 activated T cells serve as negative and positive controls of these pathways activation, respectively. PBMCs from 4 different healthy volunteer donors were used. PBMCs from each donor were either used to sort T cells (by FACS) immediately after Ficoll isolation, or immediately stimulated in vitro or injected into mice.
Project description:Hematopoietic stem cells (HSCs) can regenerate the entire hematopoietic system in vivo, providing the most relevant criteria to measure candidate HSCs derived from human embryonic stem cell (hESC) or induced pluripotent stem cell (hiPSC) sources. Here, we show that unlike primitive hematopoietic cells derived from hESCs, phenotypically identical cells derived from hiPSC are more permissive to graft the bone marrow of xenotransplantation recipients. Despite establishment of bone marrow graft, hiPSC-derived cells fail to demonstrate hematopoietic differentiation in vivo. However, once removed from recipient bone marrow, hiPSC-derived grafts were capable of in vitro multilineage hematopoietic differentiation, indicating that xenograft imparts a restriction to in vivo hematopoietic progression. This failure to regenerate multilineage hematopoiesis in vivo was attributed to the inability to downregulate key microRNAs involved in hematopoiesis. Based on these analyses, our study indicates that hiPSCs provide a beneficial source of pluripotent stem cell-derived hematopoietic cells for transplantation compared with hESCs. Since use of the human-mouse xenograft models prevents detection of putative hiPSC-derived HSCs, we suggest that new preclinical models should be explored to fully evaluate cells generated from hiPSC sources. Human pluripotent stem cell-derived hematopoietic cells were isolated and qPCR-based microRNA profiling was performed.
Project description:To recapitulate the t(2;5)(p23;q35) chromosomal translocation, activated T lymphocytes from healthy donors PBMC were transfected with the RNP complex composed of the protein Cas9 with gRNA NPM1 (targeting NPM1 gene) and gRNA ALK (targeting ALK gene). Control cells were wild-type CD4+ activated lymphocytes. Cells were grown for 10-15 days in vitro prior to engraftment in vivo. Mice engrafted with NPM1-ALK+ T-cells developed T-cell lymphoma after 2-5 months. To get insights into the molecular bases of NPM1-ALK transformation, we next established the transcriptomes at an early in vitro stage and in fully transformed in vivo models. For this, we performed RNAseq analysis from 3 groups: 1-in vitro wild-type CD4+ activated lymphocytes [n=3], 2-in vitro NA-engineered CD4+ lymphocytes [n=3] and 3-in vivo derived CD4+ lymphoma cells purified by flow cytometry to distinguish CD3+ [n=3] and CD3- populations [n=3].
Project description:The study includes 14 patients with confirmed JMML and known somatic mutations (from exome data of paired tumoral and germline DNA). Bone marrow or peripheral blood mononucleated cells were injected in immundeficient mice to recapitulate the leukemia. Whole exome sequencing was performed in xenograft samples to control the persistance of patients' known mutations and look for new mutations acquired in xenograft sample.
Project description:Pre-clinical models that effectively recapitulate human disease are critical for expanding our knowledge of cancer biology and drug resistance mechanisms. For haematological malignancies, the non-obese diabetic/severe combined immunodeficient (NOD/SCID) mouse is one of the most successful models to study paediatric acute lymphoblastic leukaemia (ALL). However, for this model to be effective for studying engraftment and therapy responses at the whole genome level, careful molecular characterisation is essential.Here, we sought to validate species-specific gene expression profiling in the high engraftment continuous ALL NOD/SCID xenograft. Using the human Affymetrix whole transcript platform we analysed transcriptional profiles from engrafted tissues without prior cell separation of mouse cells and found it to return highly reproducible profiles in xenografts from individual mice. The model was further tested with experimental mixtures of human and mouse cells, demonstrating that the presence of mouse cells does not significantly skew expression profiles when xenografts contain 90% or more human cells. In addition, we present a novel in silico and experimental masking approach to identify probes and transcript clusters susceptible to cross-species hybridisation.We demonstrate species-specific transcriptional profiles can be obtained from xenografts when high levels of engraftment are achieved or with the application of transcript cluster masks. Importantly, this masking approach can be applied and adapted to other xenograft models where human tissue infiltration is lower. This model provides a powerful platform for identifying genes and pathways associated with ALL disease progression and response to therapy in vivo.
Project description:Understanding dynamics of antigen specific B cell responses and link between BCR and B cell differentiation is crucial for our ability to direct immune responses and to generate memory and plasma cells responses to protective targets. Here we have infected mice with IAV-PR8 and sacrificed them at different days after infection (7-14-28). 2 naive mice were used as controls (spleen and lungs). From individual spleen, lungs and mediastinal lymph nodes we have sorted Hemagglutinin (HA)-specific IgD- B cells and single cell sequenced RNA and BCR. We identify several known and novel B cell subpopulations forming after infection and find organ-specific difference that persist over the course of the response. We found important transcriptional differences between memory cells in lungs and immune organs and describe organ-restricted clonal expansion. Strikingly, by combining BCR mutational analysis, monoclonal antibody expression and affinity measurements we found no differences between germinal center (GC)-derived memory and plasma cells, at odds with an affinity-based selection model. These finding provide the most comprehensive picture to date of organ specific antiviral B cell responses, differentiation, clonal-proliferation and dynamics.
Project description:Despite the advanced understanding of disease mechanisms, the current therapeutic regimens fail to cure most patients with acute myeloid leukemia (AML). In the present study, we address the role of protein synthesis control in leukemia function and leukemia propagation. Using a transgenic eIF6 mouse strain that permits inducible and graded regulation of ribosomal subunit joining, we have generated a murine model of MLL-AF9 acute myeloid leukemia where the expression of transgenic eIF6 is doxycycline-inducible. Using this model system, we have performed scRNA-seq expression analysis to study the impact of eIF6 overexpression on leukemia cell function. scRNA-seq was performed using the 10x Chromium Next GEM Single Cell 3ʹ platform. BM cells were harvested from leukemia-engrafted mice that were administered doxycycline for six days and viable GFP+ leukemia cells were sorted into PBS containing 0.05 % BSA following the manusfacturer’s protocol. 5 samples: 2 control and 3 eIF6 overexpression.
Project description:Understanding dynamics of antigen specific B cell responses and link between BCR and B cell differentiation is crucial for our ability to direct immune responses and to generate memory and plasma cells responses to protective targets. Here we have infected mice with IAV-PR8 and sacrificed them at different days after infection (7-14-28). 2 naive mice were used as controls (spleen and lungs). From individual spleen, lungs and mediastinal lymph nodes we have sorted Hemagglutinin (HA)-specific IgD- B cells and single cell sequenced RNA and BCR. We identify several known and novel B cell subpopulations forming after infection and find organ-specific difference that persist over the course of the response. We found important transcriptional differences between memory cells in lungs and immune organs and describe organ-restricted clonal expansion. Strikingly, by combining BCR mutational analysis, monoclonal antibody expression and affinity measurements we found no differences between germinal center (GC)-derived memory and plasma cells, at odds with an affinity-based selection model. These finding provide the most comprehensive picture to date of organ specific antiviral B cell responses, differentiation, clonal-proliferation and dynamics.
Project description:Gene expression measurements in Thp, Th1 and Th2 cells polarised from naIve CD4+ T-cells isolated from wildtype and T-bet fl/fl x Cd4-Cre BALB/c mice or from WT and Gata-3 fl/fl x Tnfrsf4-Cre C57BL/6 mice.