Project description:We conducted single-cell gene expression analysis of neutrophils from mouse tumors with and without microbial treatment to investigate neutrophil response in the tumor microenvironment (TME). Briefly, tumor-infiltrating Ly6G+CD11b+ neutrophils were isolated from unmanipulated tumors (resting), tumors treated with a vehicle control (control), and tumors treated with S. aureus bioparticles (stimulated) 24 hours after treatment. To survey the whole spectrum of the TME, we also isolated and sequenced non-neutrophil leukocytes in each sample.
Project description:To test the contribution of distinct ST-DC populations to either autoimmunity and inflammation or sustained disease remission, we evaluated the phenotypes of their blood precursors in a human model of disease flare following treatment withdrawal in RA patients in disease remission (the BioRRA study, (Baker et al., 2019)). We investigated the frequency and phenotype of PB DC2 and PB-DC3 of RA patients (n=12) in sustained clinical and ultrasound remission achieved with cDMARDs (Baker et al., 2019) by carrying out, and analysing single cell sequencing data at baseline remission levels, and upon endpoint after treatment withdrawal. We identified that PB DCs differ transcriptionally, but not proportionally, at the baseline of patients who sustain remission, versus those who go on to flare, and at endpoint. We propose that the transcriptomic profile of these peripheral blood DCs could be used as a biomarker of flare in remission RA patients.
Project description:Immunotherapy using CD19-directed chimeric antigen receptor (CAR)-T cells has shown excellent results for treatment of B-cell leukaemia and lymphoma. To produce CAR-T cells, the patient’s own T cells are isolated from the blood and modified in a laboratory with a genetic vector to express a tumor antigen-directed CAR on its surface. The CAR-T cells are then expanded in numbers and given back to the patient with the aim to eradicate the tumors. However, some patients display primary resistance to CAR-T treatment while others relapse quickly after CAR-T treatment. In this experiment, we seek to understand whether the quality of the individual CAR-T cell product the patients were given can predict outcome to the therapy. We investigate the transcriptional profile of the individual CAR-T infusion products using single-cell RNA sequencing. In this dataset, we identified a T cell subset correlating with response that could be used as an indicator for clinical outcome. Targeted RNA and protein single-cell libraries were obtained using the BD Rhapsody platform (BD Biosciences). In total four separate targeted libraries were produced with 6 patients per library. Sequencing was performed on NovaSeq 6000 S1 sequencer at the SNP&SEQ Technology Platform (Uppsala, Sweden). The raw scRNA-seq data was pre-processed by BD Biosciences using the Rhapsody Analysis pipeline to convert the raw reads into Unique Molecular Identifier (UMI) counts. UMIs are further adjusted within Rhapsody by applying BD’s Recursive Substitution Error Correction (RSEC) and Distribution-Based Error Correction (DBEC) in order to remove false UMIs caused by sequencing or library preparation errors. Pooled samples were deconvoluted using Sample-tag reads. The scRNA-seq and AbSeq counts were loaded, processed and used for clustering and differential gene expression with Seurat v. 4.0.0.
Project description:4 groups of mice : Control, antibiotics, antibiotics + 4days of recolonization, antibiotics + 4days of recolonization + Enterocloster clostridioformis. Tumor draining lymph node were harvested after CFSE injection were harvested and CFSE+ cells were sorted and proccessed in order to generate single cell RNA-sequencing using BD Rhapsody mouse immune response targeted panel. Groups were barcoded using BD Rhapsody Multiplexing Kit.
Project description:Both iron homeostasis and erythropoiesis are known to be affected by aging. Iron needs in mammals are met primarily by iron recycling from senescent red blood cells (RBCs), a task chiefly accomplished by red pulp macrophages (RPMs) in the spleen. Given that RPMs continuously process iron, their cellular functions might be susceptible to age-dependent decline, a possibility that has been unexplored to date. In our project, we identified a formation of undegradable iron- and heme-rich extracellular aggregates in the spleens of 10-11-month-old female mice. To better understand the origin of these aggregates, here, we performed: i) protein identification and intrasample quantification (iBAQ) of proteins of magnetically-isolated red pulp macrophages from spleens of two female 8-weeks-old C57BL/6J (maintained on a standard diet) and ii) label-free quantification of proteins of the splenic protein aggregates formed in the mouse spleen 24 hours after intraperitoneal iron dextran injection, using dextran-injected mice as a control. Two 8-week-old C57BL/6J mice per group were analyzed. This dataset is related to the project PXD032900, which describes quanytitative analysis of proteins in aggregates magnetically isolated from spleen of aged (standard or iron-reduced diet) and young mice (standard diet).
Project description:Splenic white pulp (WP) structures are underpinned by fibroblastic stromal cells (FSCs) to facilitate splenic compartmentalization and execute efficient immune responses. Although distinct WP FSCs exhibit various molecular traits, the origin and the hierarchical differentiation of different cell subsets are not characterized. Here we showed, the organization of splenic WP and the differentiation of WP FSCs were governed by lymphotoxin beta receptor (LTβR) signaling pathway. Cell fate mapping analysis revealed that different WP fibroblastic stromal cells descend from a common perivascular LTβR-sensitive mesenchymal lymphoid organizer cells (mLTo) at prenatal stage. Moreover, embryonic mLTo cells required LTβR signaling to give rise to different WP stromal cell subsets, while the proliferation of these cells was devoid of LTβR signaling but followed the development of WP during ontogeny. Moreover, cell fate mapping from different time point indicated a consecutive commitment of mLTo cells initiated from the proximal region around the splenic artery. RNAseq and differentiation trajectory analysis of distinct FSCs showed that Ltbr-deficient cells and perivascular reticular cells (PRCs) from adult spleen exhibited a progenitor phenotype and revealed a closer hierarchical lineage with embryonic mLTo cells. Taken together, our results unveil that embryonic mLTo cells residing in the perivascular niches can give rise to different FSC populations in a LTβR-dependent manner during development.
Project description:SPP1 stimulation of human leukocytes drives proinflammatory monocyte activation and differentiation of dysregulated CD274(PDL-1)pos neutrophil phenotype.
Project description:Gene expression changes in metastasis associated macrophage (MAM) FACS sorted cells from mice bearing pulmonary metastasis of breast tumor cells were compared with lung and spleen resident macrophages sorted from healthy mice using same sorting protocol. A six chip study using total RNA recovered from metastasis associated macrophages from three individual mice and lungs and spleens all from three individual mice. Each chip measures the expression level of 42586 genes.