Project description:scRNAseq of HPMCs isolated ex vivo from 3 Patients. Targeted scRNASeq was applied using BD onco Panel with a additional genes. Purpose of the experiment was to characterize HPMC phenotype ex vivo and compare with the phenotype observed in RNAseq of HPMCs treated with IL17A and TNF.
Project description:SPP1 stimulation of human leukocytes drives proinflammatory monocyte activation and differentiation of dysregulated CD274(PDL-1)pos neutrophil phenotype.
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: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:Mice were infected with the pancreatic cancer cell line Pan02 (into their pancreas). 30 days later, pancreas was extracted, cells digested and BD Rhapsody RNAseq targeted against the MM immune panel was performed.
Project description:To dissect the precise roles of ST-DC2 clusters and ST-iDC3 clusters in T-cell activation, we co-cultured each cluster separately with autologous memory CD4pos T-cells. FACS-sorted ST-DC2 clusters and ST-iDC3 clusters from synovial tissue of active RA or remission RA were co-cultured for 5 days with FACS-sorted autologous blood memory CD4pos T-cells in the presence of anti-CD3 stimulation to mimic TCR activation. We investigated the frequency and phenotype of the resultant co-culture T-cell populations by carrying out single cell sequencing and analysing the output. It was observed that the DC2 and iDC3 group could differentially support distinct T-Cell populations in both frequency, and transcriptional profile.
Project description:We tested the role of distinct STM populations in the modulation of synovial tissue environment in ex vivo STM-FLS micro co-cultures. We first FACS-sorted total synovial tissue fibroblasts as we described previously from biopsies of RA patients. FLS were seeded at 3000/well alone or in contact with either 3000/well FACS-sorted MerTK/CD206neg or MerTK/CD206pos STMs from patients with active or remission RA respectively for 48h. The modulatory effect on the FLS was evaluated by comparing their expression of 446 immune/stromal genes (scRNAseq BD Rhapsody) 32,141 FLS were evaluated. FLS cells cultured alone exhibited 4 distinct activation states: FLS cluster 1 (FLS1) expressed extracellular matrix proteins (e.g. COL1A1, COL1A2) and TGFb (TGFBI, TGFB3); FLS2 expressed cell adhesion molecules (e.g. ITGB2, SELPLG); FLS3 expressed receptors for TGFb and resolvin (e.g.CMKLR1 and TGFBR1); and FLS4 expressed high levels of glycolytic enzymes and proliferation markers (e.g. LDHA, PGK1, ENO1 and PCNA). Interestingly, upon co-culture with MerTK/CD206neg an additional fifth cluster (FLS5) emerged with inflammatory properties In contrast to inflammatory effect of the MerTK/CD206neg STMs on FLS, the MerTK/CD206pos population, especially that isolated from biopsies of RA patients in sustained disease remission, induced repair response of FLS as manifested by increased expression of collagens (e.g. COL1A) and TGFb response genes (e.g. TGFBI) Importantly, MerTK/CD206neg and MerTK/CD206pos STMs isolated from the biopsies of the same patient (either with active RA or RA in remission) elicited these divergent FLS responses. These suggest that these two distinct STM populations play opposing role in synovium (pro-inflammatory versus protective).
Project description:As opposed to their well-characterized contributions to inflammatory processes, tissue eosinophils are now also thought to contribute to immune homeostasis at mucosal sites such as the gut. Yet, whether such steady-state eosinophils exist in the lung is currently unclear. In this project, we identified and characterized lung resident eosinophil and also studied what happen to these cell during a mouse model of allergic inflammation (House dust mite (HDM) exact administrations). We compared the transcriptional profile of lung resident eosinophil from naive mice (rEOS ss) or from HDM-treated mice (rEOS i) and of inflammatory eosinophil (iEOS) from HDM-treated mice.
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.