Single cell sequencing for B cell repertoire of multi-year serial blood samples from two healthy donors.
Ontology highlight
ABSTRACT: Transcriptional and clonotype analysis aimed at investigation of the structure, stability and dynamics of the human memory B cell pool, using peripheral blood samples collected from two healthy donors with an interval of 10 or 6 years.
Project description:Transcriptional and clonotype analysis aimed at investigation of the structure, stability and dynamics of the human memory B cell pool, using peripheral blood samples collected from a single healthy donor with an interval of 4 years.
Project description:We performed a study investigating donors previously infected with SARS-CoV-2 during the first wave of the COVID-19 pandemic to understand how vaccination may reshape T cell populations formed after infection. 10 donors were investigated using single-cell RNA-sequencing consisting of a) 3 mild non-hospitalised donors, b) 3 severe hospitalised donors, c) 1 mild recently vaccinated donor and d) 3 recently convalescent donors. For each of these groups we sampled at the following timepoints a) 6-9 months and 18 months after infection, b) 6-9 months and 18 months after infection, c) 13 months and 15 months after infection and d) 35 days after infection. All donors were unvaccinated at the first time point and vaccinated at the second time point. We stimulated PBMC with an overlapping peptide pool derived from the spike glycoprotein of the SARS-CoV-2 virus and sorted spike-specific CD4+ T cells (CD4+CD69+CD40L+) and spike-specific CD8+ T cells (CD8+CD69+4-1BB+) from every donor and time point using flow cytometry for 10X single-cell sequencing (5' protocol). Each time point from Dnr4868 was sequenced across two 10X reactions/libraries performed on the same day. Multiple samples were hashed and pooled together for sequencing using TotalSeq-C anti-human hashing antibodies from BioLegend. Individual library expression matrix files were generated using the CellRanger pipeline from 10X Genomics. The processed data file named immune.combined220929.rds is a Seurat Object containing all samples and generated using the Seurat package in R.
Project description:We introduced single-chain trimer (SCT) technologies into a high throughput platform for pMHC library generation that can be used for screening antigen specific CD8+ T cells. We compared the diversity of T cell receptor repertoire captured by SCT and folded peptide-MHC multimer presenting HLA-A02:01 restricted CMV peptide. We then constructed SCT libraries designed to capture SARS-CoV-2 spike specific CD8+ T cells from COVID-19 participants and healthy donors. TCR sequencing with antigen specificity was analyzed. The immunogenicity of these epitopes was validated by functional assays of T cells with cloned TCRs captured using SCT libraries.
Project description:Post-acute sequelae of COVID-19 (PASC) represent an emerging global crisis. However, quantifiable risk-factors for PASC and their biological associations are poorly resolved. We executed a deep multi-omic, longitudinal investigation of 309 COVID-19 patients from initial diagnosis to convalescence (2-3 months later), integrated with clinical data, and patient-reported symptoms. We resolved four PASC-anticipating risk factors at the time of initial COVID-19 diagnosis: type 2 diabetes, SARS-CoV-2 RNAemia, Epstein-Barr virus viremia, and specific autoantibodies. In patients with gastrointestinal PASC, SARS-CoV-2-specific and CMV-specific CD8+ T cells exhibited unique dynamics during recovery from COVID-19. Analysis of symptom-associated immunological signatures revealed coordinated immunity polarization into four endotypes exhibiting divergent acute severity and PASC. We find that immunological associations between PASC factors diminish over time leading to distinct convalescent immune states. Detectability of most PASC factors at COVID-19 diagnosis emphasizes the importance of early disease measurements for understanding emergent chronic conditions and suggests PASC treatment strategies.
Project description:This study utilizes multi-omic biological data to perform deep immunophenotyping on the major immune cell classes in COVID-19 patients. 10X Genomics Chromium Single Cell Kits were used with Biolegend TotalSeq-C human antibodies to gather single-cell transcriptomic, surface protein, and TCR/BCR sequence information from 254 COVID-19 blood draws (a draw near diagnosis (-BL) and a draw a few days later (-AC)) and 16 healthy donors.
Project description:The experiment aims at characterizing the immune responses elicited by the BNT162b2 vaccine against SARS-CoV-2, initially administered in a two dose regimen (second dose after three weeks followinf the first dose) In particular the transcriptional landscape of circulating T and B lymphocytes has been profiled longitudinnaly by scRNA-seq coupleD with CITE-seq of 19 cell surface markers to better classify T cells subpopulations, LIBRA-seq to assess the Spike-specificity of BCRs and and V(D)J seq to also track T and B cell clones dynamics. Eeach sample was profiled before vaccination (T0), 21 days after the first dose (T1), 2 months after the first dose (1 month after the second dose) (T2). The immune responses were characterized using PBMC from 3 SARS-CoV-2 experienced donors (experiencing SARS-Cov-2 at least 4 months before the first vaccinatin) and 2 SARS-CoV-2 unexperienced donors.
Project description:The spinal cord neural stem cell potential is contained within the ependymal cells lining the central canal. This neural stem cell potential is known to decline with age in the mouse. Here, we microdissected and dissociated into single cells the central canal region from the spinal cord of 4 young adult (3-to-4-month old) and 4 aged (18-to-19-month old) C57BL/6J mice to profile the transcriptomes of cells in and around the central canal using 10x Genomics technology.
Project description:In this study, we investigated somatic mutations in T cells in patients with various hematological disorders. To analyze immune cell phenotypes with somatic mutations, we performed scRNA+TCRab sequencing from 9 patients with chronic GVHD and clonal expansions of CD4+ or CD8+ T cells based on T cell receptor sequencing. CD45+ PBMCs (lymphocytes and monocytes) were sorted with BD Influx cell sorter and subjected to sequencing with Chromium VDJ and Gene Expression platform (v1.1, 10X Genomics). Sequencing was performed with Novaseq 6000 (Illumina). The immune cell phenotypes were compared to healthy controls processed in the same laboratory (accession number E-MTAB-11170). Due to data privacy concerns, the raw sequencing data is in the European Genome-Phenome Archive (EGA) under accession code [xxxx] and can be requested through the EGA Data Access Committee.