Single-cell RNA and BCR sequencing of naive B cells cultured in IgG class-switching polarising conditions
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ABSTRACT: Naive B cells (CD27-IgD+) were obtained from 3 healthy controls and cultured in vitro under anti-IgM, CD40L and IFN-gamma to polarise class-switching towards IgG isotypes. Single-cell RNA and BCR sequencing libraries were prepared at Day 0 (before addition of stimuli), Day 3 and Day 6 of the cell culture time-course. This is intended as a dataset to validate a new computational method sciCSR in enumerating productive and sterile immunoglobulin transcripts as indicators of B cell class switching and maturation dynamics.
Project description:In response to antigen challenge, human B cells clonally expand, undergo selection and differentiate within secondary lymphoid tissues to produce mature B cell subsets and high affinity antibodies necessary for an effective immune response. However, the interplay between affinity, antibody class and different B cell fates has proved challenging to decipher in primary human tissue. We have applied an integrated analysis of bulk and single-cell antibody repertoires paired with single-cell transcriptomics of human B cells from a model secondary lymphoid tissue. Specifically, here we have used single-cell sequencing of antibody repertoires using the 10X Genomics platform to profile unsorted immune cells and sorted memory B cells from paediatric tonsil tissue. Matched gene expression and bulk B cell repertoires are also available for the same patient donors.
Project description:Separation of B cells has been historically important in discovering their functional relevance, particularly in relation to infection, immune disorders and vaccination. Traditional use of phenotypic markers often poses problems in distinguishing heterogeneous populations such as the Double Negative (DN, CD19+CD27-IgD-) cells. B cells represent a small subset of PBMCs; this represents challenges to use bottom-up approaches such as single-cell transcriptomics in defining B cell subpopulations. In this study we therefore used the 10X single-cell RNAseq platform on B cell populations already defined by FACS sorting (Transitional, CD19+CD27-IgD+CD10+; Naïve, CD19+CD27-IgD+CD10-; Classical Memory, CD19+CD27+IgD-; IgM Memory, CD19+CD27+IgD+; and DN). These data match known phenotypes to transcriptionally defined B cell subpopulations, and provide a reference atlas for researchers interested in better defining B cell subsets in their data.
Project description:In response to antigen challenge, human B cells clonally expand, undergo selection and differentiate within secondary lymphoid tissues to produce mature B cell subsets and high affinity antibodies necessary for an effective immune response. However, the interplay between affinity, antibody class and different B cell fates has proved challenging to decipher in primary human tissue. We have applied an integrated analysis of bulk and single-cell antibody repertoires paired with single-cell transcriptomics of human B cells from a model secondary lymphoid tissue. Specifically, here we have performed bulk B cell repertoire sequencing of the immunoglobulin heavy chain (IgH) for sorted B cell subsets from paediatric tonsil tissue. Matched single-cell gene expression and single-cell VDJ data are also available for the same patient donors.
Project description:In response to antigen challenge, human B cells clonally expand, undergo selection and differentiate within secondary lymphoid tissues to produce mature B cell subsets and high affinity antibodies necessary for an effective immune response. However, the interplay between affinity, antibody class and different B cell fates has proved challenging to decipher in primary human tissue. We have applied an integrated analysis of bulk and single-cell antibody repertoires paired with single-cell transcriptomics of human B cells from a model secondary lymphoid tissue. Specifically, here we have used single-cell RNA-seq using the 10X Genomics platform to profile unsorted immune cells and sorted memory B cells from paediatric tonsil tissue. Matched single-cell VDJ data and bulk B cell repertoires are also available for the same patient donors.
Project description:Motivation: The B-cell receptor (BCR) performs essential functions for the adaptive immune system including recognition of pathogen-derived antigens. The vast repertoire and adaptive variation of BCR sequences due to V(D)J recombination and somatic hypermutation (SHM) necessitates single-cell characterization of BCR sequences. Single-cell RNA sequencing (scRNA-seq) presents the opportunity for simultaneous capture of paired BCR heavy and light chains and the transcriptomic signature. Results: We developed VDJPuzzle, a novel bioinformatic tool that reconstructs productive, full-length B-cell receptor sequences of both heavy and light chains. VDJPuzzle successfully reconstructed BCRs from 98.3% (n=117) human and 96.5% (n=200) murine B cells. The reconstructed BCRs were successfully validated with single-cell Sanger sequencing. Availability: VDJPuzzle is available at https://bitbucket.org/kirbyvisp/vdjpuzzle2
Project description:RNA microarray profiling analysis was performed on splenic B cell subsets: “IgD+CD27-” (naive B cells) and “IgD+CD27+” (MZB B cells) isolated from splenic samples of 6 adults
Project description:RNA microarray profiling analysis was performed on 2 different ex-vivo splenic B cell subsets: M-^SIgD+CD27+M-^T (marginal zone B cells) and switched memory M-^SIgG+CD27+M-^T cells, isolated from splenic samples of 4 children (from 4 to 6 yr of age), 3 adults and 3 (or 4) seniors of more than 75 yr.
Project description:We compared migratory DCs in draining mediastinal lymph node after intranasal administration of different vaccine adjuvants based on CTA1 subunit of cholera toxin. The goal was to determine different characteristics of dendritic cells subsets after pro-inflammatory vs tolerogenic adjuvant. We had 3 groups: CTA1-DD (pro- inflammatory), CTA1(R7K)-DD (tolerogenic) and PBS to define DC which migrate to LN. All vaccine constructs included 3Ea peptide to allow for sorting of DC which took up the vaccine.
Project description:In this experiment we generated Affymetrix gene expression data for T Follicular Helper (TFH) cells from tonsils of healthy volunteers (4 biological replicates) and naive CD4-positive helper T cells (2 biological replicates). TFH cells provide a model relevant to SLE as TFH operate upstream of the activation of pathogenic autoantibody-producing B cells during the disease. This experiment accompanies promoter capture-C and ATAC-seq experiments on the same cell types.
Project description:In this study, we investigated somatic mutations of CD4+ and CD8+ T cells in patients with immune-mediated aplastic anemia (AA). To understand the role of mutations, we performed single-cell level analysis of 6 longitudinal samples of 2 AA patients carrying STAT3 or KRAS and other mutations in CD8+ T cells. The analysis was performed using V(D)J and 5' gene expression platform (10X Genomics). STAT3 mutated clone was clearly distinguishable from other CD8+ T cells and showed a cytotoxic phenotype, attenuated by successful immunosuppressive treatment. Our results suggest that somatic mutations in T cells can alter T cell phenotype warranting further investigation of their role in the pathogenesis of immune-mediated AA.