Gene expression profiling of human Ig class switched memory B cells
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ABSTRACT: We purified 4 Ig class switched memory B cell populations from human adults to compare their gene expression profiles with each other and with those of human naive and IgM memory B cells generated in a previous experiment (E-MEXP-3767).
Project description:To study the nature of persistent polyclonal B cell lymphocytosis (PPBL), we purified CD27posIgMposIgDpos from 4 patients, as well as transitional (CD38hiCD24hi), pre-naive (CD38dimCD24dimCD5pos), naive mature (CD38dimCD24dimCD5-CD27-), natural effector (CD27posIgMposIgDpos) and IgM-only (CD27posIgMposIgDneg) B cells from healthy controls and performed genome-wide transcription profiling. We aimed to identify the control counterpart of PPBL cells and to study deregulated processes as compared to this control counterpart
Project description:We purified five subsets representing the main stages of human precursor-B-cell differentiation and CD34+lin- cord blood cells. The immunoglobulin (Ig) gene rearrangement status was determined using TaqMan quantitative PCR and GeneScan analysis. To gain more insight in the networks of genes that initiate and/or regulate the different types of Ig gene rearrangements, we analyzed their gene expression profiles by correlating the initiation of Ig gene rearrangements with specific upregulation of transcription factors. In addition to previously described transcription factors, we identified 16 candidate genes involved in initiation and/or regulation of Ig gene rearrangements.
Project description:We derived B-lineage cells by in vitro culture of neonatal cord blood CD34+ cells on MS-5 stromal cells with recombinant IL-7. These cultures yielded CD19+CD127+ and CD19+CD127- cell populations. We performed gene expression profiling on these populations and compared these with each other and with published expression profiles of freshly isolated BM precursor B-cell subsets (E-MEXP-384). These analyses yielded new insights into their different functionality and developmental stage.<br><br>A file containing statistical analysis of the normalized data is included in the file named E-MEXP-2878.additional.zip on the FTP site for this experiment.
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: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: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:Memory B cell responses are more rapid and of greater magnitude than are primary antibody responses. The mechanisms by which these secondary responses are eventually attenuated remain unknown. We demonstrate that the transcription factor ZBTB32 limits the rapidity and duration of antibody recall responses. ZBTB32 is highly expressed by mouse and human memory B cells, but not by their naïve counterparts. Zbtb32-/- mice mount normal primary antibody responses to T-dependent antigens. However, Zbtb32-/- memory B cell-mediated recall responses occur more rapidly and persist longer than do control responses. Microarray analyses demonstrate that Zbtb32-/- secondary bone marrow plasma cells display elevated expression of genes that promote cell cycle progression and mitochondrial function relative to wild-type controls. BrdU labeling and adoptive transfer experiments confirm more rapid production and a cell-intrinsic survival advantage of Zbtb32-/- secondary plasma cells relative to wild-type counterparts. ZBTB32 is therefore a novel negative regulator of antibody recall responses. CD45.2 wild type and Zbtb32-/- splenocytes from NP-CGG-immune donors were transferred into CD45.1 recipients and challenged with NP-CGG. CD45.2 donor NP-specific memory B cells were isolated from the spleen 7 days later. 5-6 biological replicates of each genotype were performed.
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:Antibodies are immune glycoproteins that represent a crucial part of the adaptive immune system as they mediate broad protection against viruses, bacteria, and cancer. Moreover, antibodies may serve as a unique source of information on past and current physiological and pathological events in the human body. The dysregulation of the antibody-dependent response can lead to a variety of abnormalities and diseases. Antibodies are found in the secreted form in the blood (antibody phenotype) and membrane-bound on B cells as B-cell receptor (antibody genotype). Thus, investigating the relationship between genomic and phenotypic antibody diversity is of decisive importance for understanding antibody-driven immune protection and disease. In this project, we aim to establish a robust high-throughput framework for single-cell (SC) genomic and proteomic characterization of antibody repertoires using high-throughput genomic sequencing and mass spectrometry. The generated data will be analyzed using a variety of bioinformatics tools.