Ig repertoire of IgG+ murine memory B cells
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE139835 | GEO | 2020/04/24
REPOSITORIES: GEO
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Project description:Single cell sequencing for analysis gene of gene transcription and Ig repertoires of isotype-switched IgG-expressing memory B cells for paired analysis with Ig repertoires in the same cells. Aim of the study was to explore heterogeneity of isotype-switched memory B cells within and between spleen and bone marrow compartments.
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Project description:Recent advancements in microfluidics and high-throughput sequencing technologies have enabled recovery of paired heavy- and light- chain of immunoglobulins (Ig) and VDJ- and VJ- chains of T cell receptors (TCR) from thousands of single cells simultaneously. Due to the complexity of these polyclonal receptors, for many species single-cell immune repertoire sequencing assays are not yet commercially available. Rhesus macaques are one of the most well-studied model organisms of the human adaptive immune response; application of these new immune repertoire sequencing assays is highly relevant to vaccine and infectious disease studies. Here we use custom designed primers to target and enrich for every known Ig and TCR chain and isotype in the rhesus macaque animal model. We sequenced more than 110,000 cell barcodes from rhesus macaque repertoires using PBMC, splenocyte, and FACS-sorted T and B cell. We were able to recover every Ig and TCR isotype, measure clonal expansion in proliferating T cells, and pair repertoires with gene expression profiles of single cells. Our results establish the ability to perform single-cell based immune repertoire analysis in rhesus macaque.
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Project description:Ig repertoire of IgG+ murine memory B cells
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Project description: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).
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Project description:Enhanced secondary Ab responses are a vital component of adaptive immunity, yet little is understood about the intrinsic and extrinsic regulators of naïve and memory B cells that results in differences in their responses to Ag. Microarray analysis, together with surface and intracellular phenotyping, revealed that memory B cells have increased expression of members of the TNF receptor, SLAM, B7 and Bcl2 families, as well as the TLR-related molecule CD180 (RP105). Accordingly, memory B cells exhibited enhanced survival, proliferation and Ig secretion, as well as entered division more rapidly than naïve B cells in response to both T-dependent and T-independent stimuli. Furthermore, both IgM and isotype switched memory B cells, but not naïve B cells, co-stimulated CD4+ T cells in vitro through a mechanism dependent on their constitutive expression of CD80 and CD86. This study demonstrates that upregulation of genes involved in activation, co-stimulation and survival provides memory B cells with a unique ability to produce enhanced immune responses and contributes to the maintenance of the memory B cell pool. Keywords: cell type comparison Four subsets of human splenic B cells (naïve, IgM-memory, Ig isotype switched memory and plasma cells); sort-purified and analysed immediately ex vivo; performed in duplicate.
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Project description:We developed an improved library prep protocol and standardized the data analysis pipeline for accurate repertoire profiling. In addition, two metrics were implemented to assess repertoire clone properties. We then studied systemically the effects of two adjuvants, CpG and Alum, on the Ig heavy chain repertoire using the ovalbumin (OVA) challenged mouse model. Ig repertoires of different tissues (spleen and bone marrow) and isotypes (IgG and IgM) were examined and compared in terms of sequence mutation frequency, IGHV gene usage, CDR3 length, rescaled Hill numbers for clonal diversity, and clone selection strength. As a result, Ig repertoires of different tissues or isotypes exhibited distinguishable profiles at the non-immunized steady state. Adjuvanted immunizations further resulted in statistically significant alterations in Ig repertoire compared with PBS or OVA alone immunized groups. Lastly, we applied unsupervised machine learning techniques – multiple factor analysis and clustering – to identify Ig repertoire signatures in different compartments and under varying immunizations.
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Project description:The immune system produces a vast repertoire of immunoglobulins (Ig) in response to the wide number of different antigens to which we are exposed. Thanks to processes such as V(D)J recombination, somatic hypermutation, and antigen selection, the immune system is able of generating the high diversity of Ig sequences. Igs are present in the body as soluble or membrane-bound proteins attached to the B-cells (similar structure to IgM) for regulating the immune system and performing the function of B-cell receptors (BCR), respectively. Specifically, the disrupted activation of the BCR appears as the responsible of the chronic lymphocytic leukemia (CLL). We have focused this study in the sequencing of cellular Ig from B cells purified from the peripheral blood of CLL patients in order to characterize their peptide profiles.
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