Single cell RNA-sequencing of PBMC derived B cells from human healthy donor
ABSTRACT: 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:The B-cell receptor (BCR) enables individual B cells to identify diverse antigens, including bacterial and viral proteins. While advances in RNA-seq have enabled high throughput profiling of transcript expression in single cells, the unique task of assembling the full-length heavy and light chain sequences from single cell RNA-sequencing (scRNA-seq) in B cells has been largely unstudied. We developed a new software tool, BASIC, which allows investigators to use scRNA-seq for assembling BCR sequences at single cell level. To demonstrate the utility of our software, we subjected single B cells from a human donor to scRNA-seq, assembled the full-length heavy and the light chains, and experimentally confirmed these results by using single cell primer based nested PCRs and Sanger sequencing.
Project description:To date nothing is known about the specificity of regulatory B cells. Here we analyzed the B cell repertoire at the Ig gene level of regulatory transitional CD24(high)CD38(high) B cells of patients with multiple sclerosis (MS) in comparison with healthy donors. 5 patients with highly active MS, 4 patients with benign MS and 6 healthy donors were examined. Mononuclear cells from peripheral blood were stained against CD19, CD24 and CD38 surface markers. Subpopulation CD19(+)CD24(high)CD38(high) and total pool of CD19(+) cells were sorted individually for subsequent RNA isolation and BCR sequences comparison. Variable heavy (VH) and light (VL) chains genes of each patient were amplified from cDNA and deep sequencing of the VH and VL genes was carried out.
Project description:Vaccination approaches have generally focused on the antigen rather than the resultant antibodies generated, which differ greatly in quality and function between individuals. The ability to replace the variable regions of the native B cell receptor (BCR) heavy and light chain loci with defined recombined sequences of a preferred monoclonal antibody could enable curative adoptive cell transfer. We report CRISPR-mediated homologous recombination (HR) into the BCR of primary human B cells. Ribonucleoprotein delivery enabled editing at the model CXCR4 locus, as demonstrated by T7E1 assay, flow cytometry, and TIDE analysis. Insertion via HR was confirmed by sequencing, cross-boundary PCR, and restriction digest. Optimized conditions were used to achieve HR at the BCR variable heavy and light chains. Insertion was confirmed at the DNA level, and transgene expression from the native BCR promoters was observed. Reprogramming the specificity of antibodies in the genomes of B cells could have clinical importance.
Project description:The B-cell receptor enables individual B cells to identify diverse antigens, including bacterial and viral proteins. While advances in RNA-sequencing (RNA-seq) have enabled high throughput profiling of transcript expression in single cells, the unique task of assembling the full-length heavy and light chain sequences from single cell RNA-seq (scRNA-seq) in B cells has been largely unstudied.We developed a new software tool, BASIC, which allows investigators to use scRNA-seq for assembling BCR sequences at single-cell resolution. To demonstrate the utility of our software, we subjected nearly 200 single human B cells to scRNA-seq, assembled the full-length heavy and the light chains, and experimentally confirmed these results by using single-cell primer-based nested PCRs and Sanger sequencing.http://ttic.uchicago.edu/?aakhan/BASICaakhan@ttic.eduSupplementary data are available at Bioinformatics online.
Project description:A unique type of combinatorial protein libraries has been constructed. These libraries are based on the pre-B cell receptor (pre-BCR). The pre-BCR is a protein that is produced during normal development of the antibody repertoire. Unlike that of canonical antibodies, the pre-BCR subunit is a trimer that is composed of an antibody heavy chain paired with two surrogate light chain (SLC) components. Combinatorial libraries based on these pre-BCR proteins in which diverse heavy chains are paired with a fixed SLC were expressed in mammalian, Escherichia coli, and phagemid systems. These libraries contain members that have nanomolar affinity for antigen. We term this type of antigen-binding protein a "surrobody" to distinguish it from the canonical antibody molecule.
Project description:VRC01-class broadly neutralizing HIV-1 antibodies protect animals from experimental infection and could contribute to an effective vaccine response. Their predicted germline forms (gl) bind Env inefficiently, which may explain why they are not elicited by HIV-1 Env-immunization. Here we show that an optimized Env immunogen can engage multiple glVRC01-class antibodies. Furthermore, this immunogen activates naive B cells expressing the human germline heavy chain of 3BNC60, paired with endogenous mouse light chains in vivo. To address whether it activates B cells expressing the fully humanized gl3BNC60 B-cell receptor (BCR), we immunized mice carrying both the heavy and light chains of gl3BNC60. B cells expressing this BCR display an autoreactive phenotype and fail to respond efficiently to soluble forms of the optimized immunogen, unless it is highly multimerized. Thus, specifically designed Env immunogens can activate naive B cells expressing human BCRs corresponding to precursors of broadly neutralizing HIV-1 antibodies even when the B cells display an autoreactive phenotype.
Project description:The prognosis of chronic lymphocytic leukemia (CLL) depends on different markers, including cytogenetic aberrations, oncogenic mutations, and mutational status of the immunoglobulin (Ig) heavy-chain variable (IGHV) gene. The number of IGHV mutations distinguishes mutated (M) CLL with a markedly superior prognosis from unmutated (UM) CLL cases. In addition, B cell antigen receptor (BCR) stereotypes as defined by IGHV usage and complementarity-determining regions (CDRs) classify ?30% of CLL cases into prognostically important subsets. Subset 2 expresses a BCR with the combination of IGHV3-21-derived heavy chains (HCs) with IGLV3-21-derived light chains (LCs), and is associated with an unfavorable prognosis. Importantly, the subset 2 LC carries a single-point mutation, termed R110, at the junction between the variable and constant LC regions. By analyzing 4 independent clinical cohorts through BCR sequencing and by immunophenotyping with antibodies specifically recognizing wild-type IGLV3-21 and R110-mutated IGLV3-21 (IGLV3-21R110), we show that IGLV3-21R110-expressing CLL represents a distinct subset with poor prognosis independent of IGHV mutations. Compared with other alleles, only IGLV3-21*01 facilitates effective homotypic BCR-BCR interaction that results in autonomous, oncogenic BCR signaling after acquiring R110 as a single-point mutation. Presumably, this mutation acts as a standalone driver that transforms IGLV3-21*01-expressing B cells to develop CLL. Thus, we propose to expand the conventional definition of CLL subset 2 to subset 2L by including all IGLV3-21R110-expressing CLL cases regardless of IGHV mutational status. Moreover, the generation of monoclonal antibodies recognizing IGLV3-21 or mutated IGLV3-21R110 facilitates the recognition of B cells carrying this mutation in CLL patients or healthy donors.
Project description:Expression of stereotyped B cell receptors (BCR), i.e. non-random combinations of immunoglobulin heavy-chain variable (IGHV) genes, complementarity-determining region-3 (HCDR3), and IGV light chains, identifies discrete clusters and represents a peculiar feature of chronic lymphocytic leukemia (CLL). Expression of IGHV3-23 characterized a CLL subset with peculiar molecular and clinical features. Overall design: Two-condition experiment, Mutated IGVH3-23 vs. Mutated non-IGVH3-23. Biological replicates: 22 control replicates, 5 Mutated IGVH3-23 replicates. Refrence design
Project description:B cell clonal expansion is vital for adaptive immunity. High-throughput BCR sequencing enables investigating this process but requires computational inference to identify clonal relationships. This inference usually relies on only the BCR H chain, as most current protocols do not preserve H:L chain pairing. The extent to which paired L chains aids inference is unknown. Using human single-cell paired BCR datasets, we assessed the ability of H chain-based clonal clustering to identify clones. Of the expanded clones identified, <20% grouped cells expressing inconsistent L chains. H chains from these misclustered clones contained more distant junction sequences and shared fewer V segment mutations than the accurate clones. This suggests that additional H chain information could be leveraged to refine clonal relationships. Conversely, L chains were insufficient to refine H chain-based clonal clusters. Overall, the BCR H chain alone is sufficient to identify clonal relationships with confidence.
Project description:Antibody diversity is generated by a random gene recombination process with the inherent risk of the production of autoreactive specificities. The current view suggests that B cells expressing such specificities are negatively selected at an early developmental stage. Using the knock-in model system of the 3-83 autoreactive B-cell antigen receptor (BCR) in combination with precursor-BCR (pre-BCR) deficiency, we show here that the 3-83 BCR mediates efficient generation of B cells in the presence, but not the absence, of a strongly recognized auto-antigen. Experiments with mixed bone marrow chimeras showed that combining the 3-83 BCR with the corresponding auto-antigen resulted in efficient reconstitution of B-cell development in immune-deficient mice. These results suggest that B cells are positively selected by recognition of self-antigens during developmental stages that precede receptor editing. Moreover, the data indicate that the pre-BCR functions as a specialized autoreactive BCR to initiate positive selection at a stage where the cells express immunoglobulin heavy but not light chains.