![]() ![]() Single B cells are sorted, the template switching activity of RT adds a unique cell-specific barcode to all cDNAs generated from an individual B cell. b | Cell barcoding via template switching. ![]() cDNAs of the IgH and IgL expressed by individual B cells are then linked by emulsion to RT-PCR, and then pooled and sequenced. Single B cells are isolated and lysed, then their RNA is captured by poly-T beads. Comprehensive characterization of the functional antibody repertoire necessitates high-throughput, full-length and error-free sequencing of IgH and IgL pairs expressed by individual B cells. Abbreviation: CDRs, complementarity determining regions FDC, follicular dendritic cell.Īpproaches for high-throughput sequencing of functional antibody repertoires. Memory B cells circulate in the blood, and enable rapid recall responses upon re-exposure to their target antigen, whereas plasma cells localize primarily in the bone marrow and lamina propria, where they secrete antibodies. , Plasmablasts transiently circulate in the blood and migrate to secondary lymphoid organs and tissues involved in the disease process, including tissues under autoimmune, infectious or malignant attack. B cells that express high-affinity antibodies respond further to growth factors and other signals that induce differentiation into plasmablasts and memory B cells. , After multiple rounds of somatic hypermutation and clonal selection, antibody-expressing B cells produce antibodies with increased affinity for their target antigen. Clonal selection involves competition of B cells for antigen and growth factors in germinal centres, resulting in B cells that express the highest-affinity antibodies being selected to expand and survive. Somatic hypermutation is a cytidine-deaminase-mediated process in which antibody CDRs are mutated ~1–2 times per cell division. , Affinity maturation involves two processes, somatic hypermutation and clonal selection. ![]() B cells undergo clonal expansion and affinity maturation after encountering antigen and T-cell help or co-stimulatory signals, a process that generally occurs in germinal centres within secondary lymphoid organs. We anticipate that antibody repertoire sequencing will provide next-generation biomarkers, diagnostic tools and therapeutic antibodies for a spectrum of diseases, including rheumatic diseases. Sequencing antibody repertoires is transforming our understanding of immune responses to autoimmunity, vaccination, infection and cancer. Integrated analysis of coexpressed functional genes provides the potential to further pinpoint the most important antibodies and clonal families generated during an immune response. ![]() Bioinformatic identification of clonal antibody families and recombinant expression of representative members produces recombinant antibodies that can be used to identify the antigen targets of functional immune responses and to investigate the mechanisms of their protective or pathogenic functions. Although focused sequencing of immunoglobulin heavy chains or complement determining regions can be utilized to monitor particular immune responses and B-cell malignancies, high-fidelity analysis of the full-length paired heavy and light chains expressed by individual B cells is critical for characterizing functional antibody repertoires. Important parameters to consider when sequencing antibody repertoires include the methodology, the B-cell population and clinical characteristics of the individuals analysed, and the bioinformatic analysis. The development of high-throughput DNA sequencing technologies has enabled large-scale characterization of functional antibody repertoires, a new method of understanding protective and pathogenic immune responses. ![]()
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