Sequencing the functional antibody repertoire--diagnostic and therapeutic discovery

Abstract

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. 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. 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. 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. Integrated analysis of coexpressed functional genes provides the potential to further pinpoint the most important antibodies and clonal families generated during an immune response. Sequencing antibody repertoires is transforming our understanding of immune responses to autoimmunity, vaccination, infection and cancer. We anticipate that antibody repertoire sequencing will provide next-generation biomarkers, diagnostic tools and therapeutic antibodies for a spectrum of diseases, including rheumatic diseases.

Figure 1

The B-cell response. 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.^, Affinity maturation involves two processes, somatic hypermutation and clonal selection. Somatic hypermutation is a cytidine-deaminase-mediated process in which antibody CDRs are mutated ~1–2 times per cell division. 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.^, After multiple rounds of somatic hypermutation and clonal selection, antibody-expressing B cells produce antibodies with increased affinity for their target antigen. B cells that express high-affinity antibodies respond further to growth factors and other signals that induce differentiation into plasmablasts and memory B cells.^,, 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. 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. Abbreviation: CDRs, complementarity determining regions; FDC, follicular dendritic cell.

Figure 2

Approaches for high-throughput sequencing of functional antibody repertoires. 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. a | Cell barcoding via linkage PCR. Single B cells are isolated and lysed, then their RNA is captured by poly-T beads. cDNAs of the IgH and IgL expressed by individual B cells are then linked by emulsion to RT-PCR, and then pooled and sequenced. b | Cell barcoding via template switching. 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. Plate-specific barcodes are then added, resulting in cDNAs that have compound cell barcodes. Finally, the compound cell-barcoded IgH and IgL genes are amplified by PCR, pooled and sequenced.^,, c | Cell barcoding by forward and reverse primer matrix. Single B cells are sorted, then V-gene forward primers and C-region reverse primers are used to add cell-specific barcodes to, and amplify by PCR, IgH and IgL cDNA generated from an individual B cell. Single-barcoded immunoglobulin genes are then pooled and sequenced. d | Microfluidic combination of beads with unique barcodes and single B cells into individual droplets. Using microfluidics, single B cells and beads with unique barcodes are combined in individual droplets, followed by lysis of the B cell, PCR and sequencing.^, Abbreviations: C, constant; IgH, immunoglobulin heavy chain; IgL, immunoglobulin light chain; PCR, polymerase chain reaction; RT, reverse transcriptase; V, variable.

Figure 3

Cell barcodes enable robust antibody repertoire sequencing. Cell barcodes (represented by AAA, CCC and GGG) added with the template switching activity of RT^,, have many advantages, including preservation of the IgH and IgL pairs expressed by individual B cells, enabling accurate determination of the error-corrected sequence and of clonal proportions. a | In the approach presented, all cDNA generated from each cell receives the same unique barcode; therefore, coexpressed functional genes indicative of functional subsets of B cells (or T cells) can also be characterized.^,, Both cell and molecular barcodes enable quantitative analysis of mRNA in individual cells. b | Multiple copies of each mRNA are expressed by each cell, and for each cell all mRNA receives the same cell barcode; therefore, cell barcoding enables correction of both PCR and high-throughput sequencing errors. c | Cell barcodes enable accurate determination of clonal proportions. Cell barcodes enable binning of the IgH and IgL chain reads from each B cell, thereby providing accurate determination of clonal proportions. By contrast, molecular barcodes do not enable RT-PCR error correction, IgH and IgL pairing, analysis of coexpressed genes or accurate determination of clonal proportions.

Figure 4

Phylogenetic tree of the antibody repertoire. Example of a phylogenetic tree of a human plasmablast response generated using cell barcoding. Phylogenetic trees reveal clonal families and guide the expression and screening of the identified antibodies. Each major branch represents a single IgH V(D)J, outer branching is determined by the IgL VJ sequence, and each terminal leaf represents the error-corrected, full-length, paired IgH and IgL sequences expressed by an individual B cell. Clonal families (colour coded) contain antibodies that share IgH VJ and IgL VJ sequences. Immunodominant (red) and rare (other colours) families are identified. Single nucleotide substitutions can substantially alter antibody binding specificity and affinity, and in order to evaluate the spectrum of antibodies encoded within each clonal family recombinant antibodies representing the range of variants represented within each family should be expressed and characterized. Abbreviations: D, diversity; IgH, immunoglobulin heavy chain; IgL, immunoglobulin light chain; J, joining; RT-PCR, reverse transcriptase polymerase chain reaction, V, variable.