The promise and challenge of high-throughput sequencing of the antibody repertoire

Abstract

Efforts to determine the antibody repertoire encoded by B cells in the blood or lymphoid organs using high-throughput DNA sequencing technologies have been advancing at an extremely rapid pace and are transforming our understanding of humoral immune responses. Information gained from high-throughput DNA sequencing of immunoglobulin genes (Ig-seq) can be applied to detect B-cell malignancies with high sensitivity, to discover antibodies specific for antigens of interest, to guide vaccine development and to understand autoimmunity. Rapid progress in the development of experimental protocols and informatics analysis tools is helping to reduce sequencing artifacts, to achieve more precise quantification of clonal diversity and to extract the most pertinent biological information. That said, broader application of Ig-seq, especially in clinical settings, will require the development of a standardized experimental design framework that will enable the sharing and meta-analysis of sequencing data generated by different laboratories.

Figure 1. Antibody structure and sequence diversification mechanisms.

(a) Schematic of IgG structure. In the top chains, domains encoded from germline V, D, J and C segments are indicated. Nontemplated N-nucleotides are shown in red. These top chains delineate the 5′ to 3′ genetic composition of the antibody. In the bottom chains, framework (FR) and complementarity-determining regions (CDRs) are indicated. These bottom chains delineate the N-terminal to C-terminal protein sequence. Dashed lines denote disulfide bonds. (b) Key steps in antibody diversification. The primary antibody heavy chain repertoire is created predominantly by the somatic recombination of variable (V), diversity (D) and joining (J) gene segments, and by the random nontemplated addition of N-nucleotides. The antigen-binding site of a heavy chain is formed by the juxtaposition of the hypervariable complementarity-determining regions (CDR-H1, H2 and H3) and the framework 3 region (FR3). After productive IgH rearrangement, recombination of the light chain (IgL) ensues, and the heterodimeric pairing of H and L chains forms the complete antibody of the IgM isotype that is expressed on the surface of a newly formed immature B cell. Eμ: IgM intronic enhancer; Sμ: tandem repeats critical for class-switch recombination. Numbers in parentheses refer to estimates of human germline V_H D_H and J_H segments.

Figure 2. Key steps in the development of antigen-specific B cells.

The steps of normal B-cell differentiation and diversification of the antibody repertoire are indicated in black text. Normal B cells are generated in the bone marrow, migrate to the periphery and, following developmental checkpoint selection, comprise the population of IgM⁺IgD⁺ mature naive B cells. When these cells are activated by cognate antigen in the presence of T-cell help, they enter a germinal center (GC) reaction where they rapidly proliferate; this results in clonal expansion and subsequent somatic hypermutation catalyzed by activation-induced cytidine deaminase. B cells bearing antibodies with high affinity for cognate antigen and that survive the GC reaction can undergo class-switch recombination to IgG, IgA or IgE isotypes and ultimately differentiate into memory B cells, antibody-secreting plasmablasts or plasma cells. After subsequent encounter with the same cognate antigen, memory B cells can proliferate or differentiate directly into antibody-secreting cells. Steps that proceed abnormally, leading to the development of human B-cell leukemias and lymphomas, are indicated in red text. ALL, acute lymphoblastic leukemia; CLL, chronic lymphocytic leukemia; MCL, mantle cell lymphoma; GC-DLBCL, germinal center diffuse large B cell lymphoma; FL, follicular lymphoma; ABC-DLBCL, activated B cell–like DLBCL; MGUS, monoclonal gammopathy of undetermined significance; MM, multiple myeloma. B-cell malignancies that have not been analyzed extensively by high-throughput sequencing are shown in parenthesis.

Figure 3. Methods for high-throughput sequencing of the Ig sequence repertoire.

(a) Schematic of steps involved in high-throughput sequencing of Ig genes from bulk B-cell populations of B-cell subsets sorted according to expression of indicated cell-surface markers. Either genomic DNA (gDNA) or mRNA can be used as template, and the choice of template influences the number and location of primers used for subsequent PCR amplification. gDNA amplification is performed using primers complementary to the rearranged V-region gene (VDJ recombinant); amplification of cDNA is performed either using a 5′ primer pool complementary to the leader peptides or FR1s of V-gene segments, and a single 3′ C_H1 (or Cκ,Cλ if amplifying light chain genes) primer, or alternatively by 5′ RACE. Although throughput is high, in bulk analysis information regarding which V_H and V_L chains were paired in the same cell is lost, as cells are lysed in bulk and V_H and V_L genes are amplified in separate reactions. (b) Schematic of single-cell immunoglobulin repertoire sequencing methods, which preserve information about endogenous V_H:V_L pairs. Left panel: B-cell lysis and mRNA capture in picoliter well arrays. Middle panel: single-cell PCR following limited B-cell dilution and amplification using barcoded primers. Right panel: microfluidic barcoding of V_H and V_L cDNAs. Ig, immunoglobulin.

Figure 4. Deconvoluting the serum antibody repertoire.

B cells from peripheral blood or other tissues are sorted and subjected to high-throughput immunoglobulin V-gene sequencing, resulting in generation of a personal antibody sequence database. Antigen-specific antibodies from serum are then isolated by affinity chromatography, digested into peptides, and subjected to LC-MS/MS analysis. The MS/MS data are interpreted using the antibody sequence database, thereby allowing identification of CDRH3-derived peptides and the genes encoding the repertoire of antigen-specific antibodies in the serum. Ig, immunoglobulin.