Barcode-enabled sequencing of plasmablast antibody repertoires in rheumatoid arthritis

Abstract

Objective: A hallmark of rheumatoid arthritis (RA) is the production of autoantibodies, including anti-citrullinated protein antibodies (ACPAs). Nevertheless, the specific targets of these autoantibodies remain incompletely defined. During an immune response, B cells specific for the inciting antigen(s) are activated and differentiate into plasmablasts, which are released into the blood. We undertook this study to sequence the plasmablast antibody repertoire to define the targets of the active immune response in RA.

Methods: We developed a novel DNA barcoding method to sequence the cognate heavy- and light-chain pairs of antibodies expressed by individual blood plasmablasts in RA. The method uses a universal 5' adapter that enables full-length sequencing of the antibodies' variable regions and recombinant expression of the paired antibody chains. The sequence data sets were bioinformatically analyzed to generate phylogenetic trees that identify clonal families of antibodies sharing heavy- and light-chain VJ sequences. Representative antibodies were expressed, and their binding properties were characterized using anti-cyclic citrullinated peptide 2 (anti-CCP-2) enzyme-linked immunosorbent assay (ELISA) and antigen microarrays.

Results: We used our sequencing method to generate phylogenetic trees representing the antibody repertoires of peripheral blood plasmablasts from 4 individuals with anti-CCP+ RA, and recombinantly expressed 14 antibodies that were either "singleton" antibodies or representative of clonal antibody families. Anti-CCP-2 ELISA identified 4 ACPAs, and antigen microarray analysis identified ACPAs that differentially targeted epitopes on α-enolase, citrullinated fibrinogen, and citrullinated histone H2B.

Conclusion: Our data provide evidence that autoantibodies targeting α-enolase, citrullinated fibrinogen, and citrullinated histone H2B are produced by the ongoing activated B cell response in, and thus may contribute to the pathogenesis of, RA.

Figure 1. Schematic of a DNA barcode-enabled method for high-throughput sequencing and recombinant production of endogenous antibodies

(A to C) Overview of the method. (A) Plasmablasts are single-cell sorted into 96-well plates, and cDNA is generated by reverse transcription, during which a unique well-ID barcode is added to all the cDNA in an individual well. (B) All the barcoded cDNA from one plate is pooled and tagged with a unique plate-ID. (C) cDNAs, which are now double-barcoded with well-IDs and plate-IDs, are pooled and sequenced using next-generation 454 sequencing. (D to E) Specifics for barcoding and library preparation. (D) During reverse transcription, an adaptor containing a unique well-ID barcode is incorporated at the 3’ end of the first-strand cDNA by using the 3’-tailing activity of a thermally stable RNaseH⁻ reverse transcriptase. (E) Plate-IDs and 454 Titanium Primer A are added during PCR amplification by using a barcoded adaptor. A further round of nested PCR is performed, adding 3' plate-IDs and 454 Titanium primer B. (F) For cloning and expression of an antibody, either PCR or gene synthesis is used. For PCR, a 5’ primer specific to a particular well-ID is used to amplify only the desired pair of heavy- and light-chain cDNA from the pooled cDNA of the appropriate plate. Standard molecular biology is used to insert the antibody V(D)J or VJ sequences in-frame into an expression vector containing the appropriate antibody constant region.

Figure 2. ACPA production by RA plasmablasts

(A) Fluorescence-activated gating strategy for single-cell sorting IgG-secreting plasmablasts from peripheral blood mononuclear cells of individuals with RA. Plasmablasts were first gated on live, single PBMCs and then gated on surface staining for IgG⁺ plasmablasts, as shown. SSC, side scatter (B) Antigen array screening of the binding to citrullinated peptides of antibodies produced by bulk cultured plasmablasts from patients with psoriatic arthritis (PsA), with anti-CCP⁺, or with anti-CCP⁻ RA. Antibody-binding values were normalized as described in the methods; blue represents no reactivity, yellow moderate reactivity, and orange-red high reactivity. Cit, citrullinated; Cfc, citrullinated filaggrin peptide cyclic; Fib, fibrinogen; H2A, histone 2A; HSP 60, heat shock protein 60.

Figure 3. Sequencing the plasmablast antibody repertoires in individuals with RA

DNA barcoding and next-generation sequencing were used to analyze the plasmablast antibody repertoire of 4 individuals with active, anti-CCP⁺ RA. A phylogenetic tree of the plasmablast antibody repertoire was generated by concatenating and clustering the heavy- and light-chain sequences of the plasmablast antibodies and arranging them by heavy-chain V-gene family usage. Each peripheral node represents a single antibody. Clonal families of antibodies are indicated by bolded, red lines. The letters RA followed by a number denote an antibody that was selected for cloning and expression, and red font denotes antibodies that exhibited reactivity in the CCP2 assay (Figure 4A) or on RA antigen microarrays (Figure 4B).

Figure 4. Identification of recombinant antibodies that bind to citrullinated peptides or proteins

(A) CCP2 ELISA analysis of recombinant antibody binding to CCP. Three influenza-specific antibodies (Flu1, Flu2 and Flu3) were used as isotype controls. The red line indicates three SEM above the reactivity of the three negative control influenza (Flu)-binding antibodies tested. (B) Antigen microarray analysis of recombinant antibody binding to candidate citrullinated and native synovial peptides and proteins. Reactivity of the recombinant antibodies to Cfc-1, a citrullinated filaggrin peptide used in early CCP assays, is also displayed.