Monoclonal IgG antibodies generated from joint-derived B cells of RA patients have a strong bias toward citrullinated autoantigen recognition

Abstract

Antibodies targeting citrullinated proteins (ACPAs [anticitrullinated protein antibodies]) are commonly found in patients with rheumatoid arthritis (RA), strongly associate with distinct HLA-DR alleles, and predict a more aggressive disease course as compared with seronegative patients. Still, many features of these antibodies, including their site of production and the extent of MHC class II-driven T cell help, remain unclarified. To address these questions, we have used a single B cell-based cloning technology to isolate and express immunoglobulin (Ig) genes from joint-derived B cells of active RA patients. We found ∼25% of synovial IgG-expressing B cells to be specific for citrullinated autoantigens in the investigated ACPA(+) RA patients, whereas such antibodies were not found in ACPA(-) patients. The citrulline-reactive monoclonal antibodies did not react with the unmodified arginine peptides, yet several reacted with more than one citrullinated antigen. A role for active antigen selection of the citrulline-reactive synovial B cells was supported by the strong bias toward amino acid replacement mutations in ACPA(+) antibodies and by their loss of reactivity to citrullinated autoantigens when somatic mutations were reverted to the corresponding germline sequences.

Figure 1.

Similar IgG gene characteristic in synovial B cells from ACPA⁺ and ACPA⁻ RA patients. (a) Summary of VH and JH family gene usage in seropositive (ACPA⁺) and negative (ACPA⁻) patient samples. (b) Overview of IgH (γ) CDR3 amino acid characteristics: length (left) and negatively (middle) and positively (right) charged amino acids in ACPA⁺ and ACPA⁻ patients. (c and d) Light chains data depicting Vκ/Jκ (c) and Vλ/Jλ (d) gene family usage. (e) Distribution of IgG subclasses displayed per patient with the total number of sequences analyzed represented in the middle of the pie charts. (f) IgG subclass distribution in ACPA⁺ and ACPA⁻ patient samples. Differences between individual fractions were not statistically significant (P > 0.5), as determined by Fisher’s exact test.

Figure 2.

Citrulline-reactive antibodies could be generated from ACPA⁺ but not from ACPA⁻ RA patients. (a) Pie charts summarizing the frequency of the citrulline-reactive clones (to any of the tested citrulline peptides) in individual patient samples. The top panel represents the three ACPA⁺ patient samples and the three in the bottom panel, the ACPA⁻ samples. The absolute number of tested antibodies is indicated in the center of each pie chart. (b) Frequency comparison of antigen reactivity in the clones generated from the different ACPA⁺ and ACPA⁻ patients (one symbol summarizes all generated antibodies from each individual; range 15–37 clones per patient sample; in total 93 antibodies were tested from ACPA⁺ and 67 from ACPA⁻ patients). CCP denotes general citrulline reactivity; polyreactivity is based on positivity of two or more of the following: insulin, LPS, and double-stranded DNA; and lastly tetanus toxoid was used as a proxy for recall response. Horizontal lines represent the mean values of reactivity for all RA patients. (c) ELISA graphs of the various ACPA fine specificities: CEP-1 (citrullinated α-enolase, amino acids 5–21), cit-fib (citrullinated fibrinogen, amino acids 36–52), and cit-vim (citrullinated vimentin, amino acids 60–75). Black lines represent individual IgG⁺ memory B cells antibodies. (c) Green lines represent the high positive control (serum pool of ACPA⁺ antibodies). (c and e) Red lines represent the negative control antibody (serum pool of ACPA⁻ antibodies). Each vertical row represents one ACPA⁺ patient sample. (d) ELISA graphs of the corresponding arginine versions of the peptides. (c–e) Dashed horizontal lines show a cutoff of OD450 for positive reactivity. (e) Sequences from four selected antibodies with high reactivity to two or more of the citrullinated peptides that were subsequently reverted into their predicted germline sequence (in both the FWRs and CDRs) and then expressed as recombinant antibodies. ELISA graphs illustrate the citrulline reactivity of the original antibodies (i.e., mutated; left) compared with those encoded by the predicted germline sequences (right). Data are representative of three to six independent experiments.

Figure 3.

Immunohistochemical localization of citrullinated proteins in synovial tissues of RA patients. (a and b) Immunohistochemistry using two of the recombinant citrulline-specific antibodies, 1276SF-D10 (a) and 1325SF-B109 (b), brown staining of both the lining (black arrows) and sublining (white arrows) layers in an inflamed synovial biopsy, obtained at the time of joint arthroplasty from a RA patient. (c) Staining of a matched irrelevant IgG2a-negative control used at similar concentration. Insets show the same samples at higher magnification. Similar results were observed in two other RA synovial tissues (not depicted). Data presented in this figure are representative of three independent experiments from four patients. Bars, 68 µm.

Figure 4.

Citrulline-reactive antibodies display a bias toward nonsynonymous mutations in the CDRs. (a) Comparison of the absolute numbers of somatic mutations in individual VH, Vκ, and Vλ genes of the antibodies generated from ACPA⁺ and ACPA⁻ patients. (b) Frequencies of replacement (R) and silent (S) mutations in the CDRs and FWRs of VH, Vκ, and Vλ genes of synovial IgG⁺ memory B cells from ACPA⁺ and ACPA⁻ RA patients. (c) Comparison of the absolute numbers of somatic mutations between citrulline-positive versus -negative antibodies generated from ACPA⁺ patients. (d) R/S mutation ratios in the CDRs and FWRs of the VH, Vκ, and Vλ genes in the citrulline-positive and -negative clones generated from ACPA⁺ patients. (e) Absolute numbers of somatic mutations in individual VH and IgL genes of the antibodies generated from the individual patient samples. (a, c, and e) Horizontal bars indicate mean. (f and g) Frequencies of R and S mutations in the CDRs and FWRs of VH and VL of synovial IgG⁺ memory B cell from the individual ACPA⁺ (f) and ACPA⁻ (g) RA patients. The R/S ratios are indicated below the respective graphs.