Antibody responses to de novo identified citrullinated fibrinogen peptides in rheumatoid arthritis and visualization of the corresponding B cells

Serum samples were collected from 347 patients diagnosed as having established RA according to the ACR criteria [25]. All patients attended the Rheumatology unit at the Karolinska University Hospital, Stockholm, Sweden, where the serum samples were collected and stored at -80 °C until further use. All the samples were previously assayed for anti-CCP2 antibodies and antibodies against full cit-Fib (cit-Fib protein) [10]. Additionally, serum samples from 152 healthy subjects and 236 patients with psoriatic arthritis (PSA) or ankylosing spondylitis (AS) were included as healthy and disease controls, respectively (Table 1). The ethical review board of Karolinska University hospital approved this study and all the patients involved gave informed consent.

A total of 326 of the 347 patients with RA were previously genotyped for the HLA-DR SE allele [6] and 322 of the 347 patients with RA were genotyped for the PTPN22 R620W risk allele [26]. HLA-DRB1*0101, *0102, *0401, *0404, *0405 or *1001 alleles were classified as HLA-shared epitope (HLA-SE) alleles [27].

Biotinylated Fib peptides (Table 2) were synthesized by a solid-phase procedure using fluorenylmethoxycarbonyl (Fmoc) chemistry as described previously [28]. The peptides were at least 90% pure as deduced from their elution pattern on reversed-phase high performance liquid chromatography (HPLC). Streptavidin-coated high binding capacity 96-well ELISA plates (Thermo Scientific) were coated with the peptides in their native and citrullinated forms at a concentration of 2.5 μg/ml in coating buffer (0.05% Tween-20, 0.1% bovine serum albumin (BSA), Tris-buffered saline). The plates were washed with PBS containing 0.05% Tween-20 after every incubation step. For detection of the antibodies against citrullinated peptides, the serum samples were diluted 1:100 in radioimmunoassay (RIA) buffer (1% BSA, 350 mM NaCl, 10 mM Tris HCl, pH 7.6, 1% (v/v) Triton X-100, 0.5% (weight/volume) sodium deoxycholate, 0.1% sodium dodecyl sulfate). The bound antibodies were detected with horseradish peroxidase-conjugated goat anti-human IgG F(ab’)₂ (Jackson Immuno Research). Bound antibodies were visualized using the chromogenic substrate 3,3′,5,5′-tetramethylbenzidine (TMB, Sigma-Aldrich). The optical density (OD) was then measured at 450 nm with reference at 650 nm subtracted. A standard curve was included in each plate to convert the OD values into arbitrary units.

The cutoff value for each of the citrullinated antigens was set to the 98^th percentile of values from healthy subjects (n = 152). In some cases, samples displayed high reactivity to both a citrullinated peptide and to its native arginine-containing counterpart, thereby distorting the analysis. Therefore, a ratio between the OD values obtained with the peptides containing arginine and citrulline was determined for each sample and samples with a ratio greater than 0.8 were considered negative.

The B cell antigen tetramer consists of an R-phycoerythrin (PE)-labeled streptavidin (SA) core and four identical biotinylated peptides. Tetramers were prepared as previously described [29]. Briefly, the biotinylated cit-Fib peptides used in the ELISA were incubated with SA-PE (Prozyme) at a molar ratio of 10:1. The tetramer fraction was then purified using a 100-kD molecular weight cutoff Amicon Ultra filter (Millipore). The molarity of the tetramer was calculated with the supplier-determined ratio of SA to PE, after measuring the concentration of PE by Nanodrop (Thermo Fischer). The decoy tetramer was prepared, as described above, by incubating the biotinylated native (non-citrullinated) Fib peptides with SA-PE pre-conjugated to Alexa Flour 647 (Molecular Probes Invitrogen). The four α-chain derived Fib peptides were assembled as tetramers separately and then pooled at the time of sample staining. The decoy tetramers were assembled and used in the same manner with the corresponding native Fib peptides.

Tetramer-positive B cells were analyzed in two small cohorts of patients with RA, selected/recruited based on their PTPN22 risk allele status (CC - non risk vs CT - risk). The pilot cohort consisted of cryopreserved peripheral blood mononuclear cells (PBMC) (n = 5 individuals, all female, median age 49 (range 40–75), all HLA-DR SE-negative) and the validation cohort consisted of fresh PBMC (n = 10 individuals, 7 female/3 male, median age 58 (range 30–69), all HLA-DR SE-positive). PBMC isolated from patients were stained with the decoy and cit-Fib tetramers in buffer containing Fc blocking solution (FcR Blocker Miltenyi Biotec), then passed over a magnetized LS column (Miltenyi Biotec) to enrich for tetramer-binding cells. Both the bound and flow-through fractions were stained with APC-H7-labeled anti-CD3 (SK7), APC-H7-labeled anti-CD14 (MφP9), APC-H7-labeled anti-CD16 (3G8), BV421-labeled anti-CD19 (HIB19), V500-C-labeled anti-CD20 (L27), PE-Cy7-labeled anti-CD27 (M-T271) and FITC-labeled anti-IgD (IA6-2) (all antibodies from BD Biosciences). All the incubation and wash steps were performed using MACS buffer containing 1 mM EDTA and 0.5% BSA in PBS.

Flow cytometry was performed using a 4-laser (405 nm, 488 nm, 561 nm and 640 nm) LSR Fortessa (BD Biosciences) and analyzed with FlowJo software (Tree Star). Fluorescent AccuCheck counting beads (Invitrogen) were used to calculate total numbers of live lymphocytes in the column-bound and flow-through suspensions. The gating strategy for tetramer staining was based on a forward scatter (FSC)/side scatter (SSC) lymphocyte gate and removal of doublets followed by dumping CD3, CD14 and CD16 as depicted in Fig. 3a, before focusing on the B cell subset.

Using mass spectrometry analysis [24], fibrinogen peptides containing the citrulline sites α-35, α-263,271 and α-425,426 had a spectral count 2.5 times higher than controls and were present in the synovial fluid of more patients with RA than controls. All the peptides had a Mascot score greater than 40 and have also been identified by in-vitro citrullination of fibrinogen using human and rabbit PAD enzymes (summarized in [16]). To assess the detailed anti cit-Fib B cell responses, we used ELISA to test for the presence of antibodies against the four different cit-fibrinogen peptides [16, 24]. A cohort of healthy subjects was used to determine the cutoff at the 98^th percentile for the ELISA and based upon this cutoff, the cit-Fib reactivity in serum from 347 patients with RA was analyzed. We found relatively weak, though frequently present, reactivity in the RA cohort, with 20.2% towards the cit-Fib α-35, 12.5% towards cit-Fib α-216,218, 21.0% towards cit-Fib α-263,271 and 17.0% towards cit-Fib α-425,426 (Fig. 1).

To understand if the presence of antibodies against these cit-Fib targets were specific for RA, we then analyzed serum from a cohort of patients with non-RA arthritis. For this purpose, we analyzed serum from a cohort of 236 patients with PSA and AS: we only identified a few reactive serum samples, mostly with low levels of the different antibodies. Reactivity against the cit- Fib α-35, cit- Fib α-216,218, cit- Fib α-263,271 and cit- Fib α-425,426 in this cohort was found to be 1.0% (n = 2), 0.5% (n = 1), 0.5% (n = 1) and 1.0% (n = 2), respectively (Fig. 1).

With regard to overlap between the different epitope reactivity, i.e. whether the same patients presented with more than one anti-cit-Fib antibody, and to the distribution compared to anti-CCP2, we observed that most patients were reactive to a single cit-Fib peptide, although some overlap was seen (Fig. 2). Still, the cit-Fib response was predominantly in the anti-CCP2-positive patient subset (p < 0.0001).

It has been previously shown that the presence of ACPA is associated with the HLA-DR SE alleles and with the PTPN22 R620W risk allele [30]. We examined the association between these two genotypes and antibodies against the four cit-Fib peptides, antibodies against whole cit-Fib protein and anti-CCP2 antibodies. The association was significant (p ≤ 0.05) for antibodies against the four cit-Fib peptides and anti-CCP2 antibodies (Table 3). Also a significant association was observed between the presence of antibodies against all cit-Fib peptides and those against whole cit-Fib protein. No association was observed between the presence of antibodies against any of the cit-Fib peptides and HLA-DR SE. In contrast, an association was observed between the PTPN22 risk allele and seropositivity for cit-Fib α-35 and cit-fibrinogen α-263,271 (Table 3). PTPN22 risk allele carriers had a significant odds ratio ( (OR) 95% CI) for the presence of antibodies against cit-Fib α-35, OR = 1.8 (1.0–3.1) and cit-Fib α-263,271, OR = 2.0 (1.1–3.4) (Table 4).

Based on data suggesting a role for PTPN22 in the negative selection of autoreactive B cells [31], we hypothesized that PTPN22 risk allele carriers in our cohort may have an expanded population of cit-Fib reactive B cells relative to non-risk allele carriers. To test this, we constructed B cell antigen tetramers for quantification and comparison of tetramer-positive B cells in PTPN22 risk allele non-carrier (CC) and carrier (CT, TT) patients with RA. Only CD19⁺ CD20⁺ B cells were included in the tetramer analyses (Fig. 3a). Analysis of frozen PBMC from five patients with RA (three CC and two CT) showed a trend in increased frequency of tetramer-positive B cells in the patients carrying the PTPN22 risk allele (Fig. 3b). To validate this finding, we recruited ten additional patients with RA (five CC and five CT PTPN22 allele carriers) and found a similar trend for tetramer-positive B cells with individuals carrying the PTPN22 risk allele (Fig. 3c).