The OPN-305 monoclonal antibody is a fully humanized antibody derived from the OPN-301 monoclonal antibody, a murine IgG1 anti-TLR2 antibody (mouse Toll-like receptor 2 (TLR2) antibody, clone T2.5; http://www.faqs.org/patents/app/20120164159) (22). Murine antihuman CD209 (DDX0208, clone 120C11) and antihuman DCIR (clone 8H8 103.3) antibodies were developed by Dendritics (Lyon, France). The anti-TLR2 scFv was combined with either the anti-DC-SIGN or anti-DCIR scFvs to form the tandem scFvs. The tandem scFvs were designed as follows: VL_[TLR2]-(G₄S)₃-VH_[TLR2]-(G₄S)_x -VH_{[DC-SIGN or DCIR]}-(G₄S)₃-VL_{[DC-SIGN or DCIR]}-G₃AS-HHHHHH. Bic03 corresponds to anti-TLR2x anti-CD209 with a long linker (x = 3). Bic05 corresponds to the anti-TLR2x anti-DCIR with a short linker (x = 1). The VL sequence of anti-TLR2 was mutated so that it could interact with the L protein (23). For this purpose, the VL_[TLR2] sequence was modified at residues 9–22 and 90 according to IMGT nomenclature (9–22: ATLSLSPGERATLS-SSLSASVGDRVTIT and K90T).

The genes were then synthesized and cloned into the pcDNA3.4 plasmid by GeneArt (ThermoFisher Scientific, Waltham, MA, USA). Tandem scFvs were produced by transitory transfection of ExpiCHO™ cells with the max titer protocol as described in ThermoFisher’s manufacturer’s protocols. Briefly, cells were grown in ExpiCHO™ Expression Medium and maintained in a humidified incubator with 8% CO₂ at 37°C under shaking. Prior to transfection (Day −1), cell concentration was adjusted at 4·10⁶ viable cells/mL and incubated overnight in culture-grown conditions. On the next day (Day 0), the cell culture was diluted to 6·10⁶ cells/mL and transfected by 0.8 µg/mL of plasmid encoding Bic03 or Bic05, previously mixed with ExpiFectamine CHO reagent. The expression enhancer was added at 18 h and left until 22 h posttransfection, and the flask was placed at 32°C with 5% of CO₂. An additional expression feed was added on day 5, and cells were harvested around 10 days posttransfection. Cell viability was measured with CytoSMART™ (Corning, NY 14831 USA) and centrifuged at 10,000×g for 10 min. Clarified supernatants were stored at −20°C until purification.

For purification of antibodies, samples were deep-frozen, centrifuged at 10,000×g for 20 min, and passed over a 0.22-µm filter. All supernatants were passed over HiScreen™ Capto™ L column (Cytiva, Velizy-Villacoublay, France) equilibrated with PBS buffer (2.7 mM of KCl, 0.10 M of NaCl, 2 mM of KH₂PO₄, 8 mM of Na₂HPO₄, pH 7.4) in the ÄKTA pure protein purification system. Tandem scFv was eluted by a linear pH gradient in 0.1 M glycine buffer running from pH 6 to pH 2, and the buffer was removed by a desalting column in PBS. The antibodies are concentrated by Amicon (Merk Millipore, Darmstadt, Germany) and filtered (0.2 µM—Merck Millipore, Darmstadt, Germany). Antibody concentration was determined with a UV detector at 280 nm. Tandem scFvs molecular mass and molar extinction coefficient data were all generated by the Protparam tool from http://web.expasy.org/protparam/. Integrities of all purified proteins were assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on homogeneous 10% polyacrylamide gel under reducing or nonreducing conditions. Samples were all loaded at 0.5 µg for Coomassie blue staining. ProSieve QuadColor Protein Markers (Lonza, Fribourg, Switzerland) were used. Each tandem and scFv were centrifuged at 15,000×g at 4°C for 20 min before use.

Endotoxin levels in the purified antibodies were determined by the Limulus amebocyte lysate test according to the manufacturer’s instructions (Thermo Scientific™ Pierce™ LAL Chromogenic Endotoxin Quantitation Kit, Thermo Scientific, Illkirch, France). Antibody concentration measures were performed using the BCA method (BCA Protein Assay Kit, Sigma-Aldrich, Germany) and optical density at 208 nm by Nanodrop (Thermo Scientific™ NanoDrop™ One/One^C, Illkirch, France).

Cytapheresis products were obtained from the Centre Atlantic Transfusion Department (EFS-CA, Tours, France) according to the research agreements CPDL-PLER 2016 004 and CDPL-PLER 2019 188. They were issued from the healthy adult volunteers who had given their written informed consent, and the EFS ethics committee approved the procedure.

To prepare peripheral blood mononuclear cells (PBMCs), whole blood was diluted one-half with PBS. Density gradient separation of blood involved using Lymphoprep (Eurobio, Les Ulis, France). Tubes were centrifuged at 450×g for 25 min at 25°C, then cell layers (buffy coat) were immediately collected and transferred to 50 mL conical tubes, resuspended with PBS, and centrifuged at 300×g for 10 min at 25°C. Monocytes were then purified from PBMCs by a positive selection using CD14 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) (> 90% purity). For immature monocyte-derived DCs (moDCs), monocytes were differentiated for 6 days in RPMI 1640 medium (Dominique Dutscher, Bernolscheim - France) medium supplemented with 10% FCS (Dominique Dutscher, France), 66 ng/mL of granulocyte-macrophage colony-stimulating factor (GM-CSF), and 25 ng/mL of IL-4 (Miltenyi Biotec, Bergisch Gladbach, Germany). On day 6, cells were collected, and flow cytometry analysis was performed for moDC qualification.

MoDCs were treated with different maturation agents, such as zymosan from Saccharomyces cerevisiae (Sigma-Aldrich, St. Louis, MO, USA) at 5 μg/mL, or antibodies: neutralizing anti-TLR2 antibody (Bio-Techne, Minneapolis, MN, USA), anti-CD209 (clone 120C11.01, Dendritics, Lyon, France), anti-DCIR (clone 108H8.3, Dendritic, Lyon, France), the corresponding scFvs (αTLR2, αCD209, αDCIR), or the bsAbs (Bic03, Bic05) at different concentrations at 37°C in a 5% CO₂ atmosphere. The DC maturation and secretion of cytokines were assessed after 48 h by flow cytometry and ELISA.

After isolation, PBMC (1·10⁶ cells/mL) was placed in culture in the presence of zym (5 μg/mL), Bic03 at different concentrations, or controls scFvs for 48 h. The percentage of CD4⁺/CD25⁺/CTLA-4⁺/PD-1⁺ (Miltenyi Biotec, Bergisch Gladbach, Germany) was then determined by flow cytometry. Longer PBMC cultures were performed with Bic05, zym (5 μg/mL), or scFvs with 1·10⁶ cells/mL for 2, 6, and 9 days. At day 5, IL-2 was added at 100 UI/mL (Miltenyi Biotec, Bergisch Gladbach, Germany) to all culture conditions. Within these PBMC cultures, the phenotypic profiles of CD4⁺ regulatory T cells and DC subsets were analyzed at D2, D6, and D9 by flow cytometry according to previously described panels.

The T lymphocytes were obtained from PBMCs by positive, then negative, bead selection to obtain the CD4⁺/CD25⁻ fraction (Miltenyi Biotech, Bergisch Gladbach, Germany). For T-cell differentiation, 1·10⁶ CD4⁺ T cells were cultured with 1·10⁵ allogeneic DC (10:1, T:DC). After 10 days, primed T cells were collected and purified using CD4 microbeads (Miltenyi Biotech, Bergisch Gladbach, Germany). For recall response proliferation, primed CD4⁺ T cells were stained with Cell Proliferation Dye eFluor R 670 (eBioscience, CA, USA, Villebon sur Yvette, France) and plated with DCs treated with Bics from the same donor used for priming (T:DC, 10:1). After 2, 4, and 6 days, the percentages of divided responder T cells were calculated by proliferation dye dilution by flow cytometry, and the percentage of the CD4⁺/CD127^low /CD25⁺/Foxp3⁺/IL-10⁺ population was evaluated by flow cytometry.

For the evaluation of the inhibition properties of Bic05-PBMC populations, allogenic CD4⁺/CD25⁻ were stained with Cell Proliferation Dye eFluor R 670 (eBioscience, CA, USA) and then stimulated with CD3/C28 Dynabeads (Corning, NY, USA) in the presence of D9-cultured PBMC cells in a ratio 1:1. The CD4⁺ proliferation was evaluated at D5 and D6 by flow cytometry.

Ethics approval and consent to participate: this study was a noninterventional clinical trial (ID RCB: 2017-A02678-45) and approved by the institutional review board—”Comité de Protection des Personnes - Ile de France VIII” (CPP: 17 11 76). The patients included received information on the use of their samples and medical data for research purposes. The present study was registered at ClinicalTrials.gov (NCT03416543). Patient demographics for the clinical trial analyzed in this study are provided in Supplementary Table 1. All patients gave informed consent, and the studies were approved by their respective ethical review committees.

Aspiration of SF was performed in each patient after strict aseptic techniques. Samples were stored at 4°C until they were analyzed in the laboratory. SF dilutions (one-third) were performed in RPMI 1640 (Dominique Dutcher, Brumath, France) and centrifuged at 700×g for 10 min. Cell pellets were counted in the presence of trypan blue. For flow cytometry, cells were washed with PBS (Gibco, ThermoFisher, Waltham, MA, USA) containing 1% SVF and 0.01% N₃Na in 96-well plates. Next, cells (1·10⁶/100 μL) were stained for 30 min at 4°C with the appropriate antihuman panel at the appropriate concentration or with the relevant isotypes (see “Flow cytometry analysis” paragraph).

The cells (1·10⁶/mL) from SF were maintained in culture in the presence of Bic03, Bic05, Zymosan (5 μg/mL), or medium (RPMI 1640, 10% FCS, 1% glutamine) as control at 37°C for 48 h. After 48 h, samples were centrifuged at 300×g. Culture supernatants were then stored at 20°C until ELISA analysis.

Epitope prediction was made using the software MAbTope (24). The 3D structures used for the targets were 2Z7X for TLR2, 1XPH for DC-SIGN, and 5B1X for DCIR. The anti-TLR2 scFv was modeled by homology, using 7Q4Q (VH) and 7MW5 (VL) as templates. The anti-DC-SIGN scFv was modeled by homology, using 7KYO (VH) and 4S2S (VL) as templates. The anti-DCIR was modeled by homology, using 3WII (VH) and 1A6V (VL) as templates. All models were built using MODELLER (25).

Flow cytometry was performed on a Cytoflex S flow cytometer (Beckman Coulter, Brea, CA, USA) with FlowLogic software (Miltenyi Biotec, Bergisch Gladbach, Germany). Results were expressed as the ratio of mean of fluorescence (MFI) of the marker to the MFI of the isotype control and referred to as MFI ratio, either as the MFI of positive cells minus the isotypic control MFI or referred to as MFI.

Cells (1·10⁶–2·10⁵/100 μL) were stained for 30 min at 4°C in cold PBS containing 1% SVF and 0.01% NaN₃ (Sigma-Aldrich, St. Louis, MO, USA) in the presence of appropriate VIO-Dyes (ThermoFisher, Waltham, MA, USA) with the following antihuman antibodies at the appropriate concentration or with the relevant isotypes according to the following panels:

For DC qualification at D6 of CD14⁺ culture: antihuman CD45-PEvio770, CD209-PE, and CD14-PercP (Miltenyi Biotec, Bergisch Gladbach, Germany).

For DC maturation: CD83-FITC, CD86-PE, CD25-APC, PD-L1-FITC (BD Biosciences, San Jose, CA, USA), CD14-PE, ILT3-PE-Cy7, ILT4-PE, (Beckman Coulter, Brea, CA, USA), HLA-DR–Percp (Miltenyi Biotec, Bergisch Gladbach, Germany), IL-10 receptor alpha-PE, IL-10 receptor beta-FITC (R&D Systems, Minneapolis, MN, USA) in three different panels.

In PBMC culture: for CD4⁺ cell identification, the following antibody pattern were used: CD4-viogreen, CD25-FITC, CD152-PE-CF594, Lap-APC (Lap (TGF-β1), IL-10-PE, CD73-APC750, CD127-PEcy7 (BD Biosciences, Minneapolis, MN, USA), Foxp3-Bv421 (BioLegend, San Diego, CA, USA), and PD1-PE-Cy7 (R&D).

For DC subset identification, the following antibody patterns were used:

HLA-DR-FITC, CD123-PE, BDCA-2-PEvio770, CD11c-PE, CD1c-PEvio700, CD141-PEvio700, IL-10-APC, (Miltenyi Biotec, Bergisch Gladbach, Germany), and TGFb1-Bv421 (BioLegend, San Diego, CA, USA).

- In synovial cells: CD45-PEvio770, CD3-PE/CD56-FITC, CD14-PE/CD19-FITC, and CD66b-PE (Miltenyi Biotec, Bergisch Gladbach, Germany).

For the DC subset identification in synovial cells, the following panel was used: HLA-DR-FITC, CD123-PE, CD303-PEVio700, CD11c-PE, CD1c-PEvio770, and CD141-PE-vio770. All cells were labeled by TLR2-APC, DCIR-APCvio770, and CD209-PE-Vio700 (Miltenyi Biotec, Bergisch Gladbach, Germany).

The cells were then washed once using cold PBS containing 1% SVF and 0.01% NaN₃, and viable cells were then analyzed by flow cytometry.

MoDCs (1·10⁶ cells/100μL) were incubated with different concentrations of Bic03 or Bic05 at 37°C for at least 1 h. The cells were washed two times with cold PBS containing 1% SVF and 0.01% NaN₃ and incubated with anti-histidine-PE (R&D Systems, Minneapolis, MN, USA) or anti-histidine–APC antibody (Miltenyi Biotech, Bergisch Gladbach, Germany) for 1 h. For binding inhibition experiments, anti-TLR2 (clone T2.5, Invivogen Toulouse, France), anti-CD209, anti-DCIR antibodies (Dendritics, Lyon, France) in the presence of Fc block (Miltenyi Biotech, Bergisch Gladbach, Germany) or anti-TLR2, anti-CD209, anti-DCIR corresponding scFvs were incubated at least 30 min (Bic05) or 1 h (Bic03) before adding Bic03 or Bic05. The binding of both bsAbs was demonstrated thanks anti-Hist-PE/-APC antibody as described above.

MoDCs or PBMC (1·10⁶/mL) were stimulated for 48 h by medium, Zymosan (5 μg/mL), Bic03 or Bic05. The culture supernatants were collected, and centrifuged at 10,000×g for 10 min at 4°C, and then IL-10, IL-12-p70, TNF-α, and IFN-γ ELISA measurement was performed on culture supernatants of each condition according to the manufacturer’s instructions (ThermoFisher, Waltham, MA, USA). Data were expressed as means ± SD of donors. LegendPlex™ was used to measure the secretion of cytokines from PBMC cells (IL-4, IL-2, CXCL10 (IP-10), IL-1β, TNF-α, CCL2 (MCP-1), IL-17A, IL-6, IL-10, IFN-γ, IL-12p70, CXCL8 (IL-8), and Free Active TGF-β1. The protocol was performed per the manufacturer’s directions. Briefly, detection beads were sonicated and incubated with media from purified cells. Beads bound to target cytokines were then washed, incubated with detection antibodies, and washed again, and the cytokine concentrations were then determined through flow cytometric analysis and a standard curve. The Cytoflex S (Beckman Coulter) was used for data acquisition, and accompanying LegendPlex™ software was used for analysis.

Adult patients with known or newly diagnosed RA, primitive OA, or gout disease who presented with mono-, oligo-, or polyarthritis and underwent aspiration of synovial fluid were included. Patients with microcrystalline arthritis other than gout, spondylo-arthritis, septic arthritis, or receiving therapeutic antibodies were excluded. Patient’s consent forms were collected before each inclusion after they were given loyal and complete information. The diagnosis of RA was established using the ACR/EULAR criteria 2010. Gout disease was diagnosed in the presence of multiple criteria, such as tophus, monosodium urate crystals found in the SF, and hyperuricemia.

All data are presented as mean ± SD unless otherwise stated. Statistical and graphical analyses were made using GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA). Statistical significance was determined by a one-way ANOVA to compare differences among multiple groups. p < 0.05 was considered statistically significant.

To mimic zym, we designed a bsAb to crosslink TLR2 and a CLR on the DC surface. We selected a murine monoclonal Ab (mAb) against CD209 (DC-SIGN) that does not activate human moDCs, induce their IL-10 secretion, or inhibit zym-induced IL-10 secretion (Supplementary Figure 1A). All bsAbs and single-chain fragment variables (scFvs) were analyzed by SDS-PAGE, size-exclusion chromatography, and mass spectrometry (Supplementary Figures 2B–D). The sample productions were checked for endotoxin contamination (Supplementary Figure 2E). We also evaluated the presence and expression of bsAb targets on moDCs, showing a low level of TLR2 and a high level of CD209 (Supplementary Figure 3A).

The mAbs were directed against different domains of CD209 leading to different DC maturation types (26). We determined the putative epitope of our anti-CD209 Ab by applying a new and robust in silico method (24). The epitope was located in the carbohydrate recognition domain (Supplementary Figure 4A), a location that does not inhibit endocytosis (26). We also selected an anti-TLR2 mAb that had been designed under an scFv format (22) and whose epitope was predicted to be in the 152-169 aa region of TLR2 (Supplementary Figure 4B). The anti-CD209 mAb was derived under the same scFv format and attached to the anti-TLR2 scFv via a (G4S)₃ linker (Supplementary Figure 2A). Such a construct was named “bispecific anti-C-lectin” (Bic). None of the scFvs alone could induce moDC IL-10 secretion (Supplementary Figure 1B).

The binding of Bic03 to donor moDCs was dose-dependent (Supplementary Figure 5A) and could be inhibited by both anti-TLR2 and/or anti-CD209 scFvs (Figure 1A). MoDC treatment with Bic03 induced a semimature phenotype different from that with zym (Figure 1B; Supplementary Figure 6A) as well as the expression of regulatory molecules such as immunoglobulin-like transcript (ILT3; CD85k) or ILT4 (CD85d) and programmed death-ligand 1 (PD-L1) (CD274) (Figure 1C). Bic03 also induced the expression of CCR7 to the same level as with zym (Supplementary Figure 7A). In parallel, Bic03 dose-dependently induced IL-10 secretion by moDCs (Figure 1D), but the corresponding scFvs did not, even in combination (Supplementary Figure 1B). This effect could be inhibited by the anti-TLR2 scFv alone (Figure 1E). However, this semimature profile and the amount of IL-10 secretion were not sufficient to consider Bic03-treated DCs as tolDCs (27), and we further explored cytokine secretion panels. The cytokine secretion was clearly different from that obtained with zym, with a higher level of TGF-β1 and lower levels of IFN-γ, IL-6, and TNF-α (Figure 1F).

To further analyze the effect of Bic03 on human cells, donor PBMCs were treated for 48 h with different concentrations of Bic03. We found strong IL-10 secretion (Figure 1G) and dose-dependent induction of CD4⁺/CD127^low/CD25⁺/CTLA-4⁺/PD-1⁺ cells after 48 h (Figure 1H). Moreover, mixed lymphocyte reaction (MLR) 6-day cultures of naïve allogenic CD4^+/CD25⁻ cells and Bic03-treated moDCs increased CD4⁺/CD127^low/CD25⁺/Foxp3⁺ and/IL-10⁺ Treg proportion, which disappeared at higher Bic03 concentrations (Figure 1I). Thus, Bic03 seemed able to induce tolerogenic moDCs and phenotypical Treg populations.

Bic03 being difficult to produce and to extend our observations, we designed another Bic targeting TLR2 and a CLR, namely DCIR (CD367), which is an immunoreceptor tyrosine-based inhibitory motif-dependent CLR, unlike CD209, which is an immunoreceptor tyrosine-based activation motif-dependent CLR. As for Bic03, we selected a murine mAb against human DCIR that could not activate moDCs (Supplementary Figure 1A) and also recognized the carbohydrate recognition domain (Supplementary Figure 4C). As compared with Bic03, for Bic05, the scFv derived from the mAb was associated with the anti-TLR2 scFv by a shorter linker (G4S)₁ because the long linker did not work (Supplementary Figure 2A). The Bic05 long-linker version did not bind to TLR2-expressing cells and had no effect on moDCs (data not shown). Bic05 binding to donor moDCs was dose-dependent (Supplementary Figure 5B) and could be inhibited by anti-TLR2 or anti-DCIR scFvs (Figure 2A). The K_D of Bic05 on interferometry was 0.55 nM, comparable to that of the anti-TLR2 scFv (0.30 nM) (data not shown). Its antigenic target (DCIR) is expressed at a similar level to CD209 (Supplementary Figure 3B). Like Bic03, Bic05 induced a semi-mature phenotype of moDCs (Figure 2B; Supplementary Figure 6B) and a dose-dependent secretion of IL-10 and TGF-β1 (Figures 2D–F). However, Bic05 induced a lower level of PD-L1 than with zym (Figure 2C), a higher level of IFN-γ (Figure 2F), and a higher percentage of cells expressing CCR7 (Supplementary Figure 7B). Of note, in sharp contrast with zym, Bic05 increased the proportion of IL-10Rα– and IL-10Rα/IL-10Rβ−expressing cells (Supplementary Figures 8A, B). Like Bic03, Bic05 treatment of PBMCs dose-dependently induced IL-10 secretion (Figures 2G, H). Bic05 induced the secretion of TGF-β1, IL-6, IFN-γ, IL-12p70, and TNF-α at low levels; these cytokines were generally produced in lower quantities than with zym. By contrast, Bic05 did not induce IL-1β or IL-4 secretion, contrary to zym (Figure 2H).

Having phenotypically observed the differentiation of Treg populations with Bic03-treated DCs, a phenomenon that suggests the induction of tolDCs (28), we analyzed in further detail the properties of Bic05-pretreated DCs in coculture experiments. MLR with Bic05-pretreated moDCs and allogeneic CD4⁺/CD25⁻ cells led to an intermediate level of CD4⁺ T-cell proliferation as compared with inflammatory DCs and zym-pretreated DCs on day 6 (Figure 3A) as for iDCs and contrary to zym-pretreated DCs. Under the same conditions, Bic05-pretreated DCs induced IL-10 production in supernatants (Figure 3B).

In order to confirm the orientation toward Bic05-induced tolerance, we performed long-term cultures of PBMC with Bic05 concentrations of 250 nM and 500 nM at the maximal plateau of IL-10 secretion for 2, 6, and 9 days (Figure 3C). These high concentrations were chosen to anticipate Bic05 degradation during the long-term cultures. So, we demonstrated that Bic05 was able to increase the proportion of CD4⁺/CD127^low/CD25⁺/Foxp3⁺/IL-10⁺ Tregs similar to with zym. Bic05 induced Treg-expressed LAP (membrane TGF-β1) and CTLA-4 to the same extent as that induced with zym but significantly greater CD73 induction (Figure 3D). Cells from Bic05-treated PBMC cultures on day 9 strongly inhibited the proliferation of allogeneic CD4^+/CD25⁻ T cells induced by CD3⁺/CD28⁺ beads, as did zym-treated PBMCs (Figure 4A), and as expected from a mixture of tolerant DCs and functional Tregs (29).

In addition to the Treg compartment, we evaluated the ability of blood DCs to be modulated in long-term Bic05-treated PBMC cultures. Hence, pDCs were identified by CD123⁺/CD303⁺, cDC1 cells by CD11c⁺/CD141⁺, and cDC2 cells by CD11c⁺/CD1c⁺ labeling in the HLA-DR gate (30, 31). All circulating DC subsets, identified by their phenotypic profiles, secreted IL-10 and TGF-β1. The survival rate on day 9 of pDCs and cDC1⁺(cDC2) and DC CD141⁺(cDC1) number was increased immediately (Figure 4B) (32).

To directly assess inflammatory DCs, we obtained SF cells from patients in a small cohort (NCT03416543) (Supplementary Table S1) of gout and RA patients, representing two kinds of inflammatory conditions. In accordance with what was previously reported (33), all three DC subsets were equally distributed in gout and RA SF (Figure 5A). TLR2 was significantly overexpressed in all three DC subsets and monocytes of RA as compared with gout monocytes and DCs (Figure 5B). TLR2 was coexpressed with CD209 in all DC subsets and with DCIR in all DC subsets in both diseases (Supplementary Figures 9A–C). By contrast, except for IL-12p70 and IFN-γ, the levels of inflammatory cytokines in SF did not differ between gout and RA disease (Supplementary Figure 10). SF from RA patients induced a higher secretion of IFN-γ and a lower secretion of IL-12p70 than that from gout patients.

After 48 h of incubation of purified SF cells, both Bics induced a similar dose-dependent increase in IL-10 secretion from SF from both diseases, reaching a plateau above 200 nM (Figures 5C, D). SF cells from both diseases showed significant TGF-β1 secretion under the same conditions (Figure 5E).