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Background:Rheumatoid Arthritis (RA) is a chronic inflammatory disease that affects 1% of the population worldwide. Peripheral blood monocytes subsets are altered in RA, and it is well-known that macrophages play a critical pathogenic role in RA [1-3]. In fact, macrophage derived factors (IL-6, TNF, GM-CSF) are targets of currently used disease modifying antirheumatic drugs (DMARDs). JAK inhibitors (JAKi) are the first targeted synthetic DMARDs approved for treatment of RA with comparable efficacy to biologics [4]. However, the effect of JAKi on monocyte and macrophage specification and differentiation is currently unknown.Objectives:To study the transcriptional and functional effects of JAKi on monocytes and on the differentiation of monocyte-derived macrophages promoted by granulocyte-macrophage colony-stimulating factor (GM-CSF), a factor that drives the development and pathogenesis of RA.Methods:Peripheral blood from RA patients was obtained before and three months after upadacitinib (Upa) treatment. Monocyte subsets (classical, intermediate and non-classical) were studied by flow cytometry on the basis of CD14/CD16/HLA-DR expression in RA patients and sex-matched voluntary normal donors. RNASeq was also performed on CD14+ monocytes from RA samples.For in vitro experiments, human monocytes and GM-CSF-differentiating macrophages were exposed to clinically used doses of various JAKi, and transcriptional analysis (RNA-Seq), Western blot, ELISA and siRNA-mediated knockdown approaches were used to define the range of genes, function-associated markers and effector functions specifically modulated by JAKi, as well as the underlying molecular mechanism.Results:Upadacitinib treatment diminished classical CD14++CD16- monocytes and augmented non-classical CD14+CD16+ monocytes proportions in RA patients. After three months of treatment, monocyte subsets levels of RA patients were similar to those found in normal donors (Figure 1A). Monocyte subset changes correlated with a reduction in disease activity index DAS28 (Figure 1B). RNAseq confirmed these phenotypic changes. GSEA revealed that the transcriptome of monocytes exhibited a very significant over-representation of the genes that define non-classical monocytes as well as a significant reduced expression of the genes that define classical monocytes after the 3-month upadacitinib treatment (Figure 1C).In vitro, upadacitinib modified the acquisition of the transcriptional profile of GM-CSF-dependent monocyte-derived macrophages (GM-MØ) in a dose-dependent manner. Enhanced expression of genes that define anti-inflammatory macrophages (IL10, FOLR2, CD163, LGMN) with 100nM clinical dose (100Upa-GM-MØ), and reduced expression of pro-inflammatory genes (INHBA, STING, IL1B) with 10nM (10Upa-GM-MØ) and 100nM Upa dose. Discriminant Regulon Expression Analysis (DoRothEA) identified an enrichment in MAFB transcription factor in 100Upa-GM-MØ, and this result was confirmed at protein level and by the expression of the MAFB target CD163 (Figure 2A). Knock-down of MAFB significantly reduced the positive effect of upadacitinib on the expression of IL10, LGMN, CD163, FOLR2 (Figure 2B). At functional level, 100Upa-GM-MØ enhanced IL-10 in response to pathogenic signals and apoptotic-Jurkat efferocytic activity (Figure 2C). These results were shared by macrophages treated with clinical doses of others JAKi (baricitinib, tofacitinib, filgotinib and peficitinib), an effect not seen with the TYK2 inhibitor deucravacitinib.Conclusion:Upadacitinib promotes the acquisition of a more anti-inflammatory profile via enhanced expression of MAFB in macrophages in vitro and can restore the altered peripheral blood monocyte subsets in RA patients in vivo.REFERENCES:[1] Smiljanovic B, et al. Ann Rheum Dis. 2018;77(2):300-8.[2] Alivernini S, et al. Nature medicine. 2020;26(8):1295-306[3] Zhang F, et al. Nature immunology. 2019;20(7):928-42.[4] Smolen JS, et al. Ann Rheum Dis. 2023;82:3-18.Acknowledgements:This study was conducted in accordance with the Declaration of Helsinki and approved by the Ethical Committee of Hospital General Universitario Gregorio Marañón (protocol codes: JAKi-2022-v1 and ESCL_REUINM_2023-v1).This work was supported by Grant PI20/00316, PI23/00224, and Red de Enfermedades Inflamatorias (REI, RICORS RD21/0002/0034), from Instituto de Salud Carlos III and cofinanced by the European Regional Development Fund “A way to achieve Europe” (ERDF) to APK, and Dirección General de Innovación e Investigación Tecnológica de la Comunidad de Madrid (RETAR-A-COVID P2022/BMD-7274) to APK. Baltasar López-Navarro and María Teresa Schiaffino contributed equally to this work.Disclosure of Interests:None declared.

Methotrexate (MTX) remains the first line of treatment in Rheumatoid Arthritis (RA)1,2. Inhibition of AICAR transformylase by MTX results in augmented release of adenine nucleotides to the extracellular space1; these are rapidly hydrolysed by the combined action of ectonucleotidases CD39 and CD79 rendering the antiinflammatory agent adenosine1. CD39, the rate-limiting enzyme in this cascade, is highly expressed by a subset of human FoxP3+CD4+ regulatory T cells (Treg39+)2-4 and MTX may act synergistically with Tregs in the control of inflammation. To study the expression of CD39 on circulating Treg cells of untreated early Rheumatoid Arthritis (ERA) patients and its relation with the ex vivo effect of MTX. Peripheral blood was drawn from 22 DMARD- and steroid- naïve ERA patients with a disease duration < 24 weeks, 15 longstanding RA patients (LRA, disease duration > 2 years) and 37 age and gender-matched healthy controls (HC). LRA patients were receiving low-dose weekly MTX and were naïve for biologicals. 10 ERA patients who had achieved remission 12 months after initiating MTX donated blood for a second time (ERA-R). The frequency of Treg and Treg cell subsets was assessed by flow cytometry. CD4+CD25+CD127- (total T reg), CD4+CD27+CD127-CD39+ Treg (Treg39+) and CD4+CD25-CD39- responder T (Tresp39-) cells were isolated by Ficoll-Hypaque, followed by sorting. The suppressor potency of Tregs was assessed in cocultures of isolated Tregs with Tresp, established at different Treg/Tresp ratios. Proliferation was determined by CFSE dilution; cytokine secretion was measured by ELISA of culture supernatants. As previously described5, ERA but not LRA patients demonstrated a superior frequency of circulating Treg (CD4+CD25+CD127-FoxP3+) cells. In addition, the proportion of Tregs that expressed CD39 (Treg39+) was significantly increased in ERA but not LRA. Total ERA Tregs were significantly more potent suppressors of proliferation, TNFα and IFNγ secretion when compared with HC or LRA Tregs, and this difference was partially and significantly abrogated in the presence of adenosine deaminase, or the adenosine A2A receptor (A2AR) antagonists DMPX (3,7-dimethyl-1-propargylxanthine) or ZM 241385, but not in the presence of the adenosine A1 receptor antagonist DPCPX (8-cyclopentyl-dipropylxanthine). When MTX was added to the culture medium, the suppressor potency of total Tregs was further enhanced in all 3 groups of patients, and this enhancement was significantly higher in ERA total Treg/Tresp39- cocultures as compared with HC or LRA total Treg/Tresp39- cocultures. The effect of MTX was also partially and significantly abrogated by adenosine deaminase, DMPX or ZM 241385 but not by DPCPX. We then tested the suppressor potency of isolated Treg39+ together with the enhancer effect of MTX on this potency, and observed that there were no longer differences among ERA, LRA and HC; this further suggests that the differences observed in assays using total Tregs can be attributable to the increased Treg39+ proportions present in ERA. The frequency and function of ERA-R Treg cells were not different from HC or LRA Tregs. The suppressor action of CD39+Tregs is mediated at least in part by adenosine trough A2AR ligation, and the superior suppressive potency of total ERA Tregs is associated with their higher proportion of TregCD39+ cells as compared with HC or LRA. In addition, the augmented suppressor effect observed in the presence of MTX is partly mediated by an increased adenosine production acting on A2AR and is more marked in ERA patients reflecting again their higher proportion of Treg39+ cells. This indicates that MTX cooperates with Treg39+ cells in the control of inflammation. [1]Montesinos MC, et al. Arthritis Rheum. 2007. [2]Peres RS, et al. Proc Natl Acad Sci U S A. 2015. [3]Deaglio S, et al. J Exp Med. 2007. [4]Borsellino G, et al. Blood. 2007. [5]Benito-Miguel M, et al. J Immunol. 2009. FIS PI 20/00141; FIS RD16/0012/0012; Fondo Europeo de Desarrollo Regional (FEDER), unrestricted research grant from Gebro Pharma Laura Nuño: None declared, Alejandro Villalva: None declared, Marta Novella-Navarro: None declared, Irene Monjo: None declared, Diana Peiteado: None declared, Sara García-Carazo: None declared, Amaya Puig-Kröger: None declared, Alejandro Balsa Grant/research support from: BMS, Gebro Pharma, Maria-Eugenia Miranda-Carus Grant/research support from: BMS, Gebro Pharma

Background:The protagonism of regulatory B cells seems to vary along the course of the disease in murine models of inflammatory conditions. Decreased numbers of circulating regulatory CD19+CD24hiCD38hi transitional B cells (cTrB) have been described in patients with longstanding RA.Objectives:To examine the frequency and evolution of cTrB cells in the peripheral blood of early RA (ERA) patients.Methods:Freshly isolated PBMCs from 48 steroid and DMARD-naïve ERA patients with a disease duration below 24 weeks and 48 healthy controls (HC) were examined by flow cytometry. Cocultures of isolated memory B cells were established with autologous T cells, in the absence or presence of TrB cells.Results:As compared with HC, ERA patients demonstrated an increased frequency of cTrB cells. cTrBs of ERA and HC displayed an anti-inflammatory cytokine profile and were able to downregulate T cell IFNγ and IL-21 production, together with ACPA secretion in autologous B/T cell cocultures. Basal frequencies of cTrBs above the median value observed in HC were associated with a good EULAR response to MTX at 12 months (RR=2.91; 95% CI, 1.37-6.47). A significant reduction of cTrBs was observed 12 months after initiating MTX, when the cTrB cell frequency was no longer elevated but decreased, and this was independent of the degree of clinical response or the intake of prednisone.Conclusion:An increased frequency of regulatory cTrB cells is apparent in untreated ERA, and the baseline cTrB cell frequency is associated with the clinical response to MTX at 12 months.References:[1]Matsushita T, et al. J Clin Invest. 2008;118:342. Flores-Borja F, et al. Sci Transl Med. 2013;5:173ra23.Disclosure of Interests:Paula Fortea-Gordo Grant/research support from: BMS, Alejandro Villalba: None declared, Laura Nuño: None declared, Maria-Jose Santos-Bornez Grant/research support from: BMS, Diana Peiteado: None declared, Irene Monjo: None declared, Amaya Puig-Kröger: None declared, Paloma Sanchez-Mateos: None declared, Emilio Martín-Mola Grant/research support from: BMS, Roche, Alejandro Balsa Grant/research support from: BMS, Roche, Consultant of: AbbVie, Gilead, Lilly, Pfizer, UCB, Sanofi, Sandoz, Speakers bureau: AbbVie, Lilly, Sanofi, Novartis, Pfizer, UCB, Roche, Nordic, Sandoz, Maria-Eugenia Miranda-Carus Grant/research support from: BMS, Roche

BackgroundUndifferentiated arthritis (UA) is defined as an inflammatory oligo-or polyarthritis that does not fulfil criteria for a definitive diagnosis. Earlier diagnosis would permits a better functional prognosis. Synovial tissue (ST) macrophages have been associated with disease activity, radiographic erosion and response to therapy in RA. Furthermore, it has been reported that M1 polarised macrophages predominate in RA synovitis, whereas M2 predominate in SpA synovitis.1 ObjectivesTo analyse polarised macrophages (M1 proinflammatory and M2 anti-inflammatory) in ST of patients with UA which evolved to RA or PsA, after a long follow-up, to explore their diagnostic value.MethodsTo determine the polarisation state of macrophages in ST obtained by arthroscopy from patients with UA that evolved to RA (UA-RA=8) or PsA (UA-PsA=9), the expression of proteins associated to GM-CSF-driven polarisation M1(INHBA, TNFα and MMP12) and M-CSF-driven polarisation M2(CD209) was assessed in ST CD163+macrophages. Patients with established RA(n=12) or PsA(n=10) were included as control. 4 µm cryosections of ST in OCT were blocked for 10 min with 1% human immunoglobulins and incubated with primary (1–5 µg/ml) and secondary antibodies. Imaging was performed with an inverted confocal microscope (SPE, Leica Microsystems) and glycerol immersion objective (ACS-APO 20x/NA 0.60). Single cell mean fluorescence intensity(MFI) was assessed using ImageJ software (National Institutes of Health, Bethesda, MD, USA). At least three random fields were evaluated for each type of ST, quantifying the expression of INHBA, TNF-α, MMP12 and CD209 in all segmented CD163+macrophages. Macrophages density was normalised based on selected tissue area(mm2). After background subtraction, data were plotted using GraphPad software (GraphPad Software, La Jolla, CA, USA).ResultsCD163+ sublining (SL) macrophages from UA-RA expressed abundantly the INHBA-encoded activin A, whereas TNFα and MMP12 were variably detected. Regarding the M-CSF-associated marker CD209, 2 populations of CD163+ macrophages were found in the SL of UA-RA, CD163+CD209+ and the other CD163+CD209-, with higher than 100 arbitrary units (au) for CD163+CD209+ and lower than 100 au for CD163+CD209-. Similarly, INHBA, MMP12 and TNFα expression and 2 populations of CD209 were detected in CD163+ macrophages from UA-PsA. Macrophage density was also found comparable between UA, with 650/mm2 in UA-RA and 649/mm2 in UA-PsA. Quantification of the above indicated markers in CD163 +ST macrophages from established RA and PsA revealed similar levels of INHBA, TNFα, MMP12 and CD209 than those from UA-RA and UA-PsA.ConclusionsThis study shows for the first time that the polarisation state of ST CD163+ macrophages in UA progressing to RA and PsA is similar to that of established RA and PsA in terms of INHBA, MMP12, TNFα and CD209 expression. Therefore, the inflammatory polarisation state of macrophages is similar in RA and PsA and it is already detected at the earlier steps.Reference[1] Vandooren B, et al. Arthritis Rheum2009;60(4):966–75.AcknowledgementsFinanced “Fondo de Investigación Sanitaria”(PI14/00785.JD Cañete)Instituto de Salud Carlos III. Cofinanced BECA FER-2015.Disclosure of InterestNone declared

BackgroundA novel population of CD4 +T cells with B cell helping capacity has been described in the synovial tissues and peripheral blood of seropositive RA patients with an established disease, and termed ‘peripheral helper’ (Tph) cells. (Rao DA et al, Nature 2017) Tph cells are characterised by the lack of CXCR5 together with a bright expression of PD-1 (CD4 +CXCR5-PD-1hi T cells). As opposed to CD4 +CXCR5+PD-1hi follicular helper T cells (Tfh), Tph cells are not located in lymphoid organs but accumulate in inflamed tissues. Tph cell numbers have not been previously examined in early RA (eRA).ObjectivesTo study the frequency of circulating CD3 +CD4+CXCR5-PD-1hi Tph cells (cTph), in patients with eRA.MethodsPeripheral blood was drawn from DMARD-naïve early RA patients (eRA) (2010 ACR criteria) with a disease duration <24 weeks (n=42), and healthy controls (HC) matched for age and gender (n=42). For comparison, blood was also drawn from 66 patients with established RA (disease duration >2 years), 45 patients with Spondyloarthritis (SpA), and their age and gender-matched HC (one HC per patient). In addition, synovial fluid from 7 patients with established RA and 3 patients with SpA was examined. Established RA patients were receiving low-dose oral methotrexate and were naïve for biological agents. SpA patients were receiving NSAIDs, low-dose oral methotrexate and/or sulphasalazine and were naïve for biologicals. After isolation by Ficoll-Hypaque gradient, PBMCs were stained with antibodies to CD3, CD4, CXCR5, ICOS and PD-1, and examined by flow cytometry.ResultsThe frequency of circulating CXCR5- cells gated for CD4 +T cells was not different among the studied groups. In contrast, eRA patients demonstrated an increased frequency of circulating CD4 +CXCR5-PD-1hi Tph and CD4 +CXCR5-PD-1hiICOS+ T cells. When examining seropositive (RF +and/or ACPA+, n=25) and seronegative eRA patients (RF- and ACPA-, n=17) separately, it was evident that the above described alterations were only apparent in seropositive eRA. Likewise, increased cTph numbers were observed in seropositive (n=47) but not seronegative (n=19) established RA, and not in SpA patients (n=45), which is consistent with data reported by Rao et al. Interestingly, this increased cTph cell frequency was observed only in seropositive RA patients with an active disease (DAS28 >2.6, n=24), whereas the numbers of cTph cells in established RA patients who had achieved remission (DAS28 <2.6, n=23) were not different from HC. Furthermore, Tph cells were present in the synovial fluid of seropositive RA (n=4) but not of seronegative RA (n=3) or SpA (n=3).ConclusionsTph cells may play an important role in the pathogenesis of seropositive but not seronegative RA. An increased cTph cell frequency is a marker of active, seropositive RA.Reference[1] Rao DA, et al. Nature2017;542(7639):110–114.Disclosure of InterestNone declared

BackgroundCD19+CD24highCD38high B cells have been described to have a regulatory capacity and their frequency is altered in the peripheral blood of patients with various autoimmune diseases. The pathogenesis of AS is not well understood, and evidence suggesting the implication of either autoinflammatory or autoimmune mechanisms has been reported. In addition, increased frequencies of circulating B cells bearing a regulatory phenotype has recently been described in spondyloartrhitis (1).ObjectivesTo study the frequency of circulating CD19+CD24high CD38high B cells (Breg) in patients with Ankylosing Spondylitis (AS), and test the regulatory capacity of this B cell subset.MethodsPeripheral blood was drawn from AS patients naïve for TNF blockers (AS/nb) (n=37) and healthy controls (HC) (n=37), that were matched with patients for age and gender. After isolation by Ficoll-Hypaque gradient, PBMCs were stained with antibodies to CD3, CD4, CD19, CD24, and CD38, and examined by flow cytometry. For functional studies, total CD19+ B cells were isolated from PBMCs of 3 HC by magnetical sorting. Breg-depleted CD19+ B cells were obtained after total CD19+ B cells were depleted of CD19+CD24highCD38high B cells by cytometry in a FacsVantage sorter (Beckton Dickinson). Total CD19+ B cells or Breg-depleted CD19+ B cells were established in culture and stimulated through their BCR. Secretion of IFNγ was determined by ELISA in culture supernatants.ResultsWhen compared with healthy controls, AS/nb patients demonstrated a significantly increased frequency of CD19+CD24highCD38high B cells (Breg). The frequency of circulating Breg was increased not only in AS/nb patients with high or very high disease activity (ASDAS-CRP) >2.1 but also in AS patients with low activity or no activity (ASDAS-CRP<2.1). The frequency of circulating Breg cells did not correlate significantly with ASDAS-CRP, ASDAS-ESR, BASDAI, CRP or ESR values. Functional in vitro studies showed that the secretion of IFNγ was significantly higher in Breg-depleted CD19+ as compared with total CD19+ B cells, indicating that Breg have the capacity to downmodulate B cell pro-inflammatory cytokine secretion.ConclusionsAn increased frequency of circulating CD19+CD24highCD38high B cells is observed in AS/nb patients, that is not related with disease activity. Functional in vitro studies confirmed that CD19+CD24highCD38high B cells are able to downmodulate B cell pro-inflammatory cytokine secretion.ReferencesCantaert T, Doorenspleet ME, Francosalinas G, Paramarta JE, Klarenbeek PL, Tiersma Y, van der Loos CM, De Vries N, Tak PP, Baeten DL. Increased numbers of CD5+ B lymphocytes with a regulatory phenotype in spondylarthritis. Arthritis Rheum. 2012;64:1859–68.Disclosure of InterestNone declared

BackgroundCD19+CD24highCD38high B cells have been described to have a regulatory capacity and their frequency is altered in the peripheral blood of patients with various autoimmune diseases.ObjectivesTo determine the frequency of circulating CD19+CD24highCD38high B cells in patients with RA, and establish a correlation with clinical parameters.MethodsPeripheral blood was drawn from RA patients treated with non-biological DMARDs (n=48), RA patients treated with anti-TNFalpha drugs (n=15) and healthy controls (n=63) that were matched with patients for age and gender. After isolation by Ficoll-Hypaque gradient, PBMCs were stained with antibodies to CD3, CD4, CD19, CD24, and CD38, and examined by flow cytometry.ResultsA decreased frequency of CD19+CD24highCD38high B cells was apparent in in ACPA+ but not ACPA- RA patients, and this was observed not only in patients treated with non-biological DMARDs but also in patients receiving anti-TNFalpha drugs. In addition, the frequency of CD19+CD24highCD38high B cells showed a significant negative correlation with ACPA titers (r = -0.43, p=0.023). In contrast, no correlation was found between the frequency of circulating CD19+CD24highCD38high B cells and either rheumatoid factor titres or disease activity as determined by the DAS28 score.ConclusionsA decreased frequency of CD19+CD24highCD38high B cells is observed in ACPA+ but not ACPA- RA patients treated with either nonbiological DMARDs or anti-TNFalpha drugs, that is related with ACPA titres but not with rheumatoid factor titres or disease activity.Disclosure of InterestNone declared

Background Follicular helper T cells (Tfh), a CD4 T helper subset localized in lymphoid organs, help B cell differentiation and function. Circulating CD4 T cells expressing CXCR5 together with ICOS and/or PD-1 are considered as counterparts of Tfh, can function as B cell helpers, and can be subdivided into three subpopulations based on the expression of CCR6 and/or CXCR3: CXCR5+CXCR3+CCR6- (Tfh-Th1), CXCR5+CXCR3-CCR6+ (Tfh-Th17) and CXCR5+CXCR3-CCR6- (Tfh-Th2). Only Tfh-Th17 and Tfh-Th2, as opposed to Tfh-Th1, seem to display functional properties of Tfh cells. An altered proportion of these subpopulations has been associated with autoimmune diseases. Objectives To study the frequency of circulating Tfh and Tfh cell subsets together with the frequency of circulating plasmablasts (CD19+CD20-CD27+CD38high B cells) in patients with Ankylosing Spondilytis (AS). Methods Peripheral blood was drawn from healthy controls (n=50), AS patients receiving NSAIDs (n=25) and AS patients treated with TNF blocking agents (n=25). After isolation by Ficoll-Hypaque gradient, PBMCs were stained with antibodies to CD3, CD4, CXCR5, ICOS, PD-1, CCR6, CXCR3, CD19, CD20, CD27, and CD38, and examined by flow cytometry. The percentages of CXCR5+CXCR3+ CCR6- (Tfh-Th1), CXCR5+CXCR3- CCR6+ (Tfh-Th17) and CXCR5+CXCR3-CCR6- (Tfh/Th2) cells were calculated after gating for CD3, CD4 and CXCR5+. The percentage of CD20-CD38high cells was calculated after gating for CD19+ and CD27+. Results The frequency of circulating CXCR5+ cells gated for CD4+ T cells was not different among the studied groups. In contrast, AS patients receiving NSAIDs but not those on TNF blockers, demonstrated a decreased frequency of CD4+CXCR5+ICOS+ PD-1+ cells. Furthermore, in AS patients receiving NSAIDs, the frequency of Tfh-Th1 cells was significantly increased and the frequency of Tfh-Th17 and Tfh-Th2 cells was significantly decreased as compared with controls. Subsequently, the ratio (Tfh-Th17+Tfh-Th2)/Tfh-Th1 was decreased in these patients. At the same time, the frequency of circulating plasmablasts was decreased in AS patients on NSAIDs. Interestingly, there was a significantly positive correlation between the percentage of circulating plasmablasts and the frequency of CXCR5+ICOS+PD-1+CD4+ T cells. In addition, the frequency of circulating plasmablasts showed a positive correlation with the ratio (Tfh-Th17+Tfh-Th2)/Tfh-Th1. Conclusions AS patients treated with NSAIDS, but not those receiving TNF blockers, demonstrate a decreased frequency of circulating Tfh counterparts (CXCR5+ICOS+PD-1+CD4+ T cells) and altered proportions of circulating Tfh subpopulations, with underrepresentation of subsets bearing a phenotype associated with B cell helping capacity. At the same time, a decreased proportion of circulating plasmablasts is apparent in AS patients treated with NSAIDs. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.1753

Objectives To examine the frequency and phenotype of Th17 cells in the peripheral blood of early RA (eRA) patients. Methods CD4+ T cells were isolated from the peripheral blood of 33 eRA patients and 33 healthy controls (HC), and from the synovial fluid of 20 established RA patients (RASF), by ficoll-hypaque gradient and magnetical negative selection. After polyclonal stimulation, the frequency of Th17 and Th1 cells was determined by flow cytometry and concentrations of IL-17, IFN-g, TNF-a and IL-10 were measured by ELISA in cell-free supernatants. Results When all of our eRA patients were analyzed together, a significantly lower percentage of circulating Th17 cells and a lower CD4-derived IL-17 secretion were observed in comparison with HC. However, after stratifying by anti-CCP antibody status, circulating Th17 cells were decreased in anti-CCP(+) but not in anti-CCP(–)-eRA. All Th17 cells were CD45RO+CD45RA- and CCR6+. Dual Th17/Th1 cells were also exclusively decreased in anti-CCP(+)-eRA. Circulating Th17 and Th17/Th1 cells were negatively correlated with anti-CCP titres. When anti-CCP(+)-eRA patients were retested one year after initiating treatment with oral methotrexate, their circulating Th17 frequency was no longer different from HC. Of note, the percentage of circulating Th1 cells and the secretion of CD4-derived IFN-g, TNF-a and IL-10 were not different between eRA patients and HC. In RASF, both Th17 and Th1 cells were increased when compared with blood. Conclusions Decreased circulating Th17 levels in eRA seem to be a marker of anti-CCP seropositivity, and return to levels observed in healthy controls after treatment with methotrexate. Disclosure of Interest None Declared

BackgroundIL-17-expressing FoxP3 regulatory T cells have been described, and their suppressive capacity has been questioned. An inflammatory environment seems to favor IL-17 secretion by regulatory CD4+CD25+FoxP3+ T cells.ObjectivesTo assess the suppressive function and IL-17 producing capacity of CD4+CD25+CD127-FoxP3+ T cells in the synovial fluid of RA patients (RASFd).MethodsSynovial fluid was drawn from 35 patients with established RA who were receiving methotrexate and low-dose oral prednisone. The frequency of CD4+CD25+CD127-FoxP3+ T cells was assessed by flow cytometry. Total CD4+ T cells, CD4+CD25+CD127- T reg cells and CD4+CD25- Tresp cells were isolated by Ficoll-Hypaque gradient, followed by sorting. After isolation, cells were stimulated for 5 hours with PMA+ionomycin or cultured for 5 days in flat-bottom 96-well plates coated with an anti-CD3 monoclonal antibody. Treg cell function was assessed using two different approaches: A.The regulatory function of natural proportions of Tregs was inferred by comparing the proliferative and cytokine responses of total CD4+ T cells (TCD4T) versus CD25+ depleted CD4+ T cells (CD4+CD25-T cells or Tresp cells); B. The per cell suppressor potency of Tregs was assessed in cocultures of isolated Tregs with Tresp, established at different Treg/Tresp ratios. Proliferation was determined by 3Hthymidine incorporation and CFSE dilution; cytokine secretion was measured by ELISA of culture supernatants.ResultsA high proportion of CD4+CD25+CD127- T cells was present in RASFd (mean ± SD, 21.1%±6.2), which is significantly higher than reported frequencies of this cell population in the peripheral blood of both RA and healthy subjects. These RASFd CD4+CD25+CD127- T cells expressed FoxP3 but did not express CD69. The proliferation rate, TNFα and IFNγ secretion were significantly higher for isolated Tresp as compared with TCD4T cells, indicating that natural proportions of Treg cells present in the synovial fluid of RA are funcionally suppressive. Surprisingly, TCD4T cells secreted higher amounts of IL-17 as compared with Tresp cells, although the difference did not reach statistical significance. On a per cell basis, RAPB Tregs were potent suppressors of Tresp proliferation, TNFα and IFNγ but not of IL-17 secretion; in fact IL-17 secretion did not decrease but was enhanced in the presence of increasing proportions of Treg cells. Isolated Treg cells did not proliferate or produce cytokines when cultured alone in anti-CD3 coated plates. However, in the presence of plate-bound anti-CD3 plus anti-CD28 and recombinant human IL-2, isolated Treg cells secreted significant amounts of IL-17 whereas no TNFα or IFNγ could be detected in supernatants.ConclusionsCD4+CD27+CD127- FoxP3+ Treg cells present in the synovial fluid of RA patients are potent suppressors of Tresp proliferation, TNFα and IFNγ secretion, and at the same time produce significant amounts of IL-17.References Voo KS, et al. Proc Natl Acad Sci U S A. 2009;106:4793.Beriou G et al. Blood. 2009;113:4240.Wang T, et al. Ann Rheum Dis. 2015;74:1293.Komatsu N, et al. Nat Med. 2014;20:62.Yang BH, et al. Mucosal Immunol. 2016;9:444. Disclosure of InterestNone declared