Abstract Systemic autoimmune connective tissue diseases (CTDs) are characterized by anti-nuclear antibodies, shared HLA-associated genetic risk, and frequent disease overlap, suggesting a central role for CD4+ T cells in pathogenesis. However, defining disease-driving CD4+ T-cell responses remains challenging due to their localization within lymphoid and affected tissues and the lack of approaches linking these responses to circulating counterparts. We combined [18F]-labeled thymidine PET/CT-guided tissue sampling, ex vivo antigen stimulation, and single-cell multiomics to characterize CD4+ T-cell responses in blood, PET-avid locoregional lymph nodes (LNs), and disease-affected tissues from patients with the immunologically distinct CTDs systemic sclerosis and Sjögren’s disease. PET-avid LNs from both diseases exhibited enhanced adaptive immune activity and contained an expanded population of interferon-stimulated gene (ISG)-expressing TRAIL+ CD4+ T cells. In Sjögren’s disease, active LNs and affected tissues harbored diverse effector CD4+ T-cell populations, including follicular and peripheral helper T cells and Th2/Th17 cells. In contrast, systemic sclerosis tissues lacked effector CD4+ T cells, while active LNs were enriched for naïve, regulatory, and TRAIL+ ISG CD4+ T cells. Antigen stimulation of peripheral blood mononuclear cells enriched for expanded effector CD4+ T-cell populations that shared activation profiles and clonal relationships with cells in LNs and affected tissues, many representing autoreactive antigen-specific T cells. TRAIL+ CD4+ T cells suppressed effector T-cell differentiation, autoreactive plasma cell generation, and autoantibody production in vitro, identifying a previously unrecognized immunoregulatory population. Together, this workflow enables comprehensive characterization of pathogenic and regulatory CD4+ T-cell responses across CTDs.

Fibrosis in rheumatic connective tissue diseases is marked by CD8+ T cell and pro-fibrotic myofibroblast infiltration, though the role of CD8+ T cells in myofibroblast activity remains unexplored. To address this, we developed a 3D cell culture model of immunity-driven fibrosis by combining the classic mixed lymphocyte reaction and a 3D myofibroblast contractility model. Upon co-culture with myofibroblasts, CD8+ T cells more strongly induced myofibroblast contraction and activation than CD4+ T cells. This was not associated with cytotoxicity but with increased IL-6 production by CD8+ T cells and pSTAT3 and TGFβ signaling in myofibroblasts. Use of the JAK/STAT3-inhibitor tofacitinib or the TGFβ receptor inhibitor SB-505124 inhibited the activated myofibroblast phenotype, and combined use of both inhibitors had a clear additive effect. Our findings reveal a previously underappreciated non-canonical role of CD8+ T cells in fibrosis, providing new light to the mechanisms of the human immune system.

Chronic inflammation plays a key role in cancer pathogenesis. Aggressive thyroid cancer is associated with immune infiltration and systemic inflammation. Long pentraxin 3 (PTX3) is an inflammatory protein implicated in tumor progression. This study evaluates PTX3 plasma levels in patients with non-medullary thyroid cancer (TC) compared to benign thyroid disease and investigates its tissue expression. We prospectively included 55 TC patients: 42 papillary, 3 follicular, 4 oncocytic, 4 anaplastic (ATC), and 2 poorly differentiated (PDTC). The control group consisted of 32 patients with benign thyroid disease. PTX3 plasma concentrations were measured by ELISA, and tissue expression of PTX3 and CD68 was analyzed using immunohistochemistry. PTX3 plasma levels did not significantly differ between TC and controls, but patients with PDTC and ATC had markedly higher concentrations. Tissue analysis showed strong PTX3 expression in three of four ATC cases in tumor and stromal cells, whereas benign and differentiated thyroid tissues exhibited minimal staining. CD68 expression was positive in ATC, indicating tumor-associated macrophage infiltration, but a few cells were double-positive for PTX3 and CD68. Our findings suggest a possible association between PTX3 and aggressive TC, particularly ATC. Further studies are needed to validate these findings and elucidate the cellular origin and functional role of PTX3.

OBJECTIVES:Macrophages are key orchestrators of the osteoarthritis (OA)-associated inflammatory response. Macrophage phenotype is dependent on environmental cues like the inflammatory factor S100A8/A9. Here, we investigated how S100A9 exposure during monocyte-to-macrophage differentiation affects macrophage phenotype and function. METHODS:OA synovium cellular composition was determined using flow cytometry and multiplex immunohistochemistry. Healthy donor monocytes were differentiated towards M1- and M2-like macrophages in the presence of S100A9. Macrophage markers were measured using flow cytometry, and phagocytic activity was determined using pHrodo Red Zymosan A BioParticles. Gene expression was determined using qPCR. Protein secretion was measured using Luminex multianalyte analysis and ELISA. RESULTS:Macrophages were the dominant leucocyte subpopulation in OA synovium. They mainly presented with an M2-like phenotype, although the majority also expressed M1-like macrophage markers. Long-term exposure to S100A9 during monocyte-to-macrophage differentiation increased M2-like macrophage markers CD163 and CD206 in M1-like and M2-like differentiated cells. In addition, M1-like macrophage markers were increased in M1-like, but decreased in M2-like differentiated macrophages. In agreement with this mixed phenotype, S100A9 stimulation modestly increased expression and secretion of pro-inflammatory markers and catabolic enzymes, but also increased expression and secretion of anti-inflammatory/anabolic markers. In accordance with the upregulation of M2-like macrophage markers, S100A9 increased phagocytic activity. Finally, we indeed observed a strong association between S100A8 and S100A9 expression and the M2-like/M1-like macrophage ratio in end-stage OA synovium. CONCLUSION:Chronic S100A8/A9 exposure during monocyte-to-macrophage differentiation favours differentiation towards an M2-like macrophage phenotype. The properties of these cells could help explain the catabolic/anabolic dualism in established OA joints with low-grade inflammation.

Background: Systemic sclerosis (SSc) is an autoimmune disease characterized by vasculopathy, fibrosis, and immune dysregulation. In SSc pathogenesis, circulating monocytes can be recruited to the skin and differentiate into macrophages. This possibly contributes to the activation and conversion of resident fibroblasts into myofibroblasts, driving fibrosis and skin thickness. However, how these cells exert their functions and via which fibroinflammatory mediator(s) is not fully understood. Objectives: To investigate the role of monocytes in mediating myofibroblast contraction and activation using an innovative 3D collagen hydrogel model. Methods: For our 3D skin model we cocultured human primary dermal fibroblasts with either peripheral blood mononuclear cells (PBMCs) or MACS-sorted CD14+ monocytes in a 3D collagen type 1 hydrogel. Subsequently, monocyte-driven tissue contraction was measured over time. We performed immunostainings for CD68, fibroblast activation protein (FAP), and alpha-smooth muscle actin (α-SMA) to evaluate monocyte/macrophage and fibroblast activation. To investigate the signaling pathways involved in the observed myofibroblast activation, we measured transcription factor-driven luciferase production using reporter constructs in the same dermal fibroblasts. The activity of the following reporter constructs was determined after 24 hours: Sis-Inducible Element (SIE); SMAD-Binding Element (SBE); Nuclear Factor of Activated T-cells 5 Response Element (NFAT-5), and NFκB Response Element (NFκB). Results: After 60 hours of co-culture, hydrogel plugs containing fibroblasts + monocytes displayed a strong spontaneous contraction, seen by a (approx. 80-90%) decrease in the area of the plugs. Hydrogels containing only fibroblasts did not contract. Hydrogels with fibroblasts + monocytes contracted as fast as fibroblasts + PBMCs, but depletion of CD14+ cells from PBMCs slowed down contraction, showing that monocytes strongly activated fibroblasts. The expression of FAP and α-SMA by fibroblasts increased in monocyte-containing hydrogels, compared to fibroblasts cultured without monocytes. Furthermore, CD68 expression also increased in co-culture, indicating enhanced monocyte differentiation/activation into macrophages by fibroblasts. Evaluating which intracellular pathway lead to fibroblast activation, we observed that SIE and NFκB reporter fibroblast constructs were strongly elevated in the presence of monocytes. However, we could not observed enhanced TGF-β activity using the SBE reporter. Together, our results suggest that inflammatory mediators like IL-6, IL-1 and S100A8/9 may contribute to the monocytes-driving myofibroblast contraction. Conclusion: This study highlights the importance of monocytes and the SIE and NFκB intracellular signaling pathways in myofibroblast contraction in a 3D skin model, contributing to understanding the basic mechanisms of these cells in SSc skin fibrosis and thickness. REFERENCES: [1] van Caam, Arjan et al. "Unraveling SSc Pathophysiology; The Myofibroblast." Frontiers in immunology vol. 9 2452. 13 Nov. 2018, doi:10.3389/fimmu.2018.02452. [2] Al-Adwi, Yehya et al. "Macrophages as determinants and regulators of fibrosis in systemic sclerosis." Rheumatology (Oxford) vol. 62,2 (2023): 535-545. Acknowledgements: Grant #2023/04897-7, São Paulo Research Foundation (FAPESP). Disclosure of Interests: None declared.

OBJECTIVES:Cytotoxic T cells and natural killer (NK) cells are central effector cells in cancer and infections. Their effector response is regulated by activating and inhibitory receptors. The regulation of these cells in systemic autoimmune diseases such as systemic sclerosis (SSc) is less defined. METHODS:We conducted ex vivo analysis of affected skin and blood samples from 4 SSc patient cohorts (a total of 165 SSc vs 80 healthy individuals) using single-cell transcriptomics, flow cytometry and multiplex immunofluorescence staining. We further analysed the effects of costimulatory modulation in functional assays, and in a severely affected SSc patient who was treated on compassionate use with a novel anti-CD3/CD7 immunotoxin treatment. RESULTS:Here, we show that SSc-affected skin contains elevated numbers of proliferating T cells, cytotoxic T cells and NK cells. These cells selectively express the costimulatory molecule CD7 in association with cytotoxic, proinflammatory and profibrotic genes, especially in recent-onset and severe disease. We demonstrate that CD7 regulates the cytolytic activity of T cells and NK cells and that selective depletion of CD7+ cells prevents cytotoxic cell-induced fibroblast contraction and inhibits their profibrotic phenotype. Finally, anti-CD3/CD7 directed depletive treatment eliminated CD7+ skin cells and stabilised disease manifestations in a severely affected SSc patient. CONCLUSION:Together, the findings imply costimulatory molecules as key regulators of cytotoxicity-driven pathology in systemic autoimmune disease, yielding CD7 as a novel target for selective depletion of pathogenic cells.

Background Osteoarthritis (OA) is a progressive joint disease and a major cause of chronic pain in adults. The prevalence of OA is higher in female patients, who tend to have worse OA outcomes, partially due to pain. The association between joint pain and OA pathology is often inconclusive. Preclinical research studies have largely overlooked sex as a potential determinant in joint pain during OA. This study aimed to investigate the role of sex in joint pain in the collagenase-induced OA (CiOA) model and its link with joint pathology. Methods Multiple aspects of pain were evaluated during identically executed experiments of CiOA in male and female C57BL/6J mice. Cartilage damage, osteophyte formation, synovial thickness, and cellularity were assessed by histology on day 56. The association between pain and pathology was investigated, disaggregated by sex. Results Differences in pain behavior between sexes were found in the majority of the evaluated pain methods. Females displayed lower weight bearing ability in the affected leg compared to males during the early phase of the disease, however, the pathology at the end stage was comparable between sexes. In the second cohort, males displayed increased mechanical sensitivity in the affected joint compared to females but also showed more cartilage damage at the end stage of the model. Within this cohort, gait analysis showed varied results. Males used the affected paw less often and displayed dynamic weight-bearing compensation in the early phase of the model. These differences were not observed in females. Other evaluated parameters displayed comparable gait behavior between males and females. A detailed analysis of individual mice revealed that seven out of 10 pain measurements highly correlated with OA histopathology in females (Pearson r range: 0.642–0.934), whereas in males this measurement was only two (Pearson r range: 0.645–0.748). Conclusion Our data show that sex is a determinant in the link between pain-related behavior with OA features. Therefore, to accurately interpret pain data it is crucial to segregate data analysis by sex to draw the correct mechanistic conclusion.

Introduction: The association between metabolic syndrome (MetS) and osteoarthritis (OA) development has become increasingly recognized. In this context, the exact role of cholesterol and cholesterollowering therapies in OA development has remained elusive. Recently, we did not observe beneficial effects of intensive cholesterol-lowering treatments on spontaneous OA development in E3L.CETP mice. We postulated that in the presence of local inflammation caused by a joint lesion, cholesterol-lowering therapies may ameliorate OA pathology.Materials and methods: Female ApoE3*Leiden.CETP mice were fed a cholesterol-supplemented Western type diet. After 3 weeks, half of the mice received intensive cholesterol-lowering treatment consisting of atorvastatin and the anti-PCSK9 antibody alirocumab. Three weeks after the start of the treatment, OA was induced via intra-articular injections of collagenase. Serum levels of cholesterol and triglycerides were monitored throughout the study. Knee joints were analyzed for synovial inflammation, cartilage degeneration, subchondral bone sclerosis and ectopic bone formation using histology. Inflammatory cytokines were determined in serum and synovial washouts.Results: Cholesterol-lowering treatment strongly reduced serum cholesterol and triglyceride levels. Mice receiving cholesterol-lowering treatment showed a significant reduction in synovial inflammation (P = 0.008, WTD: 95% CI: 1.4- 2.3; WTD + AA: 95% CI: 0.8- 1.5) and synovial lining thickness (WTD: 95% CI: 3.0-4.6, WTD + AA: 95% CI: 2.1-3.2) during early-stage collagenase-induced OA. Serum levels of S100A8/A9, MCP-1 and KC were significantly reduced after cholesterol-lowering treatment (P = 0.0005, 95% CI: -46.0 to -12.0; P = 2.8 x 10-10, 95% CI: -398.3 to -152.1; P = 2.1 x 10-9, -66.8 to -30.4, respectively). However, this reduction did not reduce OA pathology, determined by ectopic bone formation, subchondral bone sclerosis and cartilage damage at end-stage disease.Conclusion: This study shows that intensive cholesterol-lowering treatment reduces joint inflammation after induction of collagenase-induced OA, but this did not reduce end stage pathology in female mice.& COPY; 2023 The Author(s). Published by Elsevier Ltd on behalf of Osteoarthritis Research Society International. This is an open access article under the CC BY license (http://creativecommons.org/ licenses/by/4.0/).

Joint pain severity in arthritic diseases differs between sexes and is often more pronounced in women. This disparity is thought to stem from biological mechanisms, particularly innate immunity, yet the understanding of sex-specific differences in arthritic pain remains incomplete. This study aims to investigate these disparities using an innate immunity-driven inflammation model induced by intra-articular injections of Streptococcus Cell Wall fragments to mimic both acute and pre-sensitized joint conditions. Nociceptive behavior was evaluated via gait analysis and static weight-bearing, and inflammation was evaluated via joint histology and the synovial gene expression involved in immune response. Although acute inflammation and pain severity were comparable between sexes, distinct associations between synovial inflammatory gene expression and static nociceptive behavior emerged. These associations delineated sex-specific relationships with pain, highlighting differential gene interactions (Il6 versus Cybb on day 1 and Cyba/Gas6 versus Nos2 on day 8) between sexes. In conclusion, our study found that, despite similar pain severity between sexes, the association of inflammatory synovial genes revealed sex-specific differences in the molecular inflammatory mechanisms underlying pain. These findings suggest a path towards more personalized treatment strategies for pain management in arthritis and other inflammatory joint diseases.

Background Systemic sclerosis is characterized by vasculopathy, fibrosis and autoimmune inflammation. Adaptive immune and NK cells express co-stimulatory molecules to regulate effector responses in inflammatory conditions. CD7 is a co-stimulatory molecule induced by IFN-γ signaling on T and NK cells that is downregulated upon residency in tissues such as skin [1]. Objectives Hypothetically, CD7 is upregulated in IFN-γ driven conditions, allowing targeted depletion of pathogenic cells. In this study, we analyzed expression, regulation and targeting of CD7 in SSc tissues and in a patient treated with anti-CD7 immunotoxin (CD7-IT). Methods To identify disease-specific activation markers we compared status of T cell activation in SSc skin compared to blood. To do so, single-cell RNA sequencing datasets containing 60,000 skin and blood immune cells from 109 SSc patients versus 65 healthy donors were analyzed. Twentyone-color flow cytometry (n=50) and 8-color multiplex skin immunofluorescence staining (mIF) (n=30) were used for validation and spatial localization. CD7-IT was used to induce subset-specific depletion in vitro, and ex vivo skin explants. Fibroblasts and immune cells were co-cultured in 3D collagen hydrogel to test effect of targeted immune cell depletion on fibroblast contractility. A single patient was treated with immunotoxin on compassionate use basis. Results Sc-RNAseq analysis showed that SSc skin is characterized by increased infiltration of CD7+ cells corresponding to CD8+GZMB+ and CD56+GZMB+ cytotoxic T and NK cells. Immunohistochemistry also showed that SSc lesional skin exhibits significantly higher infiltration of these CD7+ (T and NK) cells than non-affected skin (Figure 1). Expansion of CD7+ cytotoxic T cells was also observed in SSc blood. A CD7-targeting IT effectively depleted 85% of CD7+ cytotoxic T and NK cells in vitro, and in ex vivo skin. Strikingly, depletion of CD7+ cells prevented fibroblast activation and contraction in a 3D fibroblast-immune cells co-culture assay. Notably, also in vivo administration of CD7-IT significantly eliminated CD7+ cell subsets in patient's blood and skin. Conclusion Together, we show strong presence of CD7 on SSc immune cells and that targetting this molecule depletes T and NK cell populations with potential disease modifying results. Our results pave the way for a novel approach in halting SSc related fibrosis. Reference [1]Wang T, Huang C, Lopez-Coral A, et al. K12/SECTM1, an interferon-γ regulated molecule, synergizes with CD28 to costimulate human T cell proliferation. J Leukoc Biol. 2012;91(3):449-459. doi:10.1189/jlb.1011498 Acknowledgements: NIL. Disclosure of Interests None Declared.Figure 1Strong presence of CD7+ cytotoxic T and NK cells in systemic sclerosis skin

Abstract Objective Activated synovial fibroblasts are key effector cells in RA. Selectively depleting these based upon their expression of fibroblast activation protein (FAP) is an attractive therapeutic approach. Here we introduce FAP imaging of inflamed joints using 68Ga-FAPI-04 in a RA patient, and aim to assess feasibility of anti-FAP targeted photodynamic therapy (FAP-tPDT) ex vivo using 28H1-IRDye700DX on RA synovial explants. Methods Remnant synovial tissue from RA patients was processed into 6 mm biopsies and, from several patients, into primary fibroblast cell cultures. Both were treated using FAP-tPDT. Cell viability was measured in fibroblast cultures and biopsies were evaluated for histological markers of cell damage. Selectivity of the effect of FAP-tPDT was assessed using flow cytometry on primary fibroblasts and co-cultured macrophages. Additionally, one RA patient intravenously received 68Ga-FAPI-04 and was scanned using PET/CT imaging. Results In the RA patient, FAPI-04 PET imaging showed high accumulation of the tracer in arthritic joints with very low background signal. In vitro, FAP-tPDT induced cell death in primary RA synovial fibroblasts in a light dose-dependent manner. An upregulation of cell damage markers was observed in the synovial biopsies after FAP-tPDT. No significant effects of FAP-tPDT were noted on macrophages after FAP-tPDT of neighbouring fibroblasts. Conclusion In this study the feasibility of selective FAP-tPDT in synovium of rheumatoid arthritis patients ex vivo is demonstrated. Furthermore, this study provides the first indication that FAP-targeted PET/CT can be used to image arthritic joints, an important step towards application of FAP-tPDT as a targeted locoregional therapy for RA.

Background Systemic sclerosis (SSc) is a rare, severe auto-immune disease characterized by inflammation, vasculopathy and fibrosis. Activated (myo)fibroblasts are crucial drivers of fibrosis. By exploiting their expression of fibroblast activation protein (FAP) to perform targeted photodynamic therapy (tPDT), we can locoregionally deplete these pathogenic cells. Objectives We explored the use of FAP-tPDT to selectively target primary skin fibroblasts from SSc patients, both in 2D and 3D cultures, as well as in biopsies from fibrotic skin lesions in the murine bleomycin-induced skin fibrosis model. Methods The FAP targeting monoclonal antibody (clone 28H1) was conjugated with the photosensitizer IRDye700DX. Primary skin fibroblasts were obtained from lesional skin biopsies of SSc patients via spontaneous out-growth and subsequently cultured on plastic or collagen type I. For 2D FAP-tPDT, cells were incubated in buffer with or without the antibody-photosensitizer (Ab-PS) construct, washed after 4 h and exposed to λ = 689 nm light (50 J/cm 2 at 280 mW/cm 2 ). Cell viability was measured using CellTiter Glo. For 3D FAP-tPDT, cells were seeded in collagen plugs and underwent the same treatment procedure. Contraction of the plugs was followed over time to determine myofibroblast activity. Skin fibrosis was induced in mice by 3x/week injections of 1.5 IU bleomycin intradermally on the back of the mouse. After 5 weeks the mice were sacrificed and biopsies were taken from affected and not affected skin. These were incubated with or without the FAP targeting Ab-PS construct or a control Ab-PS. After washing, the biopsies were exposed to light, incubated for 1h and subsequently formalin fixed and paraffin embedded. Sections were immuno stained for the presence of cleaved caspase-3. Results FAP-tPDT resulted in antibody-dose dependent cytotoxicity in primary skin fibroblasts upon light exposure. Cells not exposed to light or incubated with a control Ab-PS construct did not show this response. FAP-tPDT fully prevented contraction of collagen plugs seeded with primary SSc fibroblasts. Even incubation with a very low dose of antibody (0.4 nM) inhibited contraction in 2 out of 3 donors. In fibrotic skin biopsies from mice with bleomycin-induced skin fibrosis, an upregulation of apoptosis, as evidenced by increased caspase-3 staining (Figure 1), was observed in response to FAP-tPDT. The same treatment on biopsies of control skin did not increase caspase-3 staining, nor did incubation with a control Ab-PS construct or light alone. Figure 1. Cleaved caspase-3 staining of biopsies from the skin of mice with bleomycin-induced dermal fibrosis upon treatment with FAP-tPDT. The red arrows indicate the presence of positive fibroblasts. Conclusion Here we have shown, for the first time, the potential of FAP-tPDT for the selective targeting of pathogenic fibroblasts and treatment of fibrosis in SSc skin both in relevant patient-derived culture models as well as an in vivo model of fibrosis. Disclosure of Interests Daphne Dorst: None declared, Arjan van Caam: None declared, Elly Vitters: None declared, Birgitte Walgreen: None declared, Monique Helsen: None declared, Christian Klein Employee of: CK is an employee of Roche pharmaceutics, Shreya Gudi: None declared, Tirza Wubs: None declared, Jyoti Kumari: None declared, Przemysław Błyszczuk: None declared, Madelon Vonk: None declared, Peter van der Kraan: None declared, Marije Koenders: None declared.

BackgroundSystemic sclerosis (SSc) is a rare, severe auto-immune disease characterized by inflammation, vasculopathy and fibrosis. Activated (myo)fibroblasts are crucial drivers of fibrosis. By exploiting their expression of fibroblast activation protein (FAP) to perform targeted photodynamic therapy (tPDT), we can locoregionally deplete these pathogenic cells.ObjectivesWe explored the use of FAP-tPDT to selectively target primary skin fibroblasts from SSc patients, both in 2D and 3D cultures, as well as in biopsies from fibrotic skin lesions in the murine bleomycin-induced skin fibrosis model.MethodsThe FAP targeting monoclonal antibody (clone 28H1) was conjugated with the photosensitizer IRDye700DX. Primary skin fibroblasts were obtained from lesional skin biopsies of SSc patients via spontaneous out-growth and subsequently cultured on plastic or collagen type I. For 2D FAP-tPDT, cells were incubated in buffer with or without the antibody-photosensitizer (Ab-PS) construct, washed after 4 h and exposed to λ = 689 nm light (50 J/cm2 at 280 mW/cm2). Cell viability was measured using CellTiter Glo. For 3D FAP-tPDT, cells were seeded in collagen plugs and underwent the same treatment procedure. Contraction of the plugs was followed over time to determine myofibroblast activity. Skin fibrosis was induced in mice by 3x/week injections of 1.5 IU bleomycin intradermally on the back of the mouse. After 5 weeks the mice were sacrificed and biopsies were taken from affected and not affected skin. These were incubated with or without the FAP targeting Ab-PS construct or a control Ab-PS. After washing, the biopsies were exposed to light, incubated for 1h and subsequently formalin fixed and paraffin embedded. Sections were immuno stained for the presence of cleaved caspase-3.ResultsFAP-tPDT resulted in antibody-dose dependent cytotoxicity in primary skin fibroblasts upon light exposure. Cells not exposed to light or incubated with a control Ab-PS construct did not show this response. FAP-tPDT fully prevented contraction of collagen plugs seeded with primary SSc fibroblasts. Even incubation with a very low dose of antibody (0.4 nM) inhibited contraction in 2 out of 3 donors. In fibrotic skin biopsies from mice with bleomycin-induced skin fibrosis, an upregulation of apoptosis, as evidenced by increased caspase-3 staining (Figure 1), was observed in response to FAP-tPDT. The same treatment on biopsies of control skin did not increase caspase-3 staining, nor did incubation with a control Ab-PS construct or light alone.Figure 1.Cleaved caspase-3 staining of biopsies from the skin of mice with bleomycin-induced dermal fibrosis upon treatment with FAP-tPDT. The red arrows indicate the presence of positive fibroblasts.ConclusionHere we have shown, for the first time, the potential of FAP-tPDT for the selective targeting of pathogenic fibroblasts and treatment of fibrosis in SSc skin both in relevant patient-derived culture models as well as an in vivo model of fibrosis.Disclosure of InterestsDaphne Dorst: None declared, Arjan van Caam: None declared, Elly Vitters: None declared, Birgitte Walgreen: None declared, Monique Helsen: None declared, Christian Klein Employee of: CK is an employee of Roche pharmaceutics, Shreya Gudi: None declared, Tirza Wubs: None declared, Jyoti Kumari: None declared, Przemysław Błyszczuk: None declared, Madelon Vonk: None declared, Peter van der Kraan: None declared, Marije Koenders: None declared.

TGF-β1 is an important growth factor to promote the differentiation of T helper 17 (Th17) and regulatory T cells (Treg). The potential of TGF-β1 as therapeutic target in T cell-mediated diseases like rheumatoid arthritis (RA) is unclear. We investigated the effect of TGF-β1 inhibition on murine Th17 differentiation in vitro, on human RA synovial explants ex vivo, and on the development of experimental arthritis in vivo. Murine splenocytes were differentiated into Th17 cells, and the effect of the TGF-βRI inhibitor SB-505124 was studied. Synovial biopsies were cultured in the presence or absence of SB-505124. Experimental arthritis was induced in C57Bl6 mice and treated daily with SB-505124. Flow cytometry analysis was performed to measure different T cell subsets. Histological sections were analysed to determine joint inflammation and destruction. SB-505124 potently reduced murine Th17 differentiation by decreasing Il17a and Rorc gene expression and IL-17 protein production. SB-505124 significantly suppressed IL-6 production by synovial explants. In vivo, SB-505124 reduced Th17 numbers, while increased numbers of Tregs were observed. Despite this skewed Th17/Treg balance, SB-505124 treatment did not result in suppression of joint inflammation and destruction. Blocking TGF-β1 signalling suppresses Th17 differentiation and improves the Th17/Treg balance. However, local SB-505124 treatment does not suppress experimental arthritis.

Purpose: Cartilage damage is one of the central hallmarks of osteoarthritis (OA). Classical imaging modalities like X-ray and magnetic resonance imaging, mainly allow the identification of advanced cartilage damage. Development of cartilage destruction is highly variable among OA patients and the detection of early cartilage damage using sensitive molecular imaging tracers will likely facilitate an early diagnosis and more precise monitoring of disease progression. Moreover, this will allow for non-invasive testing of possible new therapeutics.

Objectives TGF-β is an important growth factor to promote the differentiation of T helper 17 (Th17) and regulatory T cells (Treg). The potential of TGF-β as therapeutic target in T cell-mediated diseases like rheumatoid arthritis (RA) is unclear. We investigated the effect of TGF-β inhibition on murine Th17 differentiation in vitro , on human RA synovial explants ex vivo , and on the development of experimental arthritis in vivo . Methods Murine splenocytes were differentiated into Th17 cells, and the effect of the TGF-βRI inhibitor SB-505124 was studied. Synovial biopsies were cultured in the presence or absence of SB-505124. Experimental arthritis was induced in C57Bl6 mice and treated daily with SB-505124. FACS analysis was performed to measure different T cell subsets. Histological sections were analysed to determine joint inflammation and destruction. Results SB-505124 potently reduced murine Th17 differentiation by decreasing Il7a and Rorc gene expression and IL-17 protein production. SB-505124 significantly suppressed IL-6 production by synovial explants . In vivo, SB-505124 reduced Th17 levels, while increased levels of Tregs were observed. Despite this skewed Th17/Treg balance, SB-505124 treatment did not result in suppression of joint inflammation and destruction. Conclusions Blocking TGF-β signalling suppresses Th17 differentiation and improves the Th17/Treg balance. However, SB-505124 treatment does not suppress experimental arthritis.