Clonally expanded CD38hi cytotoxic CD8 T cells define the T cell infiltrate in checkpoint inhibitor-associated arthritis.

Immune checkpoint inhibitor (ICI) therapies used to treat cancer, such as anti-PD-1 antibodies, can induce autoimmune conditions in some individuals. The T cell mechanisms mediating such iatrogenic autoimmunity and their overlap with spontaneous autoimmune diseases remain unclear. Here, we compared T cells from the joints of 20 patients with an inflammatory arthritis induced by ICI therapy (ICI-arthritis) with two archetypal autoimmune arthritides, rheumatoid arthritis (RA) and psoriatic arthritis (PsA). Single-cell transcriptomic and antigen receptor repertoire analyses highlighted clonal expansion of an activated effector CD8 T cell population in the joints and blood of patients with ICI-arthritis. These cells were identified as CD38hiCD127- CD8 T cells and were uniquely enriched in ICI-arthritis joints compared with RA and PsA and also displayed an elevated interferon signature. In vitro, type I interferon induced CD8 T cells to acquire the ICI-associated CD38hi phenotype and enhanced cytotoxic function. In a cohort of patients with advanced melanoma, ICI therapy markedly expanded circulating CD38hiCD127- T cells, which were frequently bound by the therapeutic anti-PD-1 drug. In patients with ICI-arthritis, drug-bound CD8 T cells in circulation showed marked clonal overlap with drug-bound CD8 T cells from synovial fluid. These results suggest that ICI therapy directly targets CD8 T cells in patients who develop ICI-arthritis and induces an autoimmune pathology that is distinct from prototypical spontaneous autoimmune arthritides.

Lymphocyte activation gene 3 is increased and affects cytokine production in rheumatoid arthritis.

BACKGROUND: Lymphocyte activation gene-3 (LAG-3) inhibits T cell activation and interferes with the immune response by binding to MHC-II. As antigen presentation is central in rheumatoid arthritis (RA) pathogenesis, we studied aspects of LAG-3 as a serological marker and mediator in the pathogenesis of RA. Since Galectin-3 (Gal-3) is described as an additional binding partner for LAG-3, we also aimed to study the functional importance of this interaction. METHODS: Plasma levels of soluble (s) LAG-3 were measured in early RA patients (eRA, n = 99) at baseline and after 12 months on a treat-to-target protocol, in self-reportedly healthy controls (HC, n = 32), and in paired plasma and synovial fluid (SF) from chronic RA patients (cRA, n = 38). Peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) were examined for LAG-3 expression by flow cytometry. The binding and functional outcomes of LAG-3 and Gal-3 interaction were assessed with surface plasmon resonance (SPR) and in cell cultures using rh-LAG3, an antagonistic LAG-3 antibody and a Gal-3 inhibitor. RESULTS: Baseline sLAG-3 in the plasma was increased in eRA compared to HC and remained significantly elevated throughout 12 months of treatment. A high level of sLAG-3 at baseline was associated with the presence of IgM-RF and anti-CCP as well as radiographic progression. In cRA, sLAG-3 was significantly increased in SF compared with plasma, and LAG-3 was primarily expressed by activated T cells in SFMCs compared to PBMCs. Adding recombinant human LAG-3 to RA cell cultures resulted in decreased cytokine secretion, whereas blocking LAG-3 with an antagonistic antibody resulted in increased cytokine secretion. By SPR, we found a dose-dependent binding between LAG-3 and Gal-3. However, inhibiting Gal-3 in cultures did not further change cytokine production. CONCLUSIONS: sLAG-3 in the plasma and synovial fluid is increased in both early and chronic RA patients, particularly in the inflamed joint. High levels of sLAG-3 are associated with autoantibody seropositivity and radiographic progression in eRA, and LAG-3 plays a biologically active role in cRA by decreasing inflammatory cytokine production. This functional outcome is not affected by Gal-3 interference. Our results suggest that LAG-3 is a faceted regulator of inflammation in early and chronic RA.

Validation of an indirect ELISA assay for assessment of autoantibodies against full-length TRIM21 and its individual domains.

Anti-SSA-autoantibodies are common in patients with rheumatologic disease, especially Sjögren's syndrome, systemic lupus erythematosus and rheumatoid arthritis. They consist of both autoantibodies towards Ro60 and Ro52, the latter also known as TRIM21. TRIM21 is an intracellular protein consisting of four domains; PRY/SPRY, Coiled-Coil, B-box and RING. The aim of this study was to establish an indirect ELISA detecting autoantibodies towards both the full-length TRIM21 protein and its four domains. We expressed the five constructs, created, and validated indirect ELISA protocols for each target using plasma from anti-SSA positive patients and healthy controls. Our findings were validated to the clinically used standards. We measured significantly higher levels of autoantibodies towards our full-length TRIM21, and the PRY/SPRY, Coiled-Coil and RING domains in patients compared to healthy controls. No significant difference in the level of autoantibodies were detected against the B-box domain. Our setups had a signal to noise ratio in the range of 30 to 184, and an OD between 2 and 3. Readings did not decline using NaCl of 500 mM as wash, affirming the high binding affinity of the autoantibodies measured. Our protocols allow us to further study the different autoantibodies of anti-SSA positive patients. This creates the possibility to stratify our patients into subgroups regarding autoantibody profile and specific pheno- or endotype.

Galectin-3 interacts with PD-1 and counteracts the PD-1 pathway-driven regulation of T cell and osteoclast activity in Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by joint inflammation and bone erosions. The glycosylated programmed death-1 (PD-1) receptor plays an important role in regulating immune responses and maintaining tolerance. In this study, we focus on two features observed in RA: impaired PD-1 signalling and Galectin-3 (Gal-3) upregulation. We hypothesize that Gal-3 binds PD-1 and PD-1 ligands, potentially contributing to impaired PD-1 signalling. PD-1 and Gal-3 levels in RA synovial fluid (SF) and plasma were evaluated by ELISA. PD-1 and Gal-3 interaction was examined by Surface Plasmon Resonance and ELISA. PD-1, PD-L1 and Gal-3 expression on mononuclear cells from SF and peripheral blood as well as fibroblast-like synoviocytes were examined by flow cytometry. Effects of Gal-3 and PD-L1 on osteoclast formation was evaluated by tartrate-resistant acid phosphatase assay. We show that Gal-3 binds PD-1 and PD-L1. Results demonstrated high expression of PD-1 and Gal-3 on mononuclear cells, especially from SF. Gal-3 inhibited PD-1 signalling when PD-L1 was present. Furthermore, a role of Gal-3 in osteoclast formation was observed in vitro, both directly but also through PD-1:PD-L1 inhibition. Effects of Gal-3 on the PD-1 signalling axis are proposed to be inhibitory, meaning high Gal-3 levels in the complex synovial microenvironment are not desirable in RA. Preventing Gal-3's inhibitory role on PD-1 signalling could, therefore, be a therapeutic target in RA by affecting inflammatory T cell responses and osteoclasts.

T-cell immunoglobulin and mucin domain 3 is upregulated in rheumatoid arthritis, but insufficient in controlling inflammation.

OBJECTIVES: Rheumatoid arthritis (RA) is a chronic autoimmune disease, that involves both pro- and anti-inflammatory mechanisms. The purpose of the present study is to investigate T-cell immunoglobulin and mucin domain 3 (Tim-3) in RA. METHODS: Plasma levels of soluble (s) Tim-3 in early RA (n=98), were followed, to evaluate association with treatment and disease activity, acquired from a prospective collected biobank (clinicaltrials.gov (NCT00660647)). We also investigate the influence of Tim-3 on spontaneous cytokine production in synovial fluid mononuclear cells (SFMC) from RA patients after addition of neutralizing anti-Tim-3's antibodies, either alone or in combination with neutralizing anti-Programmed Cell death protein 1 (PD-1) antibodies. RESULTS: Long-time stimulated CD4 T-cells expressed high levels of Tim-3, but tended to decrease their PD-1 expression. Tim-3 expression was exclusively seen co-expressed with PD-1 by CD3, CD4, CD45RO positive cells in the inflamed RA joint. Addition of neutralizing Tim-3 antibodies increased the secretion of IFNγ and MCP-1, in SFMC cultures from RA. Whereas neutralizing anti-PD-1 antibodies showed a broader impact on cytokine production. Finally, we observed that soluble Tim-3 is increased in plasma and is associated with disease activity in early RA. CONCLUSION: Taken together, our findings indicate disease-suppressive functions of Tim-3 in RA.

Co-Inhibitory Molecules - Their Role in Health and Autoimmunity; Highlighted by Immune Related Adverse Events.

Immune checkpoint receptors are key players in regulating the immune response. They are responsible for both generating an immune response sufficient to kill invading pathogens, balancing the same response, and protecting against tissue destruction or the development of autoimmune events. The central role of the co-inhibitory receptors also referred to as inhibitory immune checkpoints, including PD-1 and CTLA-4 has become especially evident with the cancer treatments targeting these receptors. Blocking these pathways enhances the immune activity, resulting in both an increased chance of cancer clearance, at the same time induction of immune-related adverse events (irAE). Some of these irAE progress into actual autoimmune diseases with autoantibodies and symptoms, undistinguished from the naturally occurring diseases. This review will take advantage of the lessons learned from immune checkpoint blockade and relate this knowledge to our understanding of the same pathways in naturally occurring autoimmune diseases, mainly focusing on rheumatic diseases.

Galectin-3 Decreases 4-1BBL Bioactivity by Crosslinking Soluble and Membrane Expressed 4-1BB.

4-1BB is a T cell costimulatory receptor and a member of the tumor necrosis factor receptor superfamily. Here, we show that Galectin-3 (Gal-3) decreases the cellular response to its ligand (4-1BBL). Gal-3 binds to both soluble 4-1BB (s4-1BB) and membrane-bound 4-1BB (mem4-1BB), without blocking co-binding of 4-1BBL. In plasma, we detected complexes composed of 4-1BB and Gal-3 larger than 100 nm in size; these complexes were reduced in synovial fluid from rheumatoid arthritis. Both activated 4-1BB+ T cells and 4-1BB-transfected HEK293 cells depleted these complexes from plasma, followed by increased expression of 4-1BB and Gal-3 on the cell surface. The increase was accompanied by a 4-fold decrease in TNFα production by the 4-1BBhighGal-3+ T cells, after exposure to 4-1BB/Gal-3 complexes. In RA patients, complexes containing 4-1BB/Gal-3 were dramatically reduced in both plasma and SF compared with healthy plasma. These results support that Gal-3 binds to 4-1BB without blocking the co-binding of 4-1BBL. Instead, Gal-3 leads to formation of large soluble 4-1BB/Gal-3 complexes that attach to mem4-1BB on the cell surfaces, resulting in suppression of 4-1BBL's bioactivity.

The Programmed Death-1 Pathway Counter-Regulates Inflammation-Induced Osteoclast Activity in Clinical and Experimental Settings.

Objective: The programmed death-1 (PD-1) pathway is essential for maintaining self-tolerance and plays an important role in autoimmunity, including rheumatoid arthritis (RA). Here, we investigated how membrane-bound and soluble (s)PD-1 influence bone homeostasis during chronic inflammation, exemplified in RA. Methods: Bone mineral density and bone microstructure were examined in PD-1 and PD-L1 knockout (KO) mice and compared with wild-type (WT) mice. Receptor activator of nuclear factor kappa-B ligand (RANKL) was measured in serum, and the expression examined on activated bone marrow cells. Osteoclast formation was examined in cells from murine spleen and bone marrow and from human synovial fluid cells. sPD-1 was measured in chronic and early (e)RA patients and correlated to markers of disease activity and radiographic scores. Results: PD-1 and PD-L1 KO mice showed signs of osteoporosis. This was supported by a significantly reduced trabecular bone volume fraction and deteriorated microstructure, as well as increased osteoclast formation and an increased RANKL/OPG ratio. The recombinant form of sPD-1 decreased osteoclast formation in vitro, but was closely associated with disease activity markers in eRA patients. Sustained elevated sPD-1 levels indicated ongoing inflammation and were associated with increased radiographic progression. Conclusion: The PD-1 pathway is closely associated with bone homeostasis, and lacking members of this pathway causes a deteriorated bone structure. The immunological balance in the microenvironment determines how the PD-1 pathway regulates osteoclast formation. In eRA patients, sPD-1 may serve as a biomarker, reflecting residual but clinically silent disease activity and radiographic progression.

CXCL13 predicts long-term radiographic status in early rheumatoid arthritis.

OBJECTIVES: Identification of RA patients at a high risk of joint destruction remains challenging. The C-X-C motif chemokine 13 (CXCL13) has previously been suggested as a marker of disease activity in RA. Here, we investigate the potential of plasma CXCL13 as a marker of long-term radiographic status and progression. METHODS: CXCL13 was measured in plasma from treatment-naïve RA patients (n = 158) with an 11-year follow-up. At baseline, clinical and biochemical DASs were obtained; among these CRP, ESR, DAS in 28 joints with CRP (DAS28CRP), number of swollen joints (SJC28) and radiographic status, evaluated by total Sharp score (TSS). Age- and gender-matched healthy controls (HCs) were included. RESULTS: CXCL13 was significantly increased at baseline and decreased during treatment; however, it was not reduced to the level in HCs. At baseline, CXCL13 was associated with both CRP and ESR, but not with other markers of disease activity. Baseline CXCL13 was correlated with both TSS and radiographic progression (ΔTSS) at 11 years. With an 89% probability, levels of CXCL13 above 85 pg/ml predicted the risk of a TSS of 5 or above, after 11 years of treatment. Compared with CRP, DAS28CRP, SJC28 and ACPA status, CXCL13 was superior in predicting 11-year joint destruction. CONCLUSION: In early RA, one single measurement of plasma CXCL13 at baseline is superior to currently used clinical and serological disease markers in the prediction of long-term radiographic status and progression.

Diminished Non-Classical Monocytes in the Blood Associate with Disease Severity in Alcoholic Hepatitis.

OBJECTIVE: Alcoholic hepatitis (AH) holds a high mortality, and vast macrophage infiltration of the liver is involved in the progressive liver injury. No efficient medical treatment exists, and macrophages may be a future treatment target. Here, we examine associations between non-classical monocyte subsets and cell surface markers of migration with disease activity in patients with severe AH. METHODS: We analyzed samples from two cohorts of patients with AH. Cohort 1 included 15 AH patients, followed for 30 days, and 8 healthy controls (HCs). Cohort 2 included 23 AH patients, followed for 90 days, and 9 HCs. Peripheral blood mononuclear cells (PBMCs) from both cohorts were analyzed by flow cytometry. Liver biopsies from cohort 2 were analyzed by RNA sequencing. RESULTS: Circulating non-classical monocytes in all but absent in patients with AH compared to HC in both cohorts (both p<0.0001). The frequency of non-classical monocytes was significantly associated with Maddrey's discriminant function (mDF) (r=-0.79, p=0.0008, cohort 1), Child-Pugh score (CP) (r=-0.56, p=0.03, cohort 1), Model for End-Stage Liver Disease (MELD) (r=-0.54, p=0.02, cohort 2) and C-reactive protein (CRP) (r=-0.51, p=0.027, cohort 2). The surface expression of CD11b was increased on non-classical monocytes in patients with AH compared to HC (p<0.0001) (cohort 1). The mRNA expression of CD11b was increased in liver biopsies in patients with AH compared to HC (cohort 2) (p<0.0001). CONCLUSION: In this study, we describe an almost complete depletion of circulating non-classical monocytes in the blood in two independent cohorts of patients with AH, which may be associated with a possible harmful recruitment of these cells to the liver. These results contribute to a better understanding of the disease, which hopefully can lead to therapies that target the acute inflammatory response leading to severe AH.

Juvenile idiopathic arthritis: lymphocyte activation gene-3 is a central immune receptor in children with oligoarticular subtypes.

BACKGROUND: We investigated the role of inhibitory receptors (IRs) and especially lymphocyte activation gene-3 (LAG-3) in the pathogenesis of oligoarticular juvenile idiopathic arthritis (o-JIA). METHODS: Paired samples of synovial fluid (SF) and plasma and peripheral blood (PBMCs) and synovial fluid mononuclear cells (SFMCs) were collected from o-JIA patients along with their clinical data (n = 24). Plasma from healthy controls (n = 14) and paired SF and plasma samples from five non-arthritic juvenile orthopedic patients (n = 5) served as controls. Spontaneously differentiated fibroblast-like synoviocytes (FLSs) from SFMCs were co-cultured with autologous PBMCs/SFMCs and used as ex vivo disease model. Soluble levels and cellular expressions of IRs together with their functional properties in the ex vivo model were analyzed. RESULTS: In patients with o-JIA, soluble levels of LAG-3 and expression of LAG-3 and T cell immunoglobulin mucin03 (TIM-3) on CD3+CD4+CD45RO+ T cells were increased, especially in SF. Major histocompatibility complex (MHC) class II expression was induced on FLSs when these were co-cultured with autologous PBMCs/SFMCs, together with an increased monocyte chemoattractant protein-1 (MCP-1) production. In PBMC and FLS + PBMC co-cultures, neutralizing antibodies to IRs were added. Only anti-LAG-3 antibodies significantly increased MCP-1 secretion. The addition of agonistic LAG-3 antibody resulted in decreased effector cytokine secretion. CONCLUSIONS: This is the first report comparing the effects of different IRs in o-JIA and suggests that LAG-3 might contribute to the pathogenesis of this disease. IMPACT: This is the first study addressing the role of different co-IRs in o-JIA. We showed that LAG-3 and TIM-3 seem more important in juvenile arthritis in contrast to adult rheumatoid arthritis, where cytotoxic T-lymphocyte-associated antigen-4 and programmed cell death-1 were reported to be more important. We designed an ex vivo disease model for o-JIA, examined the effects of co-IRs in this model, and demonstrated that they might contribute to the pathogenesis of the disease. LAG-3 might play a role in o-JIA pathogenesis and might be a potential therapeutic option for o-JIA patients.

Checkpoint Molecules in Rheumatology-or the Benefits of Being Exhausted.

PURPOSE OF REVIEW: This review will focus on the most common co-inhibitory molecules, emphasizing the importance of these in relation to rheumatic disease. RECENT FINDINGS: Checkpoint molecules are pivotal in determining the outcome of antigen activation. Checkpoint molecules consist of co-stimulatory and co-inhibitory molecules, where the first activates and the latter inhibits the antigen presentation process. Studies show that increased activity of co-inhibitory molecules is associated with a good prognosis in rheumatic diseases. Opposite, when cancer patients are treated with antibodies blocking the inhibitory pathways, autoimmune diseases, including arthritis, develop as immune-related adverse events (IrAE). This emphasizes the importance of these pathways in autoimmune disease. Co-inhibitory molecules are becoming increasingly interesting as future treatment targets in rheumatic conditions. Treatments with antibodies blocking these pathways result in IrAE, often manifesting as autoimmune rheumatic diseases. Therefore, a need to get acquainted with these molecules is growing so we can cope with future challenges in rheumatic diseases.

Programmed death ligand 2 - A link between inflammation and bone loss in rheumatoid arthritis.

OBJECTIVE: Active rheumatoid arthritis (RA) is accompanied by increased appendicular and axial bone loss, closely associated to the degree of inflammation. The programmed death-1 (PD-1) pathway is important for maintaining peripheral tolerance, and its ligand PD-L2 has recently been associated with bone morphogenetic protein activity. Here, we report that PD-L2 plays a central role in RA osteoimmunology. METHODS: Femoral bone mineral density (BMD) and trabecular bone microstructure were evaluated by micro-CT in wild type (WT) and PD-L2-/- mice. Osteoclasts were generated from RA synovial fluid mononuclear cells and peripheral blood monocytes. The effects of recombinant PD-L2, was evaluated by tartrate-resistant acid phosphatase (TRAP) activity and the development of bone erosions in the presence of anti-citrullinated protein antibodies (ACPA). Plasma soluble (s)PD-L2 levels were measured in patients with early (e)RA (n â€‹= â€‹103) treated with methotrexate alone or in combination with the TNF inhibitor Adalimumab. RESULTS: PD-L2-/- mice had a decreased BMD and deteriorated trabecular bone microstructure that was not related to the RANKL/OPG pathway. PD-L2 decreased TRAP activity in osteoclasts and decreased ACPA-induced erosions. In the RA synovial membrane PD-L2 was highly expressed especially in the lining layer and plasma sPD-L2 levels were increased in eRA patients and decreased with treatment. One-year sPD-L2 correlated inversely with erosive progression two years after treatment initiation with methotrexate and placebo. CONCLUSION: PD-L2 regulates bone homeostasis in RA. Our findings provide new insight into the relationship between the immune system and bone homeostasis, and suggest a potential therapeutic target for limiting inflammatory bone loss in RA.

Divergent effects on macrophage biomarkers soluble CD163 and CD206 in axial spondyloarthritis.

The chronic joint inflammation in axial spondyloarthritis (axSpA) is characterized by infiltration of activated macrophages. The haptoglobin-hemoglobin receptor CD163 and the mannose receptor CD206 are strongly expressed on M2c and M2a macrophages, respectively. We measured the soluble forms of the receptors (sCD163 and sCD206) in plasma (PL) in two axSpA cohorts. All patients fulfil the 2009 Assessment of SpondyloArthritis International Society (ASAS) classification criteria for axSpA and/or the 1984 modified New York criteria for ankylosing spondylitis. The first cohort included anti-TNF-α treated patients with active axSpA (n = 30); the second cohort included patients in early disease stages (n = 38). Plasma sCD163 and sCD206 were both within the reference interval of healthy controls (HC), but sCD163 decreased slightly during anti-TNF-α treatment [baseline: 1.49 mg/L (IQR: 1.22-1.77 mg/L, 12 weeks: 1.29 (IQR: 1.09-1.57) mg/L, 20 weeks: 1.25 (IQR: 0.99-1.75) mg/L, 52 weeks: 1.39 (IQR: 1.15-1.78) mg/L], while sCD206 increased [baseline: 0.17 (IQR: 0.13-0.21) mg/L, 12 weeks: 0.19 (0.16-0.23) mg/L, 20 weeks: 0.20 (0.14-0.24) mg/L, 52: 0.19 (IQR: 0.14-0.23) mg/L], pointing toward a shift in polarization of involved macrophages. Plasma levels of sCD206 proved significantly higher in patients with early disease stages and definite radiological sacroiliitis (n = 10). This was not the case for sCD163. A significant increase in response to anti-TNF-α treatment, could suggest sCD206 as a marker of response to anti-TNF-α treatment, however, the potential for the two macrophage markers as diagnostic and prognostic indicators of disease in axSpA is weak.

Extracellular Vesicles Transfer the Receptor Programmed Death-1 in Rheumatoid Arthritis.

INTRODUCTION: Extracellular vesicles (EVs) have been recognized as route of communication in the microenvironment. They transfer proteins and microRNAs (miRNAs) between cells, and possess immunoregulatory properties. However, their role in immune-mediated diseases remains to be elucidated. We hypothesized a role for EVs in the rheumatoid arthritis (RA) joint, potentially involving the development of T cell exhaustion and transfer of the co-inhibitory receptor programmed death 1 (PD-1). METHODS: Synovial fluid mononuclear cells (SFMCs) and peripheral blood mononuclear cells (PBMCs) from RA patients were investigated for PD-1 and other markers of T cell inhibition. EVs were isolated from RA plasma and synovial fluid. In addition, healthy control (HC) and RA PBMCs and SFMCs were cultured to produce EVs. These were isolated and investigated by immunogold electron microscopy (EM) and also co-cultured with lymphocytes and PD-1 negative cells to investigate their functions. Finally, the miRNA expression profiles were assessed in EVs isolated from RA and HC cell cultures. RESULTS: Cells from the RA joint expressed several T cell co-inhibitory receptors, including PD-1, TIM-3, and Tigit. ELISA demonstrated the presence of PD-1 in EVs from RA plasma and synovial fluid. Immunogold EM visualized PD-1 expression by EVs. Co-culturing lymphocytes and the PD-1 negative cell line, U937 with EVs resulted in an induction of PD-1 on these cells. Moreover, EVs from RA PBMCs increased proliferation in lymphocytes when co-cultured with these. All EVs contained miRNAs associated with PD-1 and other markers of T cell inhibition and the content was significantly lower in EVs from RA PBMCs than HC PBMCs. Stimulation of the cells increased the miRNA expression. However, EVs isolated from stimulated RA SFMCs did not change their miRNA expression profile to the same extend. CONCLUSION: EVs carrying both the PD-1 receptor and miRNAs associated with T cell inhibition were present in RA cell cultures. Upon stimulation, these miRNAs failed to be upregulated in EVs from RA SFMCs. This was in line with increased expression of T cell co-inhibitory markers on SFMCs. In conclusion, we suggest EVs to play a significant role in the RA microenvironment, potentially favoring the progression of T cell exhaustion.

Soluble CD206 plasma levels in rheumatoid arthritis reflect decrease in disease activity.

Rheumatoid arthritis (RA) is characterized by chronic joint inflammation and infiltration by activated macrophages. TNFα is a central mediator in this process. The mannose receptor, CD206, is a scavenger receptor expressed by M2A-macrophages and dendritic cells. It is involved in collagen internalization and degradation. The soluble form has been suggested as a biomarker of M2A-macrophage activation. The aim of this study was to investigate sCD206 plasma levels in early RA patients initiating anti-TNFα treatment. Plasma levels of sCD206 were measured by ELISA in samples from 155 early RA patients with an average symptom duration of 3 months. Patients were randomized to 12 months' methotrexate and placebo (PLA) or methotrexate and adalimumab (ADA) treatment, followed by open-label treatment with disease-modifying anti-rheumatic drugs (DMARD) and if needed, ADA. Disease activity was assessed at baseline and after 3, 6, 12 and 24 months. Baseline plasma level of sCD206 in treatment naïve RA patients was 0.33 mg/L (CI: 0.33-0.38 mg/L) corresponding to the upper part of the reference interval for healthy controls (0.10-0.43 mg/L). In the PLA group, sCD206 levels decreased after 3 months, but did not differ from baseline after 6 months. In the ADA group, however, levels remained lower than baseline throughout the treatment period. In conclusion, initially, plasma sCD206 in early RA patients decreased in accordance with disease activity and initiation of DMARD treatment. Treatment with anti-TNFα preserved this decrease throughout the study period.

Vitamin D increases programmed death receptor-1 expression in Crohn's disease.

BACKGROUND: Vitamin D modulates inflammation in Crohn's disease (CD). Programmed death (PD)-1 receptor contributes to the maintenance of immune tolerance. Vitamin D might modulate PD-1 signalling in CD. AIM: To investigate PD-1 expression on T cell subsets in CD patients treated with vitamin D or placebo. METHODS: We included 40 CD patients who received 1200 IU vitamin D3 for 26 weeks or placebo and eight healthy controls. Peripheral blood mononuclear cells (PBMCs) and plasma were isolated at baseline and week 26. The expressions of PD-1, PD-L1, and surface activation markers were analysed by flow cytometry. Soluble PD-1 plasma levels were measured by ELISA. RESULTS: PD-1 expression upon T cell stimulation was increased in CD4+CD25+int T cells in vitamin D treated CD patients from 19% (range 10 - 39%) to 29% (11 - 79%)(p = 0.03) compared with placebo-treated patients. Vitamin D treatment, but not placebo, decreased the expression of the T cell activation marker CD69 from 42% (31 - 62%) to 33% (19 - 54%)(p = 0.01). Soluble PD-1 levels were not influenced by vitamin D treatment. CONCLUSIONS: Vitamin D treatment increases CD4+CD25+int T cells ability to up-regulate PD-1 in response to activation and reduces the CD69 expression in CD patients.

Soluble programmed death-1 levels are associated with disease activity and treatment response in patients with autoimmune hepatitis.

PURPOSE: Autoimmune hepatitis (AIH) is a chronic liver disease caused by impaired immune regulation. Programmed death-1 (PD-1) is an inhibitory receptor mainly expressed by T cells and with its ligands, PD-L1 and PD-L2 present on antigen-presenting cells. We hypothesised the PD-1 axis to be impaired in AIH and investigated systemic levels of soluble(s) PD-1 and T cells ability to up-regulate PD-1 following in vitro activation in AIH patients. MATERIALS AND METHODS: We included 67 AIH patients; 9 with active disease, 31 responders and 27 incomplete-responders to standard therapy. Forty-seven healthy controls (HC) were included for comparison. Soluble PD-1 was measured by enzyme-linked immunosorbent assay. The PD-1 expression on T cells was measured using flow cytometry before and after 48-h stimulation in vitro with CD3/CD28 in 13 AIH patients and 10 HC. RESULTS: Soluble PD-1 was significantly elevated in AIH patients with active disease [0.24 ng/mL (range 0.16-0.28)] and in incomplete responders to standard therapy [0.17 (0.11-0.22)] compared with responders [0.11 (0.08-0.16), p = .008 and p = .01, respectively] and HC [0.12 (0.05-0.16), p = .02, both]. Following in vitro activation, PD-1 was significantly up-regulated (3.3-fold) on CD4+ T cells from AIH patients compared with HC (1.5-fold) (p = .0006). CONCLUSIONS: AIH patients with active disease and incomplete response to standard treatment have similarly increased sPD-1 levels. Further, AIH patients have increased ability to up-regulate PD-1 following in vitro activation. Together these data suggests an impaired PD-1 axis in AIH.

The interleukin-20 receptor axis in early rheumatoid arthritis: novel links between disease-associated autoantibodies and radiographic progression.

BACKGROUND: Rheumatoid arthritis (RA) is often characterized by the presence of rheumatoid factor, anti-citrullinated protein antibodies, and bone erosions. Current therapies can compromise immunity, leading to risk of infection. The interleukin-20 receptor (IL-20R) axis comprising IL-19, IL-20, and IL-24 and their shared receptors activates tissue homeostasis processes but not the immune system. Consequently, modulation of the IL-20R axis may not lead to immunosuppression, making it an interesting drug target. We evaluated the role of the IL-20R axis in RA and associations between plasma cytokine levels and clinical disease. METHODS: Plasma IL-19, IL-20, and IL-24 levels were measured in early RA patients during a treat-to-target strategy by enzyme-linked immunosorbent assays. The IL-20R1 and IL-22R1 levels in paired peripheral blood mononuclear cells and synovial fluid mononuclear cells from a different cohort of RA patients were evaluated by flow cytometry and confocal microscopy. Monocytes/macrophages were stimulated with heat-aggregated human immunoglobulin immune complexes and immune complexes containing citrullinated fibrinogen, and osteoclasts were incubated with IL-19, IL-20, and IL-24. RESULTS: The plasma concentrations of IL-20 and IL-24 (but not IL-19) were increased in early RA patients compared with healthy controls (both P < 0.002) and decreased after 6 months of treatment (both P < 0.0001). The expression of IL-22R1 (but not IL-20R1) was increased on monocytes from RA synovial fluid compared with monocytes from both RA and healthy control peripheral blood. The plasma concentrations of IL-20 and IL-24 were increased in rheumatoid factor and anti-citrullinated protein antibody positive compared with negative early RA patients (all P < 0.0001). Immune complexes stimulated the production of the IL-20R cytokines by monocytes/macrophages. Increased baseline plasma concentrations of IL-20 and IL-24 were associated with Sharp-van der Heijde score progression after 24 months (Spearman's rho = 0.19 and 0.26, both P < 0.05) in the early RA patients. The IL-22R1 was expressed by osteoclast precursors and in multinucleated osteoclasts. IL-20 and IL-24 increased the secretion of monocyte chemoattractant protein 1 by these cells. CONCLUSIONS: This study suggests that IL-20 and IL-24 link RA-associated autoantibodies with radiographic progression via the IL-22R1. Modulation of this axis holds promise as feasible anti-erosive treatment modalities in seropositive RA.