Expression of Lectin-Like Transcript 1, the Ligand for CD161, in Rheumatoid Arthritis

Precursor Th17 lineage cells expressing CD161 are implicated in Rheumatoid Arthritis (RA) pathogenesis. CD4+CD161+ T-cells accumulate in RA joints and may acquire a non classical Th1 phenotype. The endogenous ligand for CD161 is lectin-like transcript 1 (LLT1). CD161/LLT1 ligation may co-stimulate T-cell IFN-γ production. We investigated the presence and identity of LLT1-expressing cells in RA synovial fluid (SF) and synovial tissue (ST). We also assessed levels of soluble LLT1 (sLLT1) in different phases of RA development.Paired samples of peripheral blood mononuclear cells (MC) and SFMC (n = 14), digested ST cells (n = 4) and ST paraffin sections (n = 6) from late-stage RA were analyzed for LLT1 expression by flow cytometry and immunohistochemistry. sLLT1 was measured using a sandwich ELISA. Sera and SF from late-stage RA (n = 26), recently diagnosed RA patients (n = 39), seropositive arthralgia patients (SAP, n = 31), spondyloarthropathy patients (SpA, n = 26) and healthy controls (HC, n = 31) were assayed.In RA SF, LLT1 was expressed by a small proportion of monocytes. In RA ST, LLT1-expressing cells were detected in the lining, sublining layer and in areas with infiltrates. The LLT1 staining pattern overlapped with the CD68 staining pattern. FACS analysis of digested ST confirmed LLT1 expression by CD68+ cells. Elevated systemic sLLT1 was found in all patient groups.In RA joints, LLT1 is expressed by cells of the monocyte/macrophage lineage. Serum levels of sLLT1 were increased in all patient groups (patients with early- and late-stage RA, seropositive arthralgia and spondyloarthropathy) when compared to healthy subjects

T cell subsets: An immunological biomarker to predict progression to clinical arthritis in ACPA-positive individuals

Objectives Anticitrullinated protein antibody (ACPA)+ individuals with non-specific musculoskeletal symptoms are at risk of inflammatory arthritis (IA). This study aims to demonstrate the predictive value of T cell subset quantification for progression towards IA and compare it with previously identified clinical predictors of progression. Methods 103 ACPA+ individuals without clinical synovitis were observed 3-monthly for 12 months and then as clinically indicated. The end point was the development of IA. Naïve, regulatory T cells (Treg) and inflammation related cells (IRCs) were quantified by flow cytometry. Areas under the ROC curve (AUC) were calculated. Adjusted logistic regressions and Cox proportional hazards models for time to progression to IA were constructed. Results Compared with healthy controls (age adjusted where appropriate), ACPA+ individuals demonstrated reduced naïve (22.1% of subjects) and Treg (35.8%) frequencies and elevated IRC (29.5%). Of the 103 subjects, 48(46.6%) progressed. Individually, T cell subsets were weakly predictive (AUC between 0.63 and 0.66), although the presence of 2 T cell abnormalities had high specificity. Three models were compared: model-1 used T cell subsets only, model-2 used previously published clinical parameters, model-3 combined clinical data and T cell data. Model-3 performed the best (AUC 0.79 (95% CI 0.70 to 0.89)) compared with model-1 (0.75 (0.65 to 0.86)) and particularly with model-2 (0.62 (0.54 to 0.76)) demonstrating the added value of T cell subsets. Time to progression differed significantly between high-risk, moderate-risk and low-risk groups from model-3 (p=0.001, median 15.4 months, 25.8 months and 63.4 months, respectively). Conclusions T cell subset dysregulation in ACPA+ individuals predates the onset of IA, predicts the risk and faster progression to IA, with added value over previously published clinical predictors of progression

Selective, autoantibody-immune complex mediated proportional and functional changes of specific NK-cell subsets in early seropositive but not seronegative rheumatoid arthritis

Background: Despite substantial data demonstrating NK-cell impairment in rheumatoid arthritis (RA), the exact role of NK-cells in RA immunopathogenesis remains unclear. Objectives: We studied the involvement of CD56dim and CD56bright NK-cells in the early stages of RA development to elucidate their role in the pathogenesis of RA. Methods: The study included newly diagnosed RA patients and seropositive arthralgia patients (SAP), representing subjects at risk of progression to RA. Whole blood samples obtained from disease modifying anti rheumatic drug (DMARD)-free RA patients at the time of diagnosis (autoantibody -positive [SP RA, n=45] and -negative [SN RA, n=12]); SAP (n=30) and healthy controls (HC, n=41), were studied to assess absolute numbers and proportions of T-cells, B-cells and NK-cells. Peripheral blood mononuclear cells (PBMC) were used for both phenotypical and functional analyses of NK-cells in the studied groups. Results: Patients (SP RA and SAP) positive for anti-cyclic citrullinated protein antibodies (ACPA) and/or rheumatoid factor (RF) showed decreased total NKcell numbers. Also, NK-cells from SP RA showed a decreased potency for IFN-γ production. NK-cell subset analysis demonstrated a specific decrease of CD56dim, but not CD56bright NK-cells in SP RA and SAP. Differential NK-cell alterations in seropositive and seronegative patients prompted the investigation of the role of CD16 (FcγRIIIa) triggering in apoptosis induction and cytokine expression by CD56dim and CD56bright NK-cell subsets. CD16 triggering in vitro induced apoptosis of CD56dim, but not CD56bright NK-cells, and was augmented by the addition of IL-2. Also, CD16 triggering in the presence of IL-2 stimulated IFN-γ expression by CD56dim NK-cells. Conclusions: The decline of CD56dim NK-cells is an early feature in the development of seropositive, but not seronegative RA. The decline of CD56dim NK-cells in SAP and SP RA and observed in vitro apoptosis of CD56dim NK-cells upon CD16 triggering, suggests a functional role of IgG-containing autoantibody (ACPA and/or RF)-immune complexes in CD16-mediated apoptosis. Moreover, CD16 triggering leads to an increased cytokine response by CD56dim NK-cells, which may contribute to the proinflammatory state in both SP RA and SAP

Expression of lectin-like transcript 1, the ligand for CD161, in rheumatoid arthritis

Background: Precursor Th17 lineage cells expressing CD161 are implicated in rheumatoid arthritis pathogenesis. CD4+CD161+ T cells were found to accumulate in RA synovial fluid and tissue where they may acquire a non-classical T-helper 1 phenotype. The sole endogenous ligand for CD161 is lectin-like transcript 1 (LLT1). Previously, the LLT1-CD161 interaction was reported to co-stimulate T cell effector functions and to enhance IFN-γ production. This prompted us to investigate whether LLT1 is upregulated in the disease-affected joints. Objectives: We investigated the presence and identity of LLT1-expressing cells in RA synovial fluid and synovial tissue. Methods: Paired samples of peripheral blood and synovial fluid mononuclear cells (n=14) and digested synovial tissue cells (n=4) from late-stage rheumatoid arthritis patients were analyzed for LLT1 expression by flow cytometry. Paraffinembedded synovial tissue sections (n=6) were used for the immunohistochemical detection of LLT1. Results: In rheumatoid arthritis synovial fluid LLT1 expression was found upregulated in a subset of monocytes with a more differentiated, mature phenotype. In rheumatoid arthritis synovial tissue, LLT1-expressing cells were detected in the lining layer, sublining layer and in areas with lymphoid infiltrates. The LLT1 staining pattern resembled the pattern of CD68 staining. Flow cytometric analysis of digested synovial tissue confirmed LLT1 expression on CD68+ cells. Conclusions: This is the first study showing that surface-expressed LLT1 is present at the site of local inflammation in RA. The finding of LLT1 expression by macrophages in synovial tissue suggests potential crosstalk with CD161+ T-cells. Ligation of CD161-LLT1 on CD4 T cells and macrophages respectively, may contribute to modulation of their function at the level of the joint