Additional file 5 of Lymphocyte activation gene 3 is increased and affects cytokine production in rheumatoid arthritis

Additional file 5: Fig. S5. Effects of rhLAG-3 and anti-LAG3-mAb on cytokine production in the presence of a Gal-3 inhibitor. Production of cytokines in recombinant human LAG-3 treated (A) and anti-LAG3-mAb (B) treated PBMC and SFMC cultures from RA patients (n = 11) with or without addition of Galectin-3 inhibitor. Level of significance is indicated by * < 0.05. Differences were analyzed using the Mann-Whitney test to compare two groups

Additional file 3 of Lymphocyte activation gene 3 is increased and affects cytokine production in rheumatoid arthritis

Additional file 3: Fig. S3. Cellular expression of LAG-3 in PBMCs and SFMCs. A. Cellular expression of LAG-3 on CD3+CD4- T cells from PBMCs and SFMCs in HC (n = 6) and cRA (n = 9) B. Distribution of LAG-3+ cells in relation to CD45R0 presented in the bar graph (n = 9)

Additional file 1 of Lymphocyte activation gene 3 is increased and affects cytokine production in rheumatoid arthritis

Additional file 1: Fig. S1. Gating strategy. A. Gating strategy for CD4/LAG-3, CD45R0/ LAG-3 and CD19/ LAG-3 measurement. B. Gating from FMO LAG-3

Additional file 2 of Lymphocyte activation gene 3 is increased and affects cytokine production in rheumatoid arthritis

Additional file 2: Fig. S2. Correlations of sLAG-3. A. The linear correlation between sLAG-3 levels at baseline and at 12 months (r = 0.7,

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

Abstract 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

Additional file 5 of Lymphocyte activation gene 3 is increased and affects cytokine production in rheumatoid arthritis

Additional file 5: Fig. S5. Effects of rhLAG-3 and anti-LAG3-mAb on cytokine production in the presence of a Gal-3 inhibitor. Production of cytokines in recombinant human LAG-3 treated (A) and anti-LAG3-mAb (B) treated PBMC and SFMC cultures from RA patients (n = 11) with or without addition of Galectin-3 inhibitor. Level of significance is indicated by * < 0.05. Differences were analyzed using the Mann-Whitney test to compare two groups

Additional file 4 of Lymphocyte activation gene 3 is increased and affects cytokine production in rheumatoid arthritis

Additional file 4: Fig. S4. Cellular expression of LAG-3 in CD19+ B cells. A. Cellular expression of LAG-3 on CD19+ B cells on PBMCs and SFMCs from chronic RA patients. B. A representative flow plot from one of the patients

Additional file 4 of Lymphocyte activation gene 3 is increased and affects cytokine production in rheumatoid arthritis

Additional file 4: Fig. S4. Cellular expression of LAG-3 in CD19+ B cells. A. Cellular expression of LAG-3 on CD19+ B cells on PBMCs and SFMCs from chronic RA patients. B. A representative flow plot from one of the patients