Fig 2: sLLT1 is increased in the serum of SAP, early and late-stage RA and SpA patients.A) Sera from HC (n = 31), SAP (n = 31), early RA patients (n = 39) and late RA patients (n = 26) and SpA patients (n = 26) were used to quantify the levels of soluble LLT1 using sandwich ELISA. Horizontal lines represent the mean value. Unpaired t test was used. B) Paired SF samples were used to compare the level of soluble LLT1 in PB and SF of long-standing RA (n = 26; Wilcoxon matched pairs test). Statistical significance is indicated as * for p <0.05, ** for p <0.001, and *** for p <0.0001. Rabbit polyclonal anti-LLT1 antibodies provided with a commercially available ELISA (MyBiosource) were used.

Fig 3: Flow-cytometric detection of LLT1 expression in RA ST cells.A) Percentages of CD3+ T-cells, CD19+ B-cells and CD68+ macrophages detected within the live cells gate of digested ST cells with flow cytometry. Briefly, necrotic cells were gated out based on the staining with the Fixable Viability Stain dye. Within the live cells gate lymphocytes and macrophages were gated based on FSC/SSC characteristics and CD68 expression, respectively. Within the lymphocyte gate T-cells and B-cells were gated based on CD3 and CD19 expression, respectively. Representative histogram overlays showing frequencies of B) LLT1+ and C) CD161+ cells within the populations of CD3+, CD19+ or CD68+ cells when compared to isotype control. D) Graphs show the percentages of LLT1 and CD161+ cells. Data from 4 independent donors were pooled. Bars represent the median value ± interquartile range. Mouse monoclonal anti-LLT1 antibody, clone 402659 (R&D Systems) was used.

Fig 4: Immunohistochemical detection of LLT1 expression in RA ST cells.To study surface-expressed LLT1 at the site of inflammation, 6 synovial tissue biopsies obtained from hand, shoulder or knee joints from long-standing, treated RA patients who underwent joint replacement surgery or synovectomy were processed for immunohistochemistry. Representative pictures showing immunohistochemical staining of consecutive tissue slides from 2 late-stage RA patients stained with antibodies against LLT1, CD68, CD3 and CD20cy (A,B). Graphs in C depict the results of the semiquantitative scoring (mean + SD) performed by 3 independent researchers. Scoring of the staining of all the markers was performed according to a 4-point scale: 0 = no positive cells; 1 = <5% positive cells; 2 = 5–50% positive cells; 3 = >50% positive cells. Three to five different pictures of each slide section (n = 6 different sections per biopsy) were taken. In each picture the lining, sublining, lymphoid infiltrate area’s (defined based on CD3 and CD20cy staining) and blood vessels were scored separately for the expression of LLT1, CD68, CD3 and CD20cy. Mouse monoclonal anti-LLT1, clone 4C7 (Abnova) was used.