Surface-expressed LLT1 is found on SF monocytes.

<p>Monocytes from A) peripheral blood and B) synovial fluid were gated based on forward and side scatter characteristics. After excluding CD3+ and CD56+ lymphocytes, monocytes were gated based on CD14 and CD16 expression. The frequency of LLT1+ cells was assessed within the total monocyte population. C) The frequency of LLT1+ monocytes and D) LLT1 MFI from paired samples of PB and SF (n = 14). Mouse monoclonal anti-LLT1 antibody, clone 402659 (R&D Systems) was used.</p

sLLT1 is increased in the serum of SAP, early and late-stage RA and SpA patients.

<p>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.</p

Immunohistochemical detection of LLT1 expression in RA ST cells.

<p>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.</p

Clinical and demographical characteristics of the subjects included in the study.

<p>HC = healthy controls; SAP = seropositive arthralgia patients; RA = rheumatoid arthritis; SpA = spondyloarthropathy; CRP = C-reactive protein; ESR = erythrocyte sedimentation rate; DAS28 = disease activity score 28; RF = rheumatoid factor; anti-CCP = anti-cyclic citrullinated proteins antibodies; BASDAI = bath ankylosing spondylitis disease activity index; ASDAS = ankylosing spondylitis disease activity score; nd = not defined; na = not applicable.</p><p>Clinical and demographical characteristics of the subjects included in the study.</p

Flow-cytometric detection of LLT1 expression in RA ST cells.

<p>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.</p

Altered dynamics of circulating CD4+CD161+ T-cells in seropositive arthralgia patients and in newly diagnosed RA patients.

<p>(A) Representative dot plots showing proportions of CD4+CD161+ T-cells in the study groups. The absolute number (B) and the frequency (C) of CD161+ cells within CD4+ T-cells from healthy donors (n=20), SAP (n=26) and early RA patients (n=35; Mann-Whitney test). Horizontal line in the box represents the median value. Boxes represent interquartile range and whiskers represent the actual range. Symbols outwith the boxes represent outliers. The correlation between the absolute number of CD161+ cells within CD4+ T-cells and (D) CRP (mg/l), (E) DAS28, (F) SJC28 and (G) SJC66 in RA patients (n=30; Spearman coefficient analysis). Frequency of CD4+CD161+ -cells expressing (H) IL-17, (I) IL-17 and IFN-γ or (J) IFN-γ alone within total CD4+ from HC (n=6), SAP (n=6) and early RA patients (n=6; Mann-Whitney test). Statistical significance is indicated as * p ≤ 0.05, ** p ≤ 0.001, CRP= C-reactive protein, DAS= disease activity score, SJC= swollen joint count.</p

Synovial fluid CD4+CD161+ T-cells demonstrate a Th1 phenotype.

<p>Paired samples of PB and SF from late-stage RA patients (n=6) were stimulated using PMA/ionomycin in the presence of BFA. The frequency of CD4+CD161+ T lymphocytes producing (A) IL-17, (B) both IL-17 and IFN-γ (C) IFN-γ, or (D) TNF-α was assessed (Wilcoxon matched pairs test). Statistical significance is indicated as * p ≤ 0.05.</p

Circulating CD4+CD161+ T-cells normalize following MTX treatment.

<p>(A) DAS28 and (B) the absolute number of CD4+CD161+ T-cells from RA patients at baseline (newly diagnosed; n=30), after 3 months (n=22) and 6 months (n=7) of MTX treatment (GEE analysis); (C) Comparison of the absolute number of CD4+CD161+ T-cells between HC (n=20) and RA patients at baseline (n=30; Mann- Whitney test) or RA patients at baseline (n=30) and RA patients after 3 months (n=26) or 6 months (n=12) of MTX treatment (Wilcoxon matched pairs test). Horizontal line in the box represents the median value. Boxes represent interquartile ranges and whiskers represent the actual range. Statistical significance is indicated as * p ≤ 0.05, ** p ≤ 0.001.</p

CD4+CD161+ T-cells are readily detected at the level of the joint in newly diagnosed and in late-stage RA.

<div><p>Detection of T cells expressing CD161 in ST obtained from a newly diagnosed RA patient using IHC on consecutive cryostat sections (Krenn score = 4 [37]). A representative example is shown (magnification 40x). Blanc (A), CD3 (B), CD4 (C), CD161 (D).</p> <p>Analysis of the number of CD4+CD161+ T-cells from paired PB and SF and non-paired PB and ST from late-stage RA. Frequency of CD161+ cells within (E, F) CD4+ and (G, H) CD8+ T-cells were compared between paired samples of PB and SF (n=6; Wilcoxon matched pairs test) or PB and enzyme-digested ST (n=4; Mann-Whitney test).</p></div

Monoclonal antibodies.

<p>Overview of fluorochrome-conjugated monoclonal antibodies applied in the study.</p><p>Monoclonal antibodies.</p