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

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

image_7_Impact of Aging on the Frequency, Phenotype, and Function of CD161-Expressing T Cells.PDF

<p>Immune-aging is associated with perturbed immune responses in the elderly. CD161-expressing T cells, i.e., the previously described subsets of CD161⁺ CD4⁺ T cells, CD161^high CD8⁺ T cells, and CD161^int CD8⁺ T cells, are highly functional, pro-inflammatory T cells. These CD161-expressing T cells are critical in immunity against microbes, while possibly contributing to autoimmune diseases. So far, little is known about the impact of aging on the frequency, phenotype, and function of these CD161-expressing T cells. In the current study, we investigated the impact of aging on CD161⁺ CD4⁺ T cells, CD161^high CD8⁺ T cells, and CD161^int CD8⁺ T cells in peripheral blood samples of 96 healthy subjects (age 20–84). Frequencies of CD161⁺ CD4⁺ T cells and CD161^int CD8⁺ T cells were stable with aging, whereas frequencies of CD161^high CD8⁺ T cells declined. Although CD161^high CD8⁺ T cells were mostly T cell receptor-Vα7.2⁺ mucosal-associated invariant T cells, CD161 expressing CD4⁺ and CD8⁺ T cells showed a limited expression of markers for gamma–delta T cells or invariant natural killer (NK) T cells, in both young and old subjects. In essence, CD161-expressing T cells showed a similar memory phenotype in young and old subjects. The expression of the inhibitory NK receptor KLRG1 was decreased on CD161⁺ CD4⁺ T cells of old subjects, whereas the expression of other NK receptors by CD161-expressing T cells was unaltered with age. The expression of cytotoxic effector molecules was similar in CD161^high and CD161^int CD8⁺ T cells of young and old subjects. The ability to produce pro-inflammatory cytokines was preserved in CD161^high and CD161^int CD8⁺ T cells of old subjects. However, the percentages of IFN-γ⁺ and interleukin-17⁺ cells were significantly lower in CD161⁺ CD4⁺ T cells of old individuals than those of young individuals. In addition, aging was associated with a decrease of nonclassic T helper 1 cells, as indicated by decreased percentages of CD161-expressing cells within the IFN-γ⁺ CD4⁺ T cell compartment of old subjects. Taken together, aging is associated with a numerical decline of circulating CD161^high CD8⁺ T cells, as well as a decreased production of pro-inflammatory cytokines by CD161⁺ CD4⁺ T cells. These aging-associated changes could contribute to perturbed immunity in the elderly.</p

image_5_Impact of Aging on the Frequency, Phenotype, and Function of CD161-Expressing T Cells.PDF

<p>Immune-aging is associated with perturbed immune responses in the elderly. CD161-expressing T cells, i.e., the previously described subsets of CD161⁺ CD4⁺ T cells, CD161^high CD8⁺ T cells, and CD161^int CD8⁺ T cells, are highly functional, pro-inflammatory T cells. These CD161-expressing T cells are critical in immunity against microbes, while possibly contributing to autoimmune diseases. So far, little is known about the impact of aging on the frequency, phenotype, and function of these CD161-expressing T cells. In the current study, we investigated the impact of aging on CD161⁺ CD4⁺ T cells, CD161^high CD8⁺ T cells, and CD161^int CD8⁺ T cells in peripheral blood samples of 96 healthy subjects (age 20–84). Frequencies of CD161⁺ CD4⁺ T cells and CD161^int CD8⁺ T cells were stable with aging, whereas frequencies of CD161^high CD8⁺ T cells declined. Although CD161^high CD8⁺ T cells were mostly T cell receptor-Vα7.2⁺ mucosal-associated invariant T cells, CD161 expressing CD4⁺ and CD8⁺ T cells showed a limited expression of markers for gamma–delta T cells or invariant natural killer (NK) T cells, in both young and old subjects. In essence, CD161-expressing T cells showed a similar memory phenotype in young and old subjects. The expression of the inhibitory NK receptor KLRG1 was decreased on CD161⁺ CD4⁺ T cells of old subjects, whereas the expression of other NK receptors by CD161-expressing T cells was unaltered with age. The expression of cytotoxic effector molecules was similar in CD161^high and CD161^int CD8⁺ T cells of young and old subjects. The ability to produce pro-inflammatory cytokines was preserved in CD161^high and CD161^int CD8⁺ T cells of old subjects. However, the percentages of IFN-γ⁺ and interleukin-17⁺ cells were significantly lower in CD161⁺ CD4⁺ T cells of old individuals than those of young individuals. In addition, aging was associated with a decrease of nonclassic T helper 1 cells, as indicated by decreased percentages of CD161-expressing cells within the IFN-γ⁺ CD4⁺ T cell compartment of old subjects. Taken together, aging is associated with a numerical decline of circulating CD161^high CD8⁺ T cells, as well as a decreased production of pro-inflammatory cytokines by CD161⁺ CD4⁺ T cells. These aging-associated changes could contribute to perturbed immunity in the elderly.</p

image_1_Impact of Aging on the Frequency, Phenotype, and Function of CD161-Expressing T Cells.PDF

<p>Immune-aging is associated with perturbed immune responses in the elderly. CD161-expressing T cells, i.e., the previously described subsets of CD161⁺ CD4⁺ T cells, CD161^high CD8⁺ T cells, and CD161^int CD8⁺ T cells, are highly functional, pro-inflammatory T cells. These CD161-expressing T cells are critical in immunity against microbes, while possibly contributing to autoimmune diseases. So far, little is known about the impact of aging on the frequency, phenotype, and function of these CD161-expressing T cells. In the current study, we investigated the impact of aging on CD161⁺ CD4⁺ T cells, CD161^high CD8⁺ T cells, and CD161^int CD8⁺ T cells in peripheral blood samples of 96 healthy subjects (age 20–84). Frequencies of CD161⁺ CD4⁺ T cells and CD161^int CD8⁺ T cells were stable with aging, whereas frequencies of CD161^high CD8⁺ T cells declined. Although CD161^high CD8⁺ T cells were mostly T cell receptor-Vα7.2⁺ mucosal-associated invariant T cells, CD161 expressing CD4⁺ and CD8⁺ T cells showed a limited expression of markers for gamma–delta T cells or invariant natural killer (NK) T cells, in both young and old subjects. In essence, CD161-expressing T cells showed a similar memory phenotype in young and old subjects. The expression of the inhibitory NK receptor KLRG1 was decreased on CD161⁺ CD4⁺ T cells of old subjects, whereas the expression of other NK receptors by CD161-expressing T cells was unaltered with age. The expression of cytotoxic effector molecules was similar in CD161^high and CD161^int CD8⁺ T cells of young and old subjects. The ability to produce pro-inflammatory cytokines was preserved in CD161^high and CD161^int CD8⁺ T cells of old subjects. However, the percentages of IFN-γ⁺ and interleukin-17⁺ cells were significantly lower in CD161⁺ CD4⁺ T cells of old individuals than those of young individuals. In addition, aging was associated with a decrease of nonclassic T helper 1 cells, as indicated by decreased percentages of CD161-expressing cells within the IFN-γ⁺ CD4⁺ T cell compartment of old subjects. Taken together, aging is associated with a numerical decline of circulating CD161^high CD8⁺ T cells, as well as a decreased production of pro-inflammatory cytokines by CD161⁺ CD4⁺ T cells. These aging-associated changes could contribute to perturbed immunity in the elderly.</p

image_2_Impact of Aging on the Frequency, Phenotype, and Function of CD161-Expressing T Cells.PDF

<p>Immune-aging is associated with perturbed immune responses in the elderly. CD161-expressing T cells, i.e., the previously described subsets of CD161⁺ CD4⁺ T cells, CD161^high CD8⁺ T cells, and CD161^int CD8⁺ T cells, are highly functional, pro-inflammatory T cells. These CD161-expressing T cells are critical in immunity against microbes, while possibly contributing to autoimmune diseases. So far, little is known about the impact of aging on the frequency, phenotype, and function of these CD161-expressing T cells. In the current study, we investigated the impact of aging on CD161⁺ CD4⁺ T cells, CD161^high CD8⁺ T cells, and CD161^int CD8⁺ T cells in peripheral blood samples of 96 healthy subjects (age 20–84). Frequencies of CD161⁺ CD4⁺ T cells and CD161^int CD8⁺ T cells were stable with aging, whereas frequencies of CD161^high CD8⁺ T cells declined. Although CD161^high CD8⁺ T cells were mostly T cell receptor-Vα7.2⁺ mucosal-associated invariant T cells, CD161 expressing CD4⁺ and CD8⁺ T cells showed a limited expression of markers for gamma–delta T cells or invariant natural killer (NK) T cells, in both young and old subjects. In essence, CD161-expressing T cells showed a similar memory phenotype in young and old subjects. The expression of the inhibitory NK receptor KLRG1 was decreased on CD161⁺ CD4⁺ T cells of old subjects, whereas the expression of other NK receptors by CD161-expressing T cells was unaltered with age. The expression of cytotoxic effector molecules was similar in CD161^high and CD161^int CD8⁺ T cells of young and old subjects. The ability to produce pro-inflammatory cytokines was preserved in CD161^high and CD161^int CD8⁺ T cells of old subjects. However, the percentages of IFN-γ⁺ and interleukin-17⁺ cells were significantly lower in CD161⁺ CD4⁺ T cells of old individuals than those of young individuals. In addition, aging was associated with a decrease of nonclassic T helper 1 cells, as indicated by decreased percentages of CD161-expressing cells within the IFN-γ⁺ CD4⁺ T cell compartment of old subjects. Taken together, aging is associated with a numerical decline of circulating CD161^high CD8⁺ T cells, as well as a decreased production of pro-inflammatory cytokines by CD161⁺ CD4⁺ T cells. These aging-associated changes could contribute to perturbed immunity in the elderly.</p

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

image_6_Impact of Aging on the Frequency, Phenotype, and Function of CD161-Expressing T Cells.PDF

<p>Immune-aging is associated with perturbed immune responses in the elderly. CD161-expressing T cells, i.e., the previously described subsets of CD161⁺ CD4⁺ T cells, CD161^high CD8⁺ T cells, and CD161^int CD8⁺ T cells, are highly functional, pro-inflammatory T cells. These CD161-expressing T cells are critical in immunity against microbes, while possibly contributing to autoimmune diseases. So far, little is known about the impact of aging on the frequency, phenotype, and function of these CD161-expressing T cells. In the current study, we investigated the impact of aging on CD161⁺ CD4⁺ T cells, CD161^high CD8⁺ T cells, and CD161^int CD8⁺ T cells in peripheral blood samples of 96 healthy subjects (age 20–84). Frequencies of CD161⁺ CD4⁺ T cells and CD161^int CD8⁺ T cells were stable with aging, whereas frequencies of CD161^high CD8⁺ T cells declined. Although CD161^high CD8⁺ T cells were mostly T cell receptor-Vα7.2⁺ mucosal-associated invariant T cells, CD161 expressing CD4⁺ and CD8⁺ T cells showed a limited expression of markers for gamma–delta T cells or invariant natural killer (NK) T cells, in both young and old subjects. In essence, CD161-expressing T cells showed a similar memory phenotype in young and old subjects. The expression of the inhibitory NK receptor KLRG1 was decreased on CD161⁺ CD4⁺ T cells of old subjects, whereas the expression of other NK receptors by CD161-expressing T cells was unaltered with age. The expression of cytotoxic effector molecules was similar in CD161^high and CD161^int CD8⁺ T cells of young and old subjects. The ability to produce pro-inflammatory cytokines was preserved in CD161^high and CD161^int CD8⁺ T cells of old subjects. However, the percentages of IFN-γ⁺ and interleukin-17⁺ cells were significantly lower in CD161⁺ CD4⁺ T cells of old individuals than those of young individuals. In addition, aging was associated with a decrease of nonclassic T helper 1 cells, as indicated by decreased percentages of CD161-expressing cells within the IFN-γ⁺ CD4⁺ T cell compartment of old subjects. Taken together, aging is associated with a numerical decline of circulating CD161^high CD8⁺ T cells, as well as a decreased production of pro-inflammatory cytokines by CD161⁺ CD4⁺ T cells. These aging-associated changes could contribute to perturbed immunity in the elderly.</p