Paulina Chalan

Disturbed B cell homeostasis in newly diagnosed giant cell arteritis and polymyalgia rheumatica

Several lines of evidence indicate that B cells may be involved in the immunopathology of giant cell arteritis (GCA) and polymyalgia rheumatica (PMR). This study was undertaken to examine the distribution of defined B cell subsets, including effector B (Beff) cells and regulatory B (Breg) cells, in patients with GCA and patients with PMR before and after corticosteroid treatment.

Circulating CD4+CD161+ T Lymphocytes Are Increased in Seropositive Arthralgia Patients but Decreased in Patients with Newly Diagnosed Rheumatoid Arthritis

Improved understanding of the immune events discriminating between seropositive arthralgia and clinical synovitis is of key importance in rheumatology research. Ample evidence suggests a role for Th17 cells in rheumatoid arthritis. We hypothesized that CD4+CD161+ cells representing Th17 lineage cells may be modulated prior to or after development of clinical synovitis. Therefore, in a cross-sectional study, we investigated the occurrence of CD4+CD161+ T-cells in seropositive arthralgia patients who are at risk for developing rheumatoid arthritis and in newly diagnosed rheumatoid arthritis patients. In a prospective study, we evaluated the effect of methotrexate treatment on circulating CD4+CD161+ T-cells. Next, we assessed if these cells can be detected at the level of the RA joints. Precursor Th17 lineage cells bearing CD161 were found to be increased in seropositive arthralgia patients. In contrast, circulating CD4+CD161+T-cells were decreased in newly diagnosed rheumatoid arthritis patients. The decrease in CD4+CD161+ T-cells correlated inversely with C-reactive protein and with the 66 swollen joint count. Methotrexate treatment led to normalization of CD4+CD161+ T-cells and reduced disease activity. CD4+CD161+ T cells were readily detected in synovial tissues from both early and late-stage rheumatoid arthritis. In addition, synovial fluid from late-stage disease was found to be enriched for CD4+CD161+ T-cells. Notably, synovial fluid accumulated CD4+CD161+T-cells showed skewing towards the Th1 phenotype as evidenced by increased interferon-γ expression. The changes in peripheral numbers of CD4+CD161+ T-cells in seropositive arthralgia and early rheumatoid arthritis and the enrichment of these cells at the level of the joint predict a role for CD4+CD161+ T-cells in the early immune events leading to clinical synovitis. Our findings may add to the development of RA prediction models and provide opportunities for early intervention.

Chronic autoimmune-mediated inflammation: a senescent immune response to injury

The increasing prevalence of chronic autoimmune-mediated inflammatory diseases (AIMIDs) in ageing western societies is a major challenge for the drug development industry. The current high medical need for more-effective treatments is at least in part caused by our limited understanding of the mechanisms that drive chronic inflammation. Here, we postulate a role for immunosenescence in the progression of acute to chronic inflammation via a dysregulated response to primary injury at the level of the damaged target organ. A corollary to this notion is that treatment of acute versus chronic phases of disease might require differential targeting strategies.

Analysis of serum immune markers in seropositive and seronegative rheumatoid arthritis and in high-risk seropositive arthralgia patients.

Presence of autoantibodies precedes development of seropositive rheumatoid arthritis (SP RA) and seropositive arthralgia patients (SAP) are at risk of developing RA. The aims of the study are to identify additional serum immune markers discriminating between SP and seronegative (SN) RA, and markers identifying high-risk SAP. Sera from SAP (n = 27), SP RA (n = 22), SN RA (n = 11) and healthy controls (n = 20) were analyzed using the Human Cytokine 25-Plex Panel. Selected markers were validated in independent cohorts of SP RA (n = 35) and SN RA (n = 12) patients. Eleven of 27 SAP developed RA within 8 months (median follow-up time, range 1–32 months), and their baseline serum markers were compared to 16 non-progressing SAP. SAP and SP RA patients showed a marked overlap in their systemic immune profiles, while SN RA showed a distinct immune profile. Three of 4 markers discriminating between SP and SN RA (IL-1β, IL-15 and Eotaxin, but not CCL5) were similarly modulated in independent cohorts. SAP progressing to RA showed trends for increases in IL-5, MIP-1β, IL-1RA and IL-12 compared to non-progressing SAP. ROC analysis showed that serum IL-5 most accurately discriminated between the two SAP groups (AUC > 0.8), suggesting that baseline IL-5 levels may aid the identification of high-risk SAP.

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

Objectives 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. Methods 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. Results 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. Conclusions 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.

CD70-expressing CD4 T cells produce IFN-γ and IL-17 in rheumatoid arthritis

OBJECTIVE CD70-expressing CD4 T cells are enriched in RA and promote autoimmunity via co-stimulatory CD70-CD27 interaction. This study aimed to explore the phenotype and cytokine production of CD70(+) CD4 T cells in RA. METHODS Peripheral blood mononuclear cells from 32 RA patients were isolated and frequencies of CD70(+) cells within different CD4 T subsets were analysed using flow cytometry. IFN-γ and IL-17 production were compared between the CD70(+) and CD70(-) cells. Expression of master transcription factors T-bet, GATA3 and retinoic acid-related orphan receptor gamma t (RORγt) were examined by real-time PCR. Results are presented as mean (s.e.m.). RESULTS CD4 T cells of healthy controls rarely expressed CD70 as compared with CD4 T cells of RA patients [mean 0.9% (s.e.m. 0.3%) vs 7.6 (0.6), P < 0.001]. In RA, CD70(+) cells were present within all CD4 T cell subsets, i.e. CD45RA(+)CCR7(+) naive, CD45RA(-)CCR7(+) central memory, CD45RA(-)CCR7(-) effector memory and CD45RA(+)CCR7(-) terminally differentiated effector memory T cells with a mean frequency of 3.9% (s.e.m. 1.1%), 4.0 (0.5), 4.2 (0.7) and 9.4 (4.3), respectively. As compared to CD70(-) CD4 T cells, CD70(+) CD4 T cells produced significantly more IFN-γ and IL-17 after short activation. CD70(+) CD4 T cells preferentially expressed transcription factor RORγt. CONCLUSION CD70(+) CD4 T cells are enriched in RA and may directly contribute to RA pathogenesis by producing IFN-γ and IL-17. Targeting CD70(+) CD4 T cells might offer new therapeutic opportunities in RA.

Altered Natural Killer Cell Subsets in Seropositive Arthralgia and Early Rheumatoid Arthritis Are Associated with Autoantibody Status

Objective. The role of natural killer (NK) cells in the immunopathogenesis of rheumatoid arthritis (RA) is unclear. Therefore, numerical and functional alterations of CD56dim and CD56bright NK cells in the early stages of RA development were studied. Methods. Whole blood samples from newly diagnosed, treatment-naive, seropositive (SP) and seronegative (SN) patients with RA (SP RA, n = 45 and SN RA, n = 12), patients with SP arthralgia (n = 30), and healthy controls (HC, n = 41) were assessed for numbers and frequencies of T cells, B cells, and NK cells. SP status was defined as positive for anticyclic citrullinated peptide antibodies (anti-CCP) and/or rheumatoid factor (RF). Peripheral blood mononuclear cells were used for further analysis of NK cell phenotype and function. Results. Total NK cell numbers were decreased in SP RA and SP arthralgia but not in SN RA. Also, NK cells from SP RA showed a decreased potency for interferon-γ (IFN-γ) production. A selective decrease of CD56dim, but not CD56bright, NK cells in SP RA and SP arthralgia was observed. This prompted investigation of CD16 (FcγRIIIa) triggering in NK cell apoptosis and cytokine expression. In vitro, CD16 triggering induced apoptosis of CD56dim but not CD56bright NK cells from HC. This apoptosis was augmented by adding interleukin 2 (IL-2). Also, CD16 triggering in the presence of IL-2 stimulated IFN-γ and tumor necrosis factor-α expression by CD56dim NK cells. Conclusion. The decline of CD56dim NK cells in SP arthralgia and SP RA and the in vitro apoptosis of CD56dim NK cells upon CD16 triggering suggest a functional role of immunoglobulin G-containing autoantibody (anti-CCP and/or RF)-immune complexes in this process. Moreover, CD16-triggered cytokine production by CD56dim NK cells may contribute to systemic inflammation as seen in SP arthralgia and SP RA.

Regulatory CD4+T-Cell Subsets and Anti-Citrullinated Protein Antibody Repertoire: Potential Biomarkers for Arthritis Development in Seropositive Arthralgia Patients?

Objective Seropositive arthralgia patients (SAP) are at high risk of developing rheumatoid arthritis (RA). This prospective study aimed to determine whether altered peripheral regulatory T-cells (Tregs) and defined subsets, besides a broadened anti-citrullinated protein antibody (ACPA) response, may qualify as biomarkers for RA development in SAP. Methods Thirty-four consecutive SAP were prospectively assessed every 6 months for minimally 2 years. At inclusion, peripheral Treg (CD4+CD25+FoxP3+) numbers and subsets, defined as CD45RA+FoxP3low naive Tregs (Fr I), CD45RA-FoxP3high activated Tregs (Fr II) and CD45RA-FoxP3low non-Tregs (Fr III), were compared to age- and sex-matched healthy controls (HC, n = 16) and treatment-naive RA patients (n = 12). SAP that developed RA were compared to non-switchers and analyzed for Treg numbers and Treg subsets at inclusion. Also, Treg numbers and subsets were compared in switched SAP before and at diagnosis. To assess the ACPA repertoire, IgG and IgA reactivity was measured against citrullinated peptides from fibrinogen, α-enolase and vimentin. Results Treg numbers were similar between HC, SAP and RA patients. Although the bonafide Treg subsets Fr I and Fr II were comparable between groups, Fr III was increased in SAP compared to HC (p = 0.01). Fourteen (41%) SAP developed RA during follow-up. Their Treg numbers and subsets were comparable to non-switched SAP. At RA diagnosis, Treg numbers in switched SAP were similar to 6 months before. Switched SAP displayed a more diverse IgG ACPA repertoire compared to non-switched SAP (p = 0.046) and showed more IgA reactivity than non-switched SAP reaching significance for Fib1 only (p = 0.047). Conclusion Numbers of Total Treg and bonafide Treg subsets are not indicative for RA development in SAP, opposed to the ACPA repertoire.

SAT0035 Regulatory CD4+ T-Cell Levels and Anti Citrullinated Protein Antibody Repertoire as Biomarkers for Arthritis Development in Seropositive Arthralgia Patients

Background Early diagnosis and treatment of rheumatoid arthritis (RA) is of paramount importance for achieving a better disease outcome. Seropositive arthralgia patients (SAP), positive for anti citrullinated protein antibody (ACPA) and/or rheumatoid factor (RF) with (a history of) arthralgia, have a “high risk” of developing RA. Therefore it is necessary to understand which preclinical immunological factors play a role in the progression towards RA in SAP since this could help identify individuals earlier and result in early intervention. Objectives This prospective study aimed at identifying whether altered regulatory T-cells (Tregs) levels and subsets, besides a broadened ACPA response, are biomarkers for RA development in SAP. Methods Thirty-four consecutive SAP were prospectively assessed every 6 months for at least 2 years. Every visit, peripheral blood mononuclear cells (PBMCs) were isolated and stored. At inclusion, peripheral Treg (CD4+CD25+FoxP3+) levels and Treg subsets, defined as CD45RA+FoxP3low naive Treg cells (Fr I), CD45RA–FoxP3high activated Treg cells (Fr II) and cytokine producing CD45RA–FoxP3low non-Treg cells (Fr III), were compared to age and sex matched healthy controls (HC, n=16) and treatment naive RA patients (n=12). SAP that developed RA during follow-up were compared to non-switchers on Treg levels and functional Treg subsets. To assess broadening of the IgG and IgA ACPA repertoire in serum, reactivity was measured against citrullinated peptides from fibrinogen, α-enolase and vimentin. Results Total Treg levels were similar between HC, SAP and RA patients. Although functional Treg subsets Fr I and Fr II were comparable between groups, Fr III was increased in SAP compared to HC (p=0.01). Fourteen (41%) SAP developed RA during follow-up. Their Treg levels were comparable to those of non-switched SAP. At RA diagnosis, Treg levels in switched SAP were not changed compared to 6 months before. Switched SAP displayed an extended IgG ACPA repertoire compared to non-switched SAP (p=0.046). Conclusions Total and functional Treg levels do not indicate development of RA in SAP, in contrast to an extended IgG and IgA ACPA repertoire. Hence, there is presumably no contribution of altered Treg levels leading to the loss in suppression of autoimmunity in RA pathology. Disclosure of Interest None declared

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. Paraffin-embedded 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. Disclosure of Interest None declared