Spondyloarthritis mass cytometry immuno-monitoring: a proof of concept study in the tight-control and treat-to target TiCoSpA trial.

OBJECTIVE: Mass cytometry (MC) immunoprofiling allows high-parameter phenotyping of immune cells. We set to investigate the potential of MC immuno-monitoring of axial spondyloarthritis (axSpA) patients enrolled in the Tight Control SpondyloArthritis (TiCoSpA) trial. METHODS: Fresh, longitudinal PBMCs samples (baseline, 24, and 48 weeks) from 9 early, untreated axSpA patients and 7 HLA-B27+ controls were analyzed using a 35-marker panel. Data were subjected to HSNE dimension reduction and Gaussian mean shift clustering (Cytosplore), followed by Cytofast analysis. Linear discriminant analyzer (LDA), based on initial HSNE clustering, was applied onto week 24 and 48 samples. RESULTS: Unsupervised analysis yielded a clear separation of baseline patients and controls including a significant difference in 9 T cell, B cell, and monocyte clusters (cl), indicating disrupted immune homeostasis. Decrease in disease activity (ASDAS score; median 1.7, range 0.6-3.2) from baseline to week 48 matched significant changes over time in five clusters: cl10 CD4 Tnai cells median 4.7 to 0.02%, cl37 CD4 Tem cells median 0.13 to 8.28%, cl8 CD4 Tcm cells median 3.2 to 0.02%, cl39 B cells median 0.12 to 2.56%, and cl5 CD38+ B cells median 2.52 to 0.64% (all p<0.05). CONCLUSIONS: Our results showed that a decrease in disease activity in axSpA coincided with normalization of peripheral T- and B-cell frequency abnormalities. This proof of concept study shows the value of MC immuno-monitoring in clinical trials and longitudinal studies in axSpA. MC immunophenotyping on a larger, multi-center scale is likely to provide crucial new insights in the effect of anti-inflammatory treatment and thereby the pathogenesis of inflammatory rheumatic diseases. Key Points • Longitudinal immuno-monitoring of axSpA patients through mass cytometry indicates that normalization of immune cell compartments coincides with decrease in disease activity. • Our proof of concept study confirms the value of immune-monitoring utilizing mass cytometry.

Performance of spectral flow cytometry and mass cytometry for the study of innate myeloid cell populations.

Introduction: Monitoring of innate myeloid cells (IMC) is broadly applied in basic and translational research, as well as in diagnostic patient care. Due to their immunophenotypic heterogeneity and biological plasticity, analysis of IMC populations typically requires large panels of markers. Currently, two cytometry-based techniques allow for the simultaneous detection of ≥40 markers: spectral flow cytometry (SFC) and mass cytometry (MC). However, little is known about the comparability of SFC and MC in studying IMC populations. Methods: We evaluated the performance of two SFC and MC panels, which contained 21 common markers, for the identification and subsetting of blood IMC populations. Based on unsupervised clustering analysis, we systematically identified 24 leukocyte populations, including 21 IMC subsets, regardless of the cytometry technique. Results: Overall, comparable results were observed between the two technologies regarding the relative distribution of these cell populations and the staining resolution of individual markers (Pearson's ρ=0.99 and 0.55, respectively). However, minor differences were observed between the two techniques regarding intra-measurement variability (median coefficient of variation of 42.5% vs. 68.0% in SFC and MC, respectively; p<0.0001) and reproducibility, which were most likely due to the significantly longer acquisition times (median 16 min vs. 159 min) and lower recovery rates (median 53.1% vs. 26.8%) associated with SFC vs. MC. Discussion: Altogether, our results show a good correlation between SFC and MC for the identification, enumeration and characterization of IMC in blood, based on large panels (>20) of antibody reagents.

Altered composition and phenotype of mucosal-associated invariant T cells in early untreated rheumatoid arthritis.

BACKGROUND: Mucosal-associated invariant T (MAIT) cells are innate-like T cells that recognise bacterial metabolites presented by MHC class I-related protein 1 (MR1). Bacterial dysbiosis has been implicated in auto-inflammatory disease development. We investigated MAIT cells in early, untreated rheumatoid arthritis (RA) and spondyloarthritis (SpA) patients. METHODS: Blood and synovial fluid mononuclear cells obtained from patients (SpA/RA) and controls were stimulated with fixed Escherichia coli to provide MAIT ligand. Cells were analysed by flow cytometry and MAIT cells were identified by MR1-5-OP-RU tetramers. Synovial biopsies were studied by confocal microscopy. RESULTS: Peripheral and synovial CD3+ MR1-tet+ MAIT cell frequencies were comparable in all groups. MAIT cells were detected in RA and SpA synovium based on CD3, CD161 and Vα7.2 expression. Peripheral RA MAIT cells were mostly CD4+ (controls 8.3%, SpA 12.3%, RA 52.6%; p < 0.001) and CD161 expression was strongly reduced (control mean fluorescence intensity (MFI) = 2348, SpA MFI = 2219, RA MFI = 226; p < 0.001). MAIT cells were hyporesponsive, shown by minimal upregulation of CD25 and CD69 to E. coli stimulation (control, CD25 MFI = 177, CD69 MFI = 1307; SpA, CD25 MFI = 95, CD69 MFI = 1257; RA, CD25 MFI = 0, CD69 MFI = 467; p < 0.001 and p = 0.01 respectively). CONCLUSION: In early untreated RA patients, the peripheral MAIT cell composition was altered, with reduced levels of CD161 expression, and cells were hyporesponsive to stimulation. MAIT cell dysfunction may provide a link between the microbiome and development of RA.

Abatacept decreases disease activity in a absence of CD4(+) T cells in a collagen-induced arthritis model.

INTRODUCTION: Abatacept is a fusion protein of human cytotoxic T-lymphocyte-associated protein (CTLA)-4 and the Fc portion of human immunoglobulin G1 (IgG1). It is believed to be effective in the treatment of rheumatoid arthritis by inhibiting costimulation of T cells via blocking CD28-B7 interactions as CTLA-4 binds to both B7.1 (CD80) and B7.2 (CD86). However, the interaction of CD28 with B7 molecules is crucial for activation of naive cells, whereas it is unclear whether the action of already activated CD4(+) T cells, which are readily present in established disease, also depends on this interaction. The aim of this study was to determine whether the mode of action of abatacept depends solely on its ability to halt T cell activation in established disease. METHODS: Arthritis was induced in thymectomized male DBA/1 mice by immunisation with bovine collagen type II. The mice were subsequently depleted for CD4(+) T cells. Abatacept or control treatment was started when 80 % of the mice showed signs of arthritis. Arthritis severity was monitored by clinical scoring of the paws, and anti-collagen antibody levels over time were determined by enzyme-linked immunosorbent assay. RESULTS: Treatment with abatacept in the absence of CD4(+) T cells resulted in lower disease activity. This was associated with decreasing levels of collagen-specific IgG1 and IgG2a antibodies, whereas the antibody levels in control or CD4(+) T cell-depleted mice increased over time. CONCLUSIONS: These results show that abatacept decreased disease activity in the absence of CD4(+) T cells, indicating that the mode of action of abatacept in established arthritis does not depend entirely on its effects on CD4(+) T cell activation.

IL-17-producing CD4+ T cells are increased in early, active axial spondyloarthritis including patients without imaging abnormalities.

OBJECTIVE: Increased numbers of IL-17-producing CD4(+) T cells have been observed in AS. However, it is not known whether these CD4(+) T cells are already present in early disease or if this is a late disease phenomenon only. Therefore we aimed to investigate whether IL-17-producing CD4(+) T cells are involved in early active axial SpA, including patients without imaging abnormalities, by determining the frequency and phenotype of IL-17-producing CD4(+) T cells in these patients. METHODS: Flow cytometry was used to analyse cytokine production and surface marker expression of peripheral blood mononuclear cells from 31 patients suffering from early active HLA-B27-positive axial SpA fulfilling the Assessment of SpondyloArthritis International Society criteria with or without MRI abnormalities and 21 healthy controls. RESULTS: Patients with early active axial SpA showed an increased percentage of IL-17-producing CD4(+) T cells compared with healthy controls (mean 1.1% vs 0.4%, respectively; P = 0.013). The percentage of IL-17-producing CD4(+) T cells was equally increased in patients with and without MRI abnormalities (1.2% vs 1.1%, respectively; P = 0.81). These IL-17-producing CD4(+)T cells expressed the αß T cell receptor but not the γδ T cell receptor, exhibited a memory phenotype and expressed CD161, but only sporadically expressed killer cell immunoglobulin-like receptor 3DL2 (KIR3DL2). CONCLUSION: IL-17-producing CD4(+) T cells are increased in patients with early active axial SpA both with and without MRI abnormalities. This finding shows that the frequency of IL-17-producing CD4(+) T cells is enhanced in the early stages of disease.

Differential Toll-like receptor recognition and induction of cytokine profile by Bifidobacterium breve and Lactobacillus strains of probiotics.

The use of probiotics as a food supplement has gained tremendous interest in the last few years as beneficial effects were reported in gut homeostasis and nutrient absorption but also in immunocompromised patients, supporting protection from colonization or infection with pathogenic bacteria or fungi. As a treatment approach for inflammatory bowel diseases, a suitable probiotic strain would ideally be one with a low immunogenic potential. Insight into the immunogenicities and types of T-cell responses induced by potentially probiotic strains allows a more rational selection of a particular strain. In the present study, the bacterial strains Bifidobacterium breve (NumRes 204), Lactobacillus rhamnosus (NumRes1), and Lactobacillus casei (DN-114 001) were compared concerning their capacity to induce inflammatory responses in terms of cytokine production by human and mouse primary immune cells. It was demonstrated that the B. breve strain induced lower levels of the proinflammatory cytokine gamma interferon (IFN-γ) than the tested L. rhamnosus and L. casei strains. Both B. breve and lactobacilli induced cytokines in a Toll-like receptor 9 (TLR9)-dependent manner, while the lower inflammatory profile of B. breve was due to inhibitory effects of TLR2. No role for TLR4, NOD2, and C-type lectin receptors was apparent. In conclusion, TLR signaling is involved in the differentiation of inflammatory responses between probiotic strains used as food supplements.

Cutting edge: TNFR-shedding by CD4+CD25+ regulatory T cells inhibits the induction of inflammatory mediators.

CD4+CD25+ regulatory T (Treg) cells play an essential role in maintaining tolerance to self and nonself. In several models of T cell-mediated (auto) immunity, Treg cells exert protective effects by the inhibition of pathogenic T cell responses. In addition, Treg cells can modulate T cell-independent inflammation. We now show that CD4+CD25+ Treg cells are able to shed large amounts of TNFRII. This is paralleled by their ability to inhibit the action of TNF-alpha both in vitro and in vivo. In vivo, Treg cells suppressed IL-6 production in response to LPS injection in mice. In contrast, Treg cells from TNFRII-deficient mice were unable to do so despite their unhampered capacity to suppress T cell proliferation in a conventional in vitro suppression assay. Thus, shedding of TNFRII represents a novel mechanism by which Treg cells can inhibit the action of TNF, a pivotal cytokine driving inflammation.

The exploration of rotenone as a toxin for inducing Parkinson's disease in rats, for application in BBB transport and PK-PD experiments.

INTRODUCTION: In search for a suitable rat model to study potentially affected blood-brain barrier (BBB) transport mechanisms in the course of Parkinsons disease (PD) progression, experiments were performed to characterise Parkinsons disease markers following subcutaneous (SC) and intracerebral (IC) infusion of the toxin rotenone in the rat. METHODS: Studies were performed using Male Lewis rats. SC infusion of rotenone (3 mg/kg/day) was performed via an osmotic minipump. IC infusion of rotenone occurred directly into the right medial forebrain bundle at three different dosages. At different times following rotenone infusion, behaviour, histopathology (tyrosine hydroxylase and alpha-synuclein immunocytochemistry), peripheral organ pathology (adrenals, heart, kidney, liver, lung, spleen and stomach) were assessed. In part of the SC and IC rats, BBB transport profiles of the permeability marker sodium fluorescein were determined using microdialysis. RESULTS: SC rotenone failed to produce dopaminergic lesions and led to extensive peripheral organ toxicity. BBB permeability for fluorescein following SC rotenone was changed, however due peripheral toxicity. In contrast, IC rotenone produced a progressive lesion of the nigrostrial dopaminergic pathway over 28 days with no associated peripheral toxicity. IC rotenone also exhibited a large increase in amphetamine induced rotational behaviour. In addition, a few IC rats showed alpha-synuclein immunoreactivity and aggregation. Following IC rotenone, no changes in BBB permeability were detected after 14 days. DISCUSSION: SC rotenone only produced peripheral toxicity. IC rotenone appeared to create a progressive lesion of the rat nigrostrial pathway, and may therefore be a more appropriate model of Parkinson's disease progression, compared with the most commonly used 6-OH-DA rat model.