Effect of anticoagulants on 162 circulating immune related proteins in healthy subjects

Diagnosis of complex disease and response to treatment is often associated with multiple indicators, both clinical and laboratorial. With the use of biomarkers, various mechanisms have been unraveled which can lead to better and faster diagnosis, predicting and monitoring of response to treatment and new drug development. With the introduction of multiplex technology for immunoassays and the growing awareness of the role of immune-monitoring during new therapeutic interventions it is now possible to test large numbers of soluble mediators in small sample volumes. However, standardization of sample collection and laboratory assessments remains suboptimal. We developed a multiplex immunoassay for detection of 162 immune related proteins in human serum and plasma. The assay was split in panels depending on natural occurring concentrations with a maximum of 60 proteins. The aim of this study was to evaluate precision, accuracy, reproducibility and stability of proteins when repeated freeze-thaw cycles are performed of this in-house developed panel, as well as assessing the protein signature in plasma and serum using various anticoagulants. Intra-assay variance of each mediator was <10%. Inter-assay variance ranged between 1.6 and 37% with an average of 12.2%. Recoveries were similar for all mediators (mean 99.8 ± 2.6%) with a range between 89-107%. Next we measured all mediators in serum, EDTA plasma and sodium heparin plasma of 43 healthy control donors. Of these markers only 19 showed similar expression profiles in the 3 different matrixes. Only 5 mediators were effected by multiple freeze-thawing cycles. Principal component analysis revealed different coagulants cluster separately and that sodium heparin shows the most consistent profile

Canonical Wnt signaling negatively modulates regulatory T cell function

Foxp3 is crucial for both the development and function of regulatory T (Treg) cells; however, the posttranslational mechanisms regulating Foxp3 transcriptional output remain poorly defined. Here, we demonstrate that Tcell factor 1 (TCF1) and Foxp3 associates in Treg cells and that active Wnt signaling disrupts Foxp3 transcriptional activity. A global chromatin immunoprecipitation sequencing comparison in Treg cells revealed considerable overlap between Foxp3 and Wnt target genes. The activation of Wnt signaling reduced Treg-mediated suppression both invitro and invivo, whereas disruption of Wnt signaling in Treg cells enhanced their suppressive capacity. The activation of effector Tcells increased Wnt3a production, and Wnt3a levels were found to be greatly increased in mononuclear cells isolated from synovial fluid versus peripheral blood of arthritis patients. We propose a model in which Wnt produced under inflammatory conditions represses Treg cell function, allowing a productive immune response, but, if uncontrolled, could lead to the development of autoimmunity

Autologous stem cell transplantation aids autoimmune patients by functional renewal and TCR diversification of regulatory T cells

Autologous hematopoietic stem cell transplantation (HSCT) is increasingly considered for patients with severe autoimmune diseases whose prognosis is poor with standard treatments. Regulatory T cells (Tregs) are thought to be important for disease remission after HSCT. However, eliciting the role of donor and host Tregs in autologous HSCT is not possible in humans due to the autologous nature of the intervention. Therefore, we investigated their role during immune reconstitution and re-establishment of immune tolerance and their therapeutic potential following congenic bone marrow transplantation (BMT)inaproteoglycan-inducedarthritis (PGIA) mousemodel. Inaddition, wedetermined Treg T-cell receptor (TCR) CDR3 diversity before and after HSCT in patients with juvenile idiopathic arthritis and juvenile dermatomyositis. In the PGIA BMT model, after an initial predominance of host Tregs, graft-derived Tregs started dominating and displayed a more stable phenotype with better suppressive capacity. Patient samples revealed a striking lack of diversity of the Treg repertoire before HSCT. This ameliorated after HSCT, confirming reset of the Treg compartment following HSCT. In the mouse model, a therapeutic approach was initiated by infusing extra Foxp3GFP+ Tregs during BMT. Infusion of Foxp3GFP+ Tregs did not elicit additional clinical improvement but conversely delayed reconstitution of the graft-derived T-cell compartment. These data indicate that HSCT-mediated amelioration of autoimmune disease involves renewal of the Treg pool. In addition, infusion of extra Tregs during BMT results in a delayed reconstitution of T-cell compartments. Therefore, Treg therapy may hamper development of long-term tolerance and should be approached with caution in the clinical autologous setting

FOXP3⁺ T cells are present in inflamed muscle of newly diagnosed, untreated patients with active JDM.

<p>(A) Representative pictures of both immunohistochemistry single stainings for CD3, a marker for T cells (left), FOXP3 (middle) on consecutive sections from muscle biopsies (all at 20× magnification, brown-red indicates positive staining), which were used to analyze cell numbers. The right panel shows an example of immunofluorescence staining (1 out of 4) of a tissue section of a JDM patient, showing FOXP3 (red) expressed in the nuclei of CD3⁺ T cells (green). The upper row shows sections of JDM muscle, the second row shows DMD muscle sections. (B) Average numbers of infiltrated CD3⁺ cells and FOXP3⁺ cells, and (C) percentages of CD3⁺ T cells expressing FOXP3 per section of 8 JDM patients and 9 DMD patients, calculated for each patient, by counting cells from the immunohistochemistry single stainings on consecutive sections, as described in the methods section. Mean ± SEM cell counts: (JDM) CD3 192.7±67.9, FOXP3 26.1±4.8, (DMD) CD3 27.7± 4.8, FOXP3 2.9±1.1, D) Paraffin embedded muscle sections of patients with active JDM were stained with anti-IL-17 (red), FOXP3 (green) and DAPI (blue). Three representative pictures (20× magnification) are shown (N = 3 active JDM patients, at the time of diagnosis, without medication). All graphs show mean percentages ± SEM. *P<0.05, **P<0.01, Mann Whitney U test was used.</p

Tregs from patients with active JDM exhibit a compromised suppressive function.

<p>(A) Representative dot plots of FOXP3 expression of gated CD4⁺CD25⁺CD127^low T cells (left dot plot) of JDM patients in remission (middle dot plot), or with active disease (right dot plot), showing means ± S.E.M. (B, C) CD4⁺CD25⁺CD127^low T cells were sorted into co-cultures with anti-CD3 activated autologous PBMC in different ratios. Proliferation was measured on day 6 by ³H thymidine incorporation. Level of suppression of PBMC proliferation was calculated by comparison to proliferation of PBMC cultured alone (suppression = 0%). (B) Suppression by CD4⁺CD25⁺CD127^low T cells from 9 patients in remission and (C) 11 patients with active disease, grey lines represent 4 patients displaying defective suppression in one or more conditions with Tregs present. Defective suppression is defined as an increased proliferation in the presence of the sorted Tregs, in at least one of the co-culture ratios.</p

Peripheral blood CD4 T cells from JDM patients and age-matched controls have similar expression of Treg markers.

<p>PBMC from JDM patients (JDM) with active (active) or remitting (remission) disease, and controls (control) were isolated and analyzed by flow cytometry (A–F). (A) Percentage of CD25^hi (control N = 13, remission N = 27, active N = 21), and (B) FOXP3 (control N = 9, remission N = 27, active N = 21) expressing CD4⁺ T cells. (C) Percentages of FOXP3 expressing CD4⁺ T cells in active JDM patients treated with corticosteroids (<0.5 mg/kg N = 12, 1 mg/kg N = 6, >1 mg/kg N = 3). Percentages of (D) GITR (control N = 5, remission N = 12, active N = 10), and (E) CTLA-4 (control N = 6, remission N = 15, active N = 13) expressing CD4⁺ T cells. (F) PBMC from patients with active JDM (at the time of diagnosis, without medication) were stimulated with PMA and ionomycin and stained for flow cytometry analysis of CD4⁺ T cells expressing FOXP3 and IL-17 (N = 5). All graphs show mean percentages ± SEM. *P<0.05, one way ANOVA analysis was used.</p