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Additional file 2: Figure S2. Titration curves of oral cladribine treatment in mice. In preliminary experiments, we analyzed the plasma [ng/ml] and brain concentrations [ng/g] of oral cladribine treatment with different doses (3.25, 5 and 10 mg/kg bodyweight) over a time period of 2.5 h. Every 30 min blood was drawn and a proportion of animals was killed to obtain brain tissue for titration analyses. Curves show the maximal concentration of cladribine in blood and brain tissue (Cmax [ng/ml or ng/g]), the time to maximal concentration (tmax [hours]) and the area under the curve (AUC [ng*h/ml or ng*h/g]).

Additional file 3: Figure S3. Gating strategy for flow cytometry. Single immune cells from the periphery (A, here exemplary shown with spleen tissue) and the central nervous system (CNS; B, here exemplary shown with spinal cord tissue) were simultaneously analyzed by flow cytometry. Total leukocytes were identified by forward scatter (FSC) and sideward scatter (SSC) and cell-doublets were removed by FSC width and FSC height gating. From these cells, we identified leukocytes subsets based on their surface marker expression: CD45R+ B cells; CD3+CD4+ T-helper cells, CD3+CD8+ cytotoxic T cells and CD4+CD8+ double positive T cells; CD3−NK1.1+ natural killer (NK) cells; CD11b+CD11c− monocytes and macrophages (M/M) and CD11b+CD11c+ dendritic cells. Regarding the CNS tissue, prior to discrimination of leukocyte subsets, we differentiated microglia cells (CD45med) from infiltrated leukocytes (CD45high).

Additional file 7: Figure S7. Proliferation assay of splenocytes from naïve wild-type mice, with and without cladribine treatment in vitro. A: Proliferated cells in % after cladribine treatment (with 0.1 or 1 μM cladribine) versus vehicle-treated cells. We compared the unstimulated to the stimulated setting (stimulated with 1 μg/mL anti-CD3 and 2 μg/mL anti-CD28 for 3 days) (n = 6 for both vehicle and cladribine treatment, two-way ANOVAs). B: Mean fluorescence intensities (MFIs) of migration and activation markers in all viable stimulated cells upon vehicle- compared to cladribine-treatment (n = 6 for both vehicle and cladribine treatment, two-way ANOVAs). p > 0.05 = ns, p < 0.05 = *, p < 0.001 = ***, p < 0.0001 = ****.

Additional file 1: Figure S1. Experimental scheme. Active MOG EAE was induced as previously described by immunization (day 0) of C57BL/6J mice with MOG35–55 peptide, followed by pertussis toxin (PTX) injections (day 0 and day 2) (15). Mice were divided into two experimental groups: Group 1 received cladribine via oral gavage (10 mg/kg from day 5 to day 9), while group 2 received only the vehicle for the same period of time. On day 10 post-EAE induction, focal EAE lesions were generated by stereotactic injection of proinflammatory cytokines (interferon gamma (INF-γ) and tumor necrosis factor alpha (TNF-α) into the auditory cortex to induce cortical grey matter lesions. Experimental read-out (electrophysiological recordings, flow cytometric immunophenotyping, histology) was performed either on dmax (defining the day of maximal clinical deterioration) or on day 27 post-EAE induction (to assess the chronical EAE state).

Additional file 6: Figure S6. Migration assay of leukocytes in organ cultures of spleens from naïve wild-type mice, with and without cladribine treatment in vitro. A: Migration ratios (number of migrated cells: total cell count) of viable (live), CD4+ or CD8+ T cells and B cells upon cladribine treatment (vehicle (migration) versus cladribine 0.1 µM (migration)) (n = 6 for both vehicle and cladribine treatment, two-way ANOVAs, p-values > 0.05). B: Flow cytometric analysis of the immune cell distribution in % in spleens (vehicle or cladribine (organ)) and the migrated immune cells (vehicle or cladribine (migration)) (n = 6 for both vehicle and cladribine treatment, two-way ANOVAs). C: Mean fluorescence intensities (MFIs) of migration and activation markers in viable CD4+ cells still located in the respective spleen (vehicle or cladribine (organ)) compared to those after egress (vehicle or cladribine ((migration)) (n = 6 for both vehicle and cladribine treatment, two-way ANOVAs). D: MFIs of migration and activation markers in viable CD8+ cells still located in the respective spleen (vehicle or cladribine (organ)) compared to those after egress (vehicle or cladribine ((migration)) (n = 6 for both vehicle and cladribine treatment, two-way ANOVAs, p-values > 0.05). E: MFIs of migration and activation markers in viable B cells still located in the respective spleen (vehicle or cladribine (organ)) compared to those after egress (vehicle or cladribine ((migration)) (n = 6 for both vehicle and cladribine treatment, two-way ANOVAs). p > 0.05 = ns, p < 0.05 = *, p < 0.01 = **, p < 0.001 = ***, p < 0.0001 = ****.

Additional file 5: Figure S5. Migration assay of leukocytes in organ cultures of cervical LNs from naïve wild-type mice, with and without cladribine treatment in vitro. A: Migration ratios (number of migrated cells: total cell count) of viable (live), CD4+ or CD8+ T cells and B cells after cladribine treatment (vehicle (migration) versus cladribine 0.1 µM (migration)) (n = 6 for both vehicle and cladribine treatment, two-way ANOVAs, p-values > 0.05). B: Flow cytometric analysis of the immune cell distribution in % in LNs (vehicle or cladribine (organ)) and the migrated immune cells (vehicle or cladribine (migration)) (n = 6 for both vehicle and cladribine treatment, two-way ANOVAs). C: Mean fluorescence intensities (MFIs) of migration and activation markers in viable CD4+ cells still located in the respective LN (vehicle or cladribine (organ)) compared to those after egress (vehicle or cladribine ((migration)) (n = 6 for both vehicle and cladribine treatment, two-way ANOVAs). D: MFIs of migration and activation markers in viable CD8+ cells still located in the respective LN (vehicle or cladribine (organ)) compared to those after egress (vehicle or cladribine ((migration)) (n = 6 for both vehicle and cladribine treatment, two-way ANOVAs, p-values > 0.05). E: MFIs of migration and activation markers in viable B cells still located in the respective LN (vehicle or cladribine (organ)) compared to those after egress (vehicle or cladribine ((migration)) (n = 6 for both vehicle and cladribine treatment, two-way ANOVAs). p > 0.05 = ns, p < 0.05 = *, p < 0.01 = **, p < 0.0001 = ****.

Additional file 4: Figure S4. Gating strategy for sorting of macrophages. CD11b+ cells were isolated from murine CNS and spinal cord via magnetic beads. Subsequently, CD11b+ cells were simultaneously analyzed and sorted by flow cytometry. Total CD11b+ cells were identified by forward scatter (FSC) and sideward scatter (SSC) (A), and cell-doublets were removed by SSC width and SSC height gating (B). From these cells, we discriminated macrophages based on their surface marker expression of CD45highCD11bhigh (C). Macrophages were sorted for downstream experiments (D). Plots (E)-(F) show the distribution of microglia (CD45intermCD11bhigh) in comparison to macrophages.

In experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, the role of each central nervous system (CNS)-resident cell type during inflammation, neurodegeneration, and remission has been frequently addressed. Although protocols for the isolation of different individual CNS-resident cell types exist, none can harvest all of them within a single experiment. In addition, isolation of individual cells is more demanding in adult mice and even more so from the inflamed CNS. Here, we present a protocol for the simultaneous purification of viable single-cell suspensions of all principal CNS-resident cell types (microglia, oligodendrocytes, astrocytes, and neurons) from adult mice—applicable in healthy mice as well as in EAE. After dissociation of the brain and spinal cord from adult mice, microglia, oligodendrocytes, astrocytes and, neurons were isolated via magnetic-activated cell sorting (MACS). Validations comprised flow cytometry, immunocytochemistry, as well as functional analyses (immunoassay and Sholl analysis). The purity of each cell isolation averaged 90%. All cells displayed cell-type-specific morphologies and expressed specific surface markers. In conclusion, this new protocol for the simultaneous isolation of all major CNS-resident cell types from one CNS offers a sophisticated and comprehensive way to investigate complex cellular networks ex vivo and simultaneously reduce mice numbers to be sacrificed.

Additional file 1:Supplementary Figure S1. Quantitative K2P5.1 expression on the protein level in cells from RA patients compared to healthy controls. Western blot analysis of five individual RA patients (one to five) compared to two healthy controls (HD). Respective DAS28 scores are indicated at the upper part of the figure. (PDF 102 KB)

Authors’ original file for figure 4

Additional file of Expression of K2P5.1 potassium channels on CD4+T lymphocytes correlates with disease activity in rheumatoid arthritis patients

Additional file 2:Supplementary Figure S2. Influence of therapeutic agents on K2P5.1 expression. A) Correlation between K2P5.1 expression levels (Δct values) on CD4+ T lymphocytes and DAS28 scores is shown for patients with convential treatments (black diamonds), TNFα inhibitors (grey diamonds) and rituximab therapy (white diamonds). B) Patient subgroups are shown with conventional treatment (left side), TNFα inhibitors (middle side) and rituximab (right side). C) The left bar graph representation shows the correlation coefficients between DAS28 and K2P5.1 expression levels. The DAS28 score for the treatment subgroups is shown on the right side. (PDF 63 KB)

Authors’ original file for figure 2

Authors’ original file for figure 1

Authors’ original file for figure 3

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). This process is characterized by the transmigration of activated T cells across brain endothelial cells (ECs), the main constituents of the BBB. However, the consequences on brain EC function upon interaction with such T cells are largely unknown. Here we describe an assay that allows for the evaluation of primary mouse brain microvascular EC (MBMEC) function and barrier integrity during the interaction with T cells over time. The assay makes use of impedance cell spectroscopy, a powerful tool for studying EC monolayer integrity and permeability, by measuring changes in transendothelial electrical resistance (TEER) and cell layer capacitance (Ccl). In direct contact with ECs, stimulated but not naïve T cells are capable of inducing EC monolayer dysfunction, as visualized by a decrease in TEER and an increase in Ccl. The assay records changes in EC monolayer integrity in a continuous and automated fashion. It is sensitive enough to distinguish between different strengths of stimuli and levels of T cell activation and it enables the investigation of the consequences of a targeted modulation of T cell-EC interaction using a wide range of substances such as antibodies, pharmacological reagents and cytokines. The technique can also be used as a quality control for EC integrity in in vitro T-cell transmigration assays. These applications make it a versatile tool for studying BBB properties under physiological and pathophysiological conditions.