Edelfosine impact on HLA-DR/DP/DQ expression on human B cells.

<p>(A) Gating strategy to identify viable CD19⁺ B cells and their IgD⁺CD27⁻, IgD⁺CD27⁺ and IgD⁻ CD27⁺ subsets after the incubation of PBMCs for 24 h in the absence of edelfosine or in the presence of 3.3 µg/ml and 10 µg/ml edelfosine, respectively. (B) Histograms, exemplary of one donor, display the edelfosine-induced downmodulation of HLA-DR/DP/DQ on the previously described B cell subsets in comparison to the respective untreated control approach. (C) Summary of MedFI values determined for each treatment within each B cell subset (n = 3 donors (represented by symbols •, ▪, ▴), + edelfosine added as indicated, − no edelfosine added). For CD19⁺, IgD⁺CD27⁻ and IgD⁺CD27⁺ populations significant reductions of HLA-DR/DP/DQ expression were observed (*P<0.05, **P<0.01, ***P<0.001 after repeated measures ANOVA and Bonferroni post-hoc analysis). Histogram legends for B: no edelfosine (black line), 3.3 µg/ml edelfosine (green line), 10 µg/ml edelfosine (red line), isotype control (blue line).</p

Edelfosine impact on HLA-DR/DP/DQ expression on human T cells.

<p>(A) Gating strategy to identify viable CD4⁺ and CD8⁺ T cells and their naïve (CD27⁺CD45RA⁺) and memory (CD27⁺CD45RA⁻) subsets after the incubation of PBMCs for 24 h in the absence of edelfosine or in the presence of 3.3 µg/ml and 10 µg/ml edelfosine, respectively. (B) Representative histograms of one donor display the considerably low expression of HLA-DR/DP/DQ on the previously described T cell subsets. (C) Summary of MedFI values determined for each treatment within each T cell subset (n = 3 donors (represented by symbols •, ▪, ▴), + edelfosine added as indicated, − no edelfosine added). For CD27⁺CD45RA⁺ and CD27⁺CD45RA⁻ populations of CD4⁺ and CD8⁺ T cells no significant reduction of HLA-DR/DP/DQ expression was observed after Bonferroni post-hoc analysis (depicted P-value: as determined by repeated measures ANOVA). Histogram legends for B: no edelfosine (black line), 3.3 µg/ml edelfosine (green line), 10 µg/ml edelfosine (red line), isotype control (blue line).</p

Edelfosine impact on T cell viability.

<p>(A) Gating strategy to determine frequencies of annexin V⁺ and/or PI⁺ CD4⁺ as well as CD8⁺ T cells. Dot plots for annexin V and PI gating of approaches in absence of edelfosine, with 10 µg/ml edelfosine, or 33.3 µg/ml edelfosine. (B) With regard to CD4⁺ T cells just 10 µg/ml edelfosine led to a decrease in annexin V⁻PI⁻ frequencies accompanied by a remarkable increase in annexin V⁺PI⁺CD4⁺ T cell frequencies. In the case of CD8⁺ T cells a marked decrease in annexin V⁻PI⁻ cells was only observed with 33.3 µg/ml edelfosine which resulted in comparable frequencies of annexin V⁺PI⁻ as well as annexin V⁺PI⁺ cells. PBMCs of a donor were seeded in triplicates and pooled before analysis.</p

Clustering of up- or downregulated genes to determine biological pathways affected by edelfosine in human CD4⁺ T cells after gene expression analysis.

<p>(A) The incubation of unstimulated cells with 10 µg/ml edelfosine resulted in the upregulation of apoptosis- and cell death-associated genes. Genes involved in immune response and antigen processing and presentation were downregulated. (B) In the case of stimulated cells which were cultured in presence of 3.3 µg/ml edelfosine the downmodulation of cell cycle progression-related genes was found. Additionally, edelfosine resulted in the upregulation of genes assigned to immune response- and virus response-pathways characterized by type I IFN-regulated genes.</p

Human T cell proliferation was affected by edelfosine.

<p>(A) Reduced PBMC proliferation upon addition of edelfosine on cell seeding was independent of the addition of PHA. Notably, PHA-activated cells appeared to be susceptible to edelfosine at 10-fold lower concentrations. (B) The inhibitory effect of edelfosine was observed if the drug was added to already activated, proliferating T cells, i.e. two days after cell seeding and PHA addition. Here, a significant reduction of proliferation in unstimulated cells was only detectable with 33.3 µg/ml edelfosine. (C) Preincubation of PBMCs with at least 3.3 µg/ml edelfosine interfered with the cells' capacity to proliferate upon PHA stimulation. No effect was detected in preconditioned, but unstimulated cells (experiments A, B, C: sample size n = 3 donors, each approach was seeded in triplicates and means for each donor are represented by symbols •, ▪, ▴). (D) 1 µg/ml edelfosine or higher concentrations profoundly diminished proliferation in MBP_(83–99)-specific TCLs. One representative TCL of two is shown. Cells were incubated in quadruplicates. • stimulated, ▪ unstimulated (E) PBMCs of one donor were cultured without addition of a stimulus. Proliferation was detectable after seven days. The presence of anti-HLA-DR- and anti-MHC class I-blocking antibodies or 3.3 µg/ml edelfosine inhibited cellular proliferation (• untreated, ▪ blocking antibodies added, ▴ 3.3 µg/ml edelfosine-treated). Bars represent mean values ± SEM, *P<0.05, **P<0.01 and ***P<0.001 after repeated measures ANOVA succeeded by Bonferroni post-hoc analysis.</p

Modulated gene expression in CD4⁺ T cells upon culture with edelfosine.

<p>Numbers of differentially up- or downregulated genes after incubation with edelfosine are shown.</p

Cytokine secretion was modulated in activated CD4⁺ T cells by edelfosine.

<p>(A) By ELISA, a significant reduction of IFN-γ-secretion was monitored upon edelfosine treatment. (B) This result was confirmed by a human 13plex kit which allowed the detection of not only reduced concentrations of IFN-γ in supernatants of edelfosine-treated cells, but also reduced concentrations of the Th1-associated cytokines IL-2 and TNF-α as well as the Th17-associated cytokines IL-17A, IL-22 and IL-6. Cells of four individuals (age-matched, two males and two females) were used, error bars indicate SEM of respective means (*P<0.05 after paired t-tests).</p

Representative immunohistochemical staining images of the investigated lymph nodes.

<p>Lymph nodes were stained using a polyclonal antibody against DKK1 (1∶100, abcam, #ab61034) and counterstained with hematoxylin. <b>a)</b> Representative DKK1 negative lymph node. Scale bar: 200 µm. <b>b)</b> Representative DKK1 positive lymph node. The subcapsular sinus is marked with (I) and the intermediary sinus and trabeculae with (II). Scale bar: 200 µm. <b>c)</b> DKK1 positive vascular endothelial cells. Scale bar: 100 µm.</p

Representative immunohistochemical staining images of the investigated lymph nodes.

<p>Lymph nodes were stained using a polyclonal antibody against DKK1 (1∶100, abcam, #ab61034) and counterstained with hematoxylin. <b>a)</b> Representative DKK1 negative lymph node. Scale bar: 200 µm. <b>b)</b> Representative DKK1 positive lymph node. The subcapsular sinus is marked with (I) and the intermediary sinus and trabeculae with (II). Scale bar: 200 µm. <b>c)</b> DKK1 positive vascular endothelial cells. Scale bar: 100 µm.</p

Network analysis.

<p>The network analysis was performed using Ingenuity Pathway Analysis software. <b>a</b>) Mechanistic network of β-catenin downstream regulators displaying predicted activation state and the regulatory relation (activation or inhibition) between the single regulators. β-catenin and a number of downstream regulators are predicted to be inactivated. <b>b)</b> The analysis shows a tight connection between the genes regulated in the pN1 samples (light grey nodes) and those regulated in the pN0 samples (dark grey nodes) linked by ERK1/2 and DKK1 embedded within the network.</p