2.3. Flow CytometryThe majority of

2.3. Flow CytometryThe majority of the extracellular flow cytometry experiments were performed directly on fresh cells, but for six donors, the T cell markers CD69, CD25, CXCR3, CCR6, CD38, HLA-DR, CD127, and PD-1 were analyzed on paired frozen samples. Staining was carried out in 96-well plates with ≤1 × 106 cells/well in 50 μl CliniMACS PBS/EDTA buffer (Miltenyi Biotech, Bergish Gladbach, Germany) supplemented with 0.1% bovine serum albumin. The cells were incubated with mAbs for 30 min at 4°C. Intracellular staining was performed after extracellular staining using the BD Cytofix/Cytoperm™ kit (BD Biosciences, Franklin Lakes, NJ) according to the manufacturer’s instructions. 7AAD staining was used to sort live and dead cells if no intracellular staining was performed. The antibodies used in this study are listed in Supplementary Table available online at https://doi.org/10.1155/2017/8010961. Data was collected using a BD FACSCanto flow cytometer and analyzed with FlowJo software (Tree Star, Ashland, OR, USA). Results from subgating were only included if the parent population consisted of ≥80 cells.2.4. Bacterial Stimulation AssayMononuclear cells were isolated by density gradient centrifugation (Lymphoprep, Axix-Shield, Dundee, Scotland) and resuspended in complete medium. Fresh cells were then cultured at 37°C and 5% CO2, at a concentration of 3 × 106 cells/ml, in 96-well plates alone or together with UV-irradiated Escherichia coli (E. coli) at a multiplicity of infection of 30 together with 1.25 μg/ml anti-CD28mAb (CD28.2, BioLegend, San Diego, CA). After 12 hours of culture, 10 μg/ml Brefeldin-A (BFA, Sigma-Aldrich, St. Louis, MO) was added, followed by an additional 4 hours of culture. After the total 16 hours of culture, cells were harvested and stained for flow cytometry.2.5. PMA/Ionomycin Stimulation AssayFrozen cells were thawed and mononuclear cells were isolated by density gradient centrifugation (Lymphoprep, Axix-Shield). Peripheral blood mononuclear cells (PBMCs) from healthy blood donors (healthy controls) were used as controls. Cells were resuspended in complete medium and cultured at 37°C and 5% CO2, at a concentration of 2 × 106 cells/ml in 96-well plates. 10 μg/ml BFA (Sigma-Aldrich) was added to all wells, and half of the samples were stimulated with 25 ng/ml PMA (Sigma-Aldrich) and 1 μg/ml ionomycin (Sigma-Aldrich). After 5 hours of culture, cells were harvested and stained for flow cytometry.2.6. StatisticsMultivariate orthogonal projection to latent structures by means of partial least squares discriminant analysis (OPLS-DA) was used to obtain a maximum separation of X-variables, that is, immune cell variables, based on class information, that is, basalis and parietalis in Figures 1(b), 2(a), and 3(b) (SIMCA software, Sartorius Stedim Biotech, Umeå, Sweden). The contribution of each X-variable, VIP values, to the OPLS-DA model in Figure 1(a) was calculated (Supplementary Figure S). X-variables with a VIP value below 0.98 were excluded, and a new model was generated based on remaining variables (Figure 1(b)). The scale presented on the y-axis of the OPLS plot is a dimensionless scale; the loading vector is normalized to unit length. The quality of OPLS analyses is based on R2, which indicates how well the variation of the variables is explained by the model, and Q2, an estimate of the model’s predictive ability. Utilizing the OPLS-DA analysis as screening for differences between the groups, the factors contributing most to the separation were further analyzed using a two-tailed Wilcoxon matched-pairs signed rank test (GraphPad Software, La Jolla, CA). An alpha value of