Fixable viability stain

We used FACS to profile B cells, T cells (and subpopulations), NK cells, NKT cells, monocytes, neutrophils, and eosinophils (Table S1). Briefly, 120 μL of blood was incubated with 30 μL of the antibody mix (Table S2) for 30 min at 4 °C. Then, erythrocytes were lysed (BD FACS Lysing Solution, San Jose, CA, USA) and cells were incubated with the Fixable Viability Stain (440UV, BD 566332) for 15 min at room temperature in the dark, washed, and fixed using PFA 4%. Fixed samples data were acquired with a LSRFortessa SORP (BD, San Jose, CA, USA). FlowJo version 10 software (FlowJo LL, Ashland, OR, USA) was used for analysis. The gating strategy to determine the populations is described in Figures S2 and S3.

At indicated times post-infection a proportion of lymphocyte cultures representing ~200,000 cells were pelleted at 400g for 3 minutes into 96-well round bottom plates. Cells were washed once with PBS and resuspended in 200μl PBS containing (0.4ng/ml) fixable viability stain (BD Cat# 564406) and incubated at room temperature for 10 minutes. Cells were pelleted and resuspended in 100μl cold PBS without calcium and magnesium containing 5% FBS, and 0.1% Sodium Azide (FACS Block) and incubated on ice for 15 minutes after which 100μl cold PBS containing 0.5% FBS and 0.1% Sodium Azide (FACS Wash) was added. Cells were pelleted and resuspended in FACS Wash containing B cell phenotype panel as follows for 15 minutes on ice: (volumes indicated were routinely used for up to 0.5(10)^6 cells and were based on titration of the individual antibodies on primary tonsil lymphocyte specimens) Ig Lambda Light Chain-V450 (2μl), CD19-PE (8μl), CD38-PECy7 (3μl, BD Cat# 560667), IgD-PerCP Cy5.5 (2.5μl, BD Cat# 561315), CD138-APC (4μl, BD Cat# 347207), CD27-APC H7 (2.5μl BD Cat# 560222), Ig Kappa Light Chain-Alexa700 (2μl). After incubation, 100μl FACS Wash was added and pelleted lymphocytes were washed with a further 200μl of FACS Wash prior to being resuspended in 200μl FACS Wash for analysis. Data was acquired on a BD LSR2 Flow Cytometer and analyzed using FlowJo software.

Expression of TREM-1 in peripheral blood cells was determined by flow cytometry. Blood (1 mL) was used, and red blood cells were lysed using RBC lysis buffer (Roche Diagnostics GmbH, Mannheim, GR). Leukocytes were washed in PBS containing 5% fetal bovine serum (FBS) (Gibco™, Thermo Fisher Scientific, Waltham, MA, USA), centrifuged, and resuspended in Hank’s balanced salt solution (Sigma-Aldrich, Merck, Darmstadt, Germany) containing 5% FBS, followed by surface antigen staining. Briefly, cells were stained with fixable viability stain (1:1000) (BD Biosciences, San Diego, CA, USA) and incubated with monoclonal antibodies specific for CD14 (1:100) (M5E2; Biolegend, San Diego, CA USA), CD16 (1:100) (BV510-3G8; Biolegend, San Diego, CA USA), and TREM-1 (1:100) (FAB1278P; R&D Systems, Minneapolis, MN, USA) for 30 min at 4 °C. The stained cells were washed and fixed with BD Cytofix™ fixation buffer (554655; BD Biosciences, San Diego, CA, USA). Data acquisition was performed using the Fortessa™ LSR flow cytometer (BD Biosciences, San Jose, CA, USA) and the FACS-Diva software (version 8.0.1) (BD Biosciences, Franklin Lakes, NJ, USA). For the analysis, 100,000 events were acquired for each sample. CD14 and CD16 positive cells were gated, and TREM-1 expression analysis was performed using FlowJo^® software (version 10.7.0-Tree Star, Ashland, OR, USA) (Figure S1).

Peripheral blood mononuclear cells were obtained using a Ficoll-density gradient. These cells were then stimulated with 50 ng/mL of phorbol 12-myristate 13-acetate, 1 μg/mL of ionomycin, and GolgiStop (BD Biosciences) for 4 hours. The Fc receptor blocking reagent (eBioscience) was initially used in all of the samples, and then sequential surface and intracellular staining were performed with the following antibodies: CD19 (HIB19; BioLegend), CD27 (M-T271; BD Biosciences), BAFF (1D6; eBioscience), and APRIL (REA347; Miltenyi Biotec). A fixable viability stain was applied to exclude dead cells (BD Biosciences). Data were acquired with an LSR Fortessa (BD Biosciences) and were analyzed with FlowJo software (FlowJo LLC).

Brain tumors were excised aseptically from mice, weighed, and mechanically diced. Single-cell suspensions were made using Brain Tumor Dissociation Kit (Miltenyi Biotech, Bergisch Gladbach, Germany) and gentleMACS^TM Octo Dissociator (Miltenyi Biotech, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. The single-cell suspensions were filtered by a 70 μm filter and resuspended in FACS stain buffer. Red blood cells were lysed using an RBC removal solution. Cells were incubated with Fixable Viability Stain (BD Pharmingen, Franklin, NJ, USA). The cells were labeled with combinations of antibodies (Supplementary Table 3) against cell surface markers. At last, the cells were resuspended in FACS staining buffer and analyzed on the Beckman CytoFLEX (Brea, CA, USA). BD CompBeads were used to optimize fluorescence settings (BD Biosciences, Franklin, NJ, USA). Fluorescence-minus-one, unstained, and single-stained cells were also used to set gates.