Igg1 isotype control

Cells at the 5th passage of culture in each oxygen tension were detached from flasks using Accutase (Sigma-Aldrich). For identification of stem cell surface markers, cells (1 × 10⁵) were labeled with antibodies against the surface markers CD105 (FITC, mouse, Abcam, Cambridge, UK), CXCR4 (FITC, mouse, R&D systems, USA), G-CSFR (FITC, mouse, R&D systems, USA), IgG1 isotype control (FITC, mouse, Biolegend, USA) and IgG2a isotype control (FITC, mouse, Santa Cruz, USA) for 1 hour, at 4 °C. Labeled cells were analyzed using FACSAria II flow cytometer (Becton Dickinson, USA). Only viable cells as determined by propidium iodide (PI) (Sigma-Aldrich) exclusion were gated and analyzed.

HEK-293A cell monolayers were transfected with constructs encoding either swCD40 (pcDNAswCD40) or boCD40 (pcDNAboCD40) using Polyethylenimine (Polyscience) as previously described [33 ]. Following 48hr. incubation, the monolayers were fixed with cold methanol, rinsed with PBS, blocked with 10% FBS/PBS solution, and incubated for 1 hr. at room temperature with 5μg/mL of the mAb 2E4E4 or 5μg/mL of an IgG1 isotype control (Biolegend). The cell monolayers were washed 3X with blocking buffer and then incubated for 1 hr. with Alkaline Phosphatase AffiniPure F(ab’)₂ fragment donkey anti-mouse IgG (H+L) (Jackson ImmunoResearch Laboratories, INC). Following washes as above, Fast Red TR–Naphthol AS-MX substrate (Sigma, F4523) was used to detect alkaline phosphatase activity. Photos were captured using Spot RT3 camera on Olympus IX70 microscope.

K562-EV or K562-CD1a were pulsed with GAS (MOI=50 or 100) for 72 hours and the extracellular bacteria were removed before coculturing with T cells. In some conditions, K562-EV or K562-CD1a cells were pulsed with lysophosphatidylcholine 18:1 (150 µM; Avanti Polar Lipids) for 16 hours and the excess lipids were removed before coculturing with T cells. Blood or skin T cells (1x10⁶) were co-cultured with control/pulsed K562-EV or K562-CD1a (0.5x10⁶) for 4-6 hrs. In indicated conditions, K562-CD1a was pretreated with anti-CD1a blocking antibody (10 µg/ml) or IgG1 isotype control (10 µg/ml; BioLegend) for 1 hr before the addition of T cells. Cytokine producing responder T cells were detected using Cytokine Secretion assays (Miltenyi Biotec) following the manufacturer’s instructions. T cells were coated with anti-cytokine (IL-22, IFNγ, GM-CSF, or IL-17A) antibody after coculture to detect CD1a dependent autocrine cytokine production using fluorochrome-conjugated detection antibodies. Antibodies against surface markers identifying T cells (anti-CD3, anti-CD4, anti-CD8, anti-TCRαβ) and their phenotypes (anti-CD45RA, anti-CD45RO, anti-CD25, anti-CD69, anti-CD137, anti-CD154, anti-CLA). Data were acquired using LSRFortessa X-50 flow cytometer (BD Biosciences) and further analyzed with FlowJo (FlowJo LLC) software.

CellEvent Caspase 3/7 Green Flow Cytometry Assay Kit (Invitrogen) was used to measure caspase acitivty in thymocytes and mature peripheral T cells as per the manufacturer's instructions. Briefly, after incubation of thymocytes with 50 ng/ml TNFα or mature T cells with 1 μg/ml anti-CD3 and 0.2 μg/ml anti-CD28 antibodies for 16 h, CellEvent Caspase 3/7 Green Detection Reagent was added and incubated with cells for 30 min at 37 °C with 5% CO₂. Samples were analyzed using an LSR II flow cytometric analyzer (BD Biosciences) and analyzed using the Flowjo software (Treestar). In some cases, SYTOX AADvanced Dead Cell Stain (Invitrogen) was used to stain for dead cells. For in vivo TNFα blockade, mice were treated every 3.5 days for 2 weeks by intraperitoneal injection with 100 μg of anti-TNFα antibody (MP6-XT22, Biolegend, San Diego, CA, USA), IgG1 isotype control (Biolegend). After 2 weeks of treatment, lymph nodes and spleen were isolated, single-cell suspensions were made, and lymphocyte populations were determined by FACS analysis using an LSR II (BD Biosciences).

A minimum of 30mL of blood was collected from pigs, cows, goats, and sheep. The blood was then processed to isolate PBMCs by Histopaque® (Sigma-Aldrich) density gradient centrifugation following the manufacturer’s protocol. Swine and bovine PBMCs were added to a 12-well plate (4 million PBMCs per well) and incubated for 24 hr. in 1mL of complete RPMI alone or in cRPMI containing LPS (10μg/mL). Half a million swine or bovine PBMCs from either treatment was added to each well of a 96 well V-bottom plate (Axygen), stained with Zombie red^TM viability kit (Biolegend) following manufacturer’s protocol, and blocked using either swine blocking buffer (cRPMI with 0.05% sodium azide/20% swine serum) or bovine blocking buffer (cRPMI with 0.05% sodium azide/20% bovine serum). The swine and bovine PBMCs were incubated for 30 min. on ice with either 5μg/mL of the mAb 2E4E4 conjugated to FITC or 5μg/mL of IgG1 isotype control (Biolegend) conjugated to FITC. The antibodies were conjugated to FITC using FluoroTag^™ FITC Conjugation Kit (Sigma-Aldrich) following manufacturer’s protocol. After incubation with the mAbs, the cells were washed 3X with blocking buffer, and fixed using FACS Fixer. Flow cytometry data was collected and analyzed as above.

Human umbilical vein endothelial cells (HUVEC) were isolated from acquired umbilical cords using a collagenase perfusion technique. HUVEC were grown in T75 flasks at 37 °C and 5% CO₂ until confluent and then seeded at a confluent density onto glutaraldehyde-crosslinked gelatin coated 30 mm glass round coverslips (Warner Instruments). Coverslips were utilized 36–48 h after seeding.
HUVEC were activated with 1 ng/mL IL-1β (Fitzgerald) in cell media 4 h before use in flow experiments. HUVEC were also activated with 100 µM histamine (Acros Organics) 2 min before use in flow experiments.
For E-selectin blocking experiments, activated HUVEC were blocked with 20 µg/mL anti-E-selectin (R&D Systems #BBA26) or IgG1 isotype control (BioLegend #400102) for 30 min before use in flow experiments to prevent leukocyte adhesion.