Ctla 4 apc

Fc-gamma receptors were blocked with a rat anti-mouse CD16/CD32 antibody (BD). Mouse-specific antibodies were the following: CD3-Brilliant violet 510, CD25-PercP Cy5.5, I-A/I-E-Brilliant violet 510, CD45R/B220-PE-Cy7, CD43-APC, CTLA-4-APC (all from BioLegend), CD4-APC-Cy7, CD69-PE, CD11c-PercP Cy5.5, CD19-APC-Cy7, CD40-FITC (all from BD Pharmingen), CD8α-PE-Cy7, FoxP3-FITC, CD86-APC, CD83-FITC, CD80-PE-Cy7, IgM-PercP-eFluor 710 (all from eBioscience).
During the experimental set-up, CD3 antibody was included in the staining panel (BV510 BioLegend clone 17A2) and used for the gating of T cells. The results obtained with gating on CD3+ CD4+ CD8- were similar to those obtained with gating on CD4+ CD8-. Due to the limitation in the maximum number of colours that can be discriminated with the FACS Canto II, CD3 staining was omitted in subsequent stainings.
Dead cells were excluded using the fixable viability dye-eFluor 450 (eBioscience), and intracellular staining was performed using the fixation/ permeabilization buffer set from eBioscience. Flow cytometry measurements were performed on a FACS Canto II instrument (BD) and the data were analyzed with FlowJo software (Tree Star).

For splenic, PP or MLN T cell flow cytometry, positively selected CD4⁺ cells were stained with combinations of antibodies (0.5 µg/ml per) to Tim3-APC (BioLegend, San Diego, CA) or Lag3-APC (BD Bioscience), PD1-APC (BioLegend, San Diego, CA), CTLA4-APC (BioLegend, San Diego, CA), Ly6A/E-APC (BD Biosciences, San Jose, CA), and GITR-APC (BD Biosciences, San Jose, CA). Cells were washed twice in MACS buffer (Miltenyi Biotec, San Diego, CA) before analysis using Attune NxT (ThermoFisher, Waltham, MA) with the support of the Cytometry & Imaging Microscopy Core Facility of the Case Comprehensive Cancer Center. Analysis of all FACS data was performed using FlowJo (Tree Star, Inc., Ashland, OR).

To analyze the content of immunomodulatory proteins on the surface of SEVs, a spectral flow cytometer with an enhanced small particle detection option (Cytek® Northern Lights™) at the FlowCore Mannheim was used. SEVs were diluted (1:20 in 100 µL PBS) and stained with 1 µL fluorescently-labeled antibodies for tetraspanins (CD9-, CD63-, CD81-FITC; BioLegend) and immunomodulatory proteins (PD-L1-PE, CTLA-4-APC, FasL-PE, TGF-β-APC, Tim3-BV421, LAG-3-BV421; BioLegend) for 30 min at 4 °C. Afterwards EVs were diluted 1:4 in PBS and 50 µL of sample was acquired. As negative controls, PBS only and PBS with antibodies were used. To prove that the signal was related to SEVs, we also disrupted the lipid bilayer of the vesicles using 0.5% sodium dodecyl sulfate (SDS). Next, EVs were gated according to ApogeeMix beads (#1527) to exclude particles or aggregates that are larger than 500 nm polystyrene beads (Suppl. Figure 2). Additionally, isotype and SDS controls were examined to exclude unspecific binding of the antibodies. The count of positive events for immunomodulatory proteins in the EV marker-positive population was calculated using FlowJo and normalized to the volume of 50 µL.