Efluortm 506

Murine monocytes were isolated from the bone marrow of C57BL/6 mice by magnetic depletion (EasySep™ Mouse Monocyte Isolation Kit, STEMCELL Technologies Inc., Vancouver, BC, Canada). 5 × 10⁴ cells were cultured in 48-well plates in sEV-free RPMI medium and treated for 8 h with 5 µg of the respective sEV fractions referred above, as determined by BCA assay. Changes in PD-L1, HLA-DR and ICAM-1 expression were evaluated by flow cytometry (BD LSR Fortessa, BD Biosciences, San Jose, CA, USA). The following antibodies were used: PD-L1-PerCP (Biolegend, San Diego, CA, USA, #46-5982-82), HLA-DR-AlexaFluor700 (eBiosciences, San Diego, CA, USA, #56-5321-82), CD54-PE (Biolegend, San Diego, CA, USA, #116108), CX3CR1-BV711 (Biolegend, San Diego, CA, USA, #149031), Ly6C-APC-Cy7 (Biolegend, San Diego, CA, USA, #128015), CD11b-PeCy7 (Biolegend, San Diego, CA, USA, #101216), F4/80-FITC (Biolegend, San Diego, CA, USA, #123107), and the viability dye eFluorTM 506 (eBiosciences, San Diego, CA, USA, #65-0866).

PBMCs were cultured in RPMI-1640 medium (Gibco) containing 10% fetal bovine serum and stimulated with anti-CD3/CD28 (2 and 2.5 μg/mL) at the presence of Plus Golgiplug (BD Pharmingen, San Diego, CA, USA) for 5 hours. Cell viability was assessed using the Fixable viability dye eFluor^TM 506 (invitrogen, Carlsbad, CA, USA). Cells were then surface stained with CD4-FITC, CD8-APC-H7, PD-1-BV785, and CD26-PE-CF594. After fixation and permeabilization, intracellular staining was performed with IL-2-PE-Cy7, TNF-α-BV421, IFN-γ-APC (BD Biosciences) antibodies.