Cd4 apc vio770

1.5 × 10^{^6} of thawed PBMCs were plated in complete RPMI containing 10% human serum supplemented with 1% Penicillin–Streptomycin–Glutamin. Overnight-rested PBMCs were stained with the appropriate antibodies for 20 min at 4 °C in the dark and acquired using FACSVerse™cytometer (BD Biosciences). Dead cells were labeled using ViobilityTM Fixable Dye (Miltenyi Biotec). Antibodies used were: CD4-APC-Vio770, CD8-APC, HLA-DR-VioBlue, CD38-PE-Vio770, Granzyme B-PE and Perforin-FITC (Miltenyi Biotec). (Representative plots are shown in Supplementary Fig. 6). Data were analyzed using FlowJo 10.7.2 (BD Biosciences).

Phenotypes and activation markers were evaluated by Miltenyi Biotec flow cytometer-MACSQuant Analyzer (8 fluorescence channels, three lasers) on freshly isolated peripheral blood mononuclear cells. Immune activation was evaluated by multiparameter flow cytofluorimetric analysis by the following anti-human monoclonal antibodies: CD3-PerCP, CD4-APC-Vio770, CD8-FITC, CD45RO-PE-Vio770, CD27-VioBlue, CD38-APC, and HLA-DR-PE (Miltenyi Biotec, Bergisch Gladbach, Germany).

Whole blood was stimulated with 10 μg/mL phytohemagglutinin (PHA, Sigma-Aldrich) for 6 h and 24 h. After 6 h, activation markers on T cells were measured using CD69-APC (clone: REA824), CD71-FITC (clone: REA902), CD154-VioBlue (REA238) and CD25-PE (clone: 3G10), CD3-VioGreen (REA613), CD4-APC-Vio770 (REA623), and CD8-PE-Vio770 (REA734) antibodies and propidium iodide as viability dye (all Miltenyi Biotec, Bergisch-Gladbach, Germany) using a MACSQuant 16 analyzer (Miltenyi Biotec). After 24 h, culture supernatants were collected for cytokine analysis.

To assess EV internalization by IECs, MSC membranes were stained with 2×10⁻⁶ M of PKH26 PKH26 Red Fluorescent dye (Sigma-Aldrich) according to manufacturer’s recommendations. Then, PKH26-labeled or -unlabeled MSCs were cultured in presence of IECs and EV uptake was assessed after 1, 2 or 4 days. At the end of co-culture, cells were detached by trypsin and stained with the following monoclonal antibodies: CD45-Vioblue (Miltenyi Biotec), CD3-V500 (BD Biosciences), CD4-APC-Vio770, CD8-FITC, CD14-FITC (Miltenyi Biotec), CD16-PercP-Cy5, CD19-PE-Cy7 (BD Biosciences) to identify the different IEC population, while TOPRO-3 was used to identify viable cells. The internalization of MSC-derived EVs by IECs was analyzed by FACS analysis.
To further confirm the transfer of EVs into IECs, cells were analyzed at the end of co-culture by confocal microscopy. Briefly, cells were detached by trypsin and stained with Viobright-FITC anti-human CD45 monoclonal antibody (Miltenyi Biotec). Then, cells were fixed using Cytofix/Cytoperm kit (BD Biosciences) and TOPRO-3 (Invitrogen Life Technologies) was used to reveal nuclei. Finally, cells were loaded into the CytoSpin centrifuge’s sample chamber and centrifuged 5 minutes at 400 rpm.
Images were obtained by LSM 710 confocal microscopy (Zeiss) at 63x magnification and elaborated with ZEN imaging software (Zeiss).

Multicolor flow cytometric analysis was performed, and the gating strategy is reported in the Supplementary Materials. The first tube was to detect B cell subsets and included the following mAbs: CD19-PE-Cy7 (SJ25C1), CD24-APC-Cy7 (ML5), CD38-FITC (HB-7), CD5-PerCP-Cy5.5 (L17F12), CD1d-PE (all from BD Biosciences, San Jose, CA, USA), CD45-APC (REA747 Miltenyi Biotec S.r.l., 40133 Bologna, Italy), and CD27-BV510 (M-T271 BioLegend, San Diego, CA, USA).
The second tube was to detect follicular T cells and included mAbs: CD4-APC-Vio770, CD45RA-PE-Vio770, CCR6-APC, CXCR3-VioBright FITC CXCR5 PerCP-Cy5.5, and CCR4-PE, all from Miltenyi Biotec (40133 Bologna, Italy).
The third tube was to phenotype regulatory T cells according to the BD Human Regulatory T Cell Cocktail (CD4-FITC (SK3), CD25-PECy7 (2A3), CD127 Alexa Fluor^® 647, BD, San Jose, CA, USA), CXCR5 PerCP-Cy5, CD8 horizon450, and CD3 APC-Cy7 (OKT3) (Biolegend, San Diego, CA, USA).
Gating was carried out using Kaluza software 2.1 (Beckman Coulter, Inc., Danvers, MA, USA): details are shown in the Supplementary Materials. The T and B cell subsets are also described in the Supplementary Materials.

PBMC were collected and aliquoted in 1 × 10⁶ cells/mL RPMI medium plus 10% Fetal Bovine Serum and then washed by centrifugation. The following anti-human monoclonal antibodies were added: CD3-PerCP, CD4⁺-APC-Vio770, CD8⁺-FITC, CD4⁺ 5RO-PEVio770, CD27-VioBlue, CD38-APC, (Miltenyi Biotec, Bergisch Gladbach, Germany). Samples were acquired by Miltenyi Biotec flow cytometer-MACSQuant Analyzer (8 fluorescence channels, 3 lasers). Gating and data analysis were performed using MACSQuantify software 2.5 (Miltenyi Biotec, Bergisch Gladbach, Germany).

T cell absolute count was performed on blood samples kept from the mandibular vein of the mouse.
For the phenotypic characterization of cell populations, the following antibodies were used: CD8-FITC (Miltenyi Biotec), CD25-APC (Pharmingen), CD3-PE-Vio770 (Miltenyi Biotec), CD4-APC-Vio770 (Milteny Biotec), CD45R (B220)-Violblu (Milteny Biotec), NK1.1-PE (Milteny Biotec).
At predetermined optimal concentrations, 100 μl of blood was stained by incubation with the antibodies. Fifty microliters of CountBright Absolute Counting Beads (Molecular Probes) was added, and, following lysis of red blood cells, cells were acquired on a CyAn Cytometer (Beckman Coulter). By comparing the ratio of bead events to cell events, absolute numbers of cells in the sample were calculated.
Some experiments were performed by acquiring the stained blood samples on the CytoFLEX cytometer (Coulter), equipped with a volumetric sample injection module, which enables volumetric sampling and provides absolute cell counts for all samples without the use of beads.