Pd l1 29e 2a3

Vesicles generated from prospective transplant donor PBMC were incubated with recipient-derived DCreg for 18–20 h (37°C; 5% CO₂) at 10–50 μg vesicles per 10⁵ DCreg. To examine their capture by DCreg, the vesicles were pre-labeled with the red membrane dye PKH26 (Sigma-Aldrich; PKH26GL), following the manufacturer’s protocol before incubation with the DCreg. Vesicle capture was then evaluated by flow cytometry.
To characterize the phenotype of DCreg loaded with donor-derived vesicles, the cells were stained with the following monoclonal antibodies (mAbs): APC-HLA-DR (G46-6), PerCP-Cy5.5-CD86 (FUN-1), V450-CD80 (L307.4), FITC-CD83 (HB15E) (all from BD Biosciences, San Jose, CA) and PE-Cy7-programed death-ligand (PD-L)1 (29E.2A3) (Biolegend, San Diego, CA), then analyzed by flow cytometry. For functional assessment, DCreg or control DC were cultured with autologous T cells at 1:40 ratio for 4–5 days. The cultures were then split; one half was used to measure T cell proliferation by ³H-thymidine incorporation and the other half rested for 3 days before re-stimulation with allogeneic PBMC from the same donor as the vesicles at 1:10 (PBMC:T cell) ratio for 3 days. Proliferation was measured by ³H-thymidine incorporation.

Blood and airway PMNs collected from human subjects and from our transmigration model were assessed by flow cytometry using standard templates for staining, acquisition and analysis, as described, enabling quantitative comparison of in vivo and in vitro samples throughout the whole study [21 ]. Reagents used for the study included Live/Dead (Invitrogen), and antibodies against Arg1 (6G3, Hycult Biotech), as well as CD11b (M1/70), CD16 (3G8), CD41a (HIP8), CD45 (HI30), CD63 (H5C6), CD66b (G10F5), CD62L (DREG-56), and PD-L1 (29E.2A3) from BioLegend, and the ROS probe CellRox (ThermoFisher), using the gating strategy in Suppl. Fig. 1A. Phosphorylated 5’ adenosine monophosphate-activated protein kinase (AMPK) α1 levels were assessed using an antibody against phospho-Thr172 (Biorbyt) using a previously published protocol [7 (link)]. For Glut1 detection, we used a specific receptor-binding domain probe (Metafora Biosciences), as detailed before [10 (link)]. Data were acquired on a LSRII cytometer (BD Biosciences) and compensated, gated and analyzed in Flowjo (Treestar). Live PMNs were counted using CountBright beads (Life Technologies), and expression of all markers is reported as median fluorescence intensities.