Canto 2 flow cytometer

To assess phenotype markers, human and murine PC cells were harvested and mechanically dissociated into a single cell suspension. Then, the cells were pelleted, resuspended in PBS and incubated for 30 min, at 4 °C, with the Abs listed in the Supplemental Methods [see Additional file 1]. Acquisition was performed using a BD Scientific Canto II Flow Cytometer (RRID:SCR_018056), and the data were analyzed using FlowJo software (RRID:SCR_008520). Dead cells were excluded by 7AAD staining.

Phenotypic analysis was performed on freshly isolated PBMC using the following monoclonal antibodies. From Fisher Scientific: PD-1 (J105), CD4 (RPA-T4), FoxP3 (236A/E7), CD127 (eBioRDR5), CD25 (4E3); from BioLegend: CD8 (SK1), CD3 (UCHT1), CD14 (M5E2), and CD19 (H1B19). The TCR Vβ repertoire was analyzed using the IOTest Beta Mark Kit (Beckman-Coulter). Staining for MART-1/Melan-A [26–35 (27L); ELAGIGILTV] specific T cells was performed using APC-bound pentamers (ProImmune). Intracellular staining (FoxP3) was performed after applying fixation/permeabilization buffer (Fisher Scientific). All samples were run on a Canto II flow cytometer and analyzed using FlowJo Ver. 9.5.8. Dominant TCR Vβ populations were identified based on any Vβ chain whose frequency was considered to be a statistical outlier in the repertoire of the 24 Vβ chains that were analyzed. An outlier test was used to define a Vβ as dominant if its frequency was at least three interquartile distances away from the third quartile of all the Vβ chains analyzed.

Peripheral blood mononuclear cells (PBMC) were isolated from Trima residuals of healthy individuals with Ficoll-Paque Plus. 500,000 PBMCs were incubated in U-bottom 96-well plates (Falcon) with anti-SIRP at a concentration of 10 ug/mL for 48 h at 37C.
For quantification of PBMC subsets by flow cytometry, cells were incubated in human FcR blocking reagent and stained with a cocktail of fluorochrome-labeled antibodies against lin—(CD3, CD14, CD16, CD19, CD56) and HLADR. Fixable viability dye was used to identify live cells. After staining, cells were washed and fixed with 0.5% paraformaldehyde in PBS. Prior to acquisition, absolute counting beads (Thermo Fisher) were added and samples were acquired with Canto II flow cytometer and analyzed using FlowJo software.

Signal transducer and activator of transcription 5 (STAT5) phosphorylation after stimulation with supernatants containing hIL-7-Fc or not was assessed on whole blood of patients with septic shock and COVID-19 and HVs. Whole blood of patients was stimulated for 10 min using diluted supernatants from MVA-hIL-7-Fc– or empty MVA–infected or –uninfected cells or directly with the rhIL-7. Whole blood cells were stained with anti-human CD3 PE-Cy7 (Clone SK7, BD Biosciences) and anti-human CD45 APC-H7 (Clone 2D1, BD Biosciences). Fixation, red blood cell lysis, and permeabilization steps were performed using Perfix-Expose kit (Beckman Coulter) as recommended by the manufacturer. After permeabilization, cells were stained with anti-human pSTAT5 (Clone 47/Stat5 (pY694), BD Biosciences). Canto II flow cytometer was used to analyze cells and data were analyzed using the FlowJo software.