Cd3 vioblue

CAR expression was assessed using biotinylated anti-Fc (Jackson ImmunoResearch, 1:100, RRID: AB_2337663) antibody followed by streptavidin-phycoerythrin (PE) (BD Biosciences, 1:20, RRID: AB_10053328) and by staining for the CD19t extracellular sequence with CD19-PE-Cy7 (BD Biosciences, clone SJ25C1, 1:100, RRID: AB_396893). Target lines were characterized by staining with IL13Rα2-PE (BioLegend, clone SHM38, 1:100, RRID: AB_11218806), IL13Rα1 (BioLegend, clone SS12B, 1:100, RRID: AB_2562552), and IL-4Rα-PE (BD Pharmingen, clone hIL4R-M57, 1:20, RRID: AB_394355). In other assays, additional antibodies were used as specified: CD107a-FITC (BD Biosciences, clone H4A3, 1:9, RRID: AB_396134), CD45 PerCP (BD Biosciences, clone 2D1, 1:20, RRID: AB_2868830), CD3-VioBlue (Miltenyi Biotec, 1:20, RRID: AB_2725961), CD8 APC-Cy7 (BD Biosciences, clone SK1, 1:50, RRID: AB_396892), and IFNγ-APC (BD Biosciences, clone B27, 1:100, RRID: AB_398580). For staining, cells were washed and resuspended in fluorescence-activated cell sorting (FACS) stain solution [Hank’s Balanced Salt Solution (HBSS), 20% (vol/vol) FBS, 0.1% (wt/vol) NaN₃ (CAS 26628-22-8)], incubated with antibodies for 30 min at 4 °C, followed by secondary stain if necessary, and then washed and run on the MACSQuant (Miltenyi Biotec, RRID: SCR_020268). Flow data were analyzed with FCS Express 4 (De Novo Software, RRID: SCR_016431).

Peripheral blood samples were collected in tubes containing ethylenediaminetetraacetic acid and Lyse/Wash Procedure was applied; lymphocytes T cells enumeration and activation were evaluated by the following anti-human monoclonal antibodies: CD3 VioBlue, CD 4 APC-Vio770, CD8 FITC, CD38 APC and HLA-DRPE (MiltenyiBiotec). 5-color flow cytometric analysis was performed with the MiltenyiBiotec flow citometer-MACSQuantAnalyzer (8 fluoresces, 3 lasers). Gating analysis was developed with MACSQuantify software 2.5 (MiltenyiBiotec) with the same gating strategy applied to all samples. We tested specificity for the expression of CD38 and HLA-DR markers, by using Fluorescence Minus One method (FMO): each cell population was stained with all the fluorescence antibodies, except one, at a time, in order to verify whether in the absence of one antibody, the labelled cells were negative for the removed one; this method allows the subset to be gated and the background level for the subset to be determined.

For FACS analysis, healthy and lesional skin (both from corresponding anatomical sites) or blood was taken from mice after induction of experimental EBA. Single cell solutions from and blood were erythrocyte lysed with RB cell lysis buffer (Miltenyi). The skin samples were cut into small pieces and digested with 345 mg/ml liberase (Roche) in RPMI for 30 min/37°C. Single cells were stained for the following surface markers using standard FACS procedures: CD45-VioGreen (clone: 30F11), CD3-VioBlue (clone: 17A2), Ly6C-FITC (clone: 1G7.G10), as well as Ly6G-APC Vio770 (clone: 1A8) and for blood CD19-APC (clone: 6D5), all from Miltenyi. For the subsequent intracellular staining, the cells were fixed in fixation buffer (BioLegend) and permeabilized using the Intracellular Staining Perm Wash Buffer (BioLegend) following the manufacturer’s protocol. Intracellular staining was performed with SYK-PE (clone: 4D10.2). Cells were first gated for scatter (SSC-A/FSC-A) and singlets (FSC-H/FSC-A). The CD45⁺ gates were further analyzed for double-positive staining of SYK with the appropriate cell markers. Measurements were performed at the Miltenyi MacsQuant10, and data were analyzed with the MACSQuantify™ Software (version 2.8).

To assess the cell-surface expression of PD-1 on distinct PBMCs subpopulations by flow cytometry, PBMCs were counted and placed in 96-well plates and washed twice with 200 μL/well cell staining buffer (Hank’s buffered salt solution, 3% fetal bovine serum and 0.1% sodium azide, 200 μL/well). Fc receptors were blocked with FcR Block (Miltenyi Biotec GmbH) in 50 μL staining buffer, 3 min at 25°C. Cells were stained with antibodies to surface markers of interest (all with corresponding isotypes) in a total volume of 100 μL: CD3-VioBlue (Miltenyi Biotec GmbH), CD8-VioGreen (Miltenyi Biotec GmbH), CD4-PerCPCy5.5 (Peridinin chlorophyll protein complex -BioLegend, San Diego, CA), anti-PD-1-APC (eBioscience, San Diego, CA). After washing and resuspending, cell surface fluorescence was analyzed using a BD LSR II Flow Cytometer (BD Biosciences, San Diego, CA). Gates of interest were defined using Flow-Jo Software V.10 (Tree Star, Inc. Ashland, OR) and encompassed either lymphocytes or large cells (Fig 1). In each gate the number and percentage of cells that expressed surface PD-1 was examined at baseline and after 24 h in culture with and without stimulus. Mean percentage expression of PD-1 was compared by one-way ANOVA on each of the PBMCs subpopulations among the 4 study groups and T1D Follow up.

Whole blood was drawn from patients and healthy controls, collected in heparin-treated tubes, treated with Ficoll Histopaque-1077, and centrifuged to isolate leukocytes. The leukocyte layer was isolated, washed with PBS, and centrifuged. Cells were counted using a hemacytometer and Trypan blue then diluted to 10⁶ cells/ml in FACS buffer (2 mM EDTA, 0.5% BSA, PBS). Cells were Fc blocked and stained with the following monoclonal anti-human antibodies: CD3-VioBlue (Miltenyi Biotec: Cat# 130-113-133, RRID : AB_2725961) and VISTA-APC (Thermo Fisher Scientific: Cat# 17-1088-42, RRID : AB_2744704). Fluorescence minus one (FMO) control was used to determine positive expression gates during analysis using FlowJo software.

Isolated T cells were stained with fluorochrome-labeled mAbs directed against human CD4/FITC (clone VIT4, MiltenyiBiotec), CD3/Vioblue (MiltenyiBiotec) and CD8/APC (clone BW 135/80, MiltenyiBiotec). For analysis of expression of GD2 on JF cells, the cells were stained using the commercial anti-GD2 mAb (Biolegend, San Diego, USA; Clone 14G2a), detected with Alexa Flour 647-labeled goat anti-mouse IgG (Life technologies, Thermo Fisher Scientific). For detection of CAR surface expression T cells were incubated with the mAb 7B6 and subsequently stained with PE-labeled goat anti-mouse IgG (Beckmann Coulter, Krefeld, Germany). Samples were analyzed using a MACSQuant^® Analyzer and MACSQuantify^® software (Miltenyi Biotec) [56 (link)].

Whole blood samples were collected from healthy donors after obtaining informed consent in accordance with the “Code for Proper Use of Human Tissues” as formulated by the Dutch Federation of Medical Scientific Organizations (www.fmwv.nl)49 (link). PBMC were isolated from whole blood using Lymphoprep™ (STEMCELL Technologies, Vancouver, Canada) density gradient centrifugation. PBMC were incubated at a concentration of 5 × 10⁶/ml in 6 well plates in Roswell Park Memorial Institute (RPMI) medium (Sigma Aldrich, Zwijndrecht, The Netherlands) containing 2% human serum albumin (HSA) overnight at 37 °C, 95% humidity, 5% CO₂ atmosphere. PBMC viability, numbers and NK cell content were counted volumetrically by FACS using a cocktail of 7-AAD (1:200) (Sigma Aldrich, Zwijndrecht, The Netherlands), CD3 VioBlue (1:11), CD56 APC-Vio 770 (1:11), CD16 APC (1:11), CD45 VioGreen (1:11), and CD25 VioBrightFITC (1:11) antibodies from Miltenyi Biotec GmbH. PBMC viability (7AAD− CD45+) and NK cell (7AAD− CD45+ CD3− CD56+) and NK CD16 (7AAD− CD3− CD56+ CD16+) percentages measured before and after overnight incubation were 7AAD− CD45 + (lymphocytes): 91% ± 4% versus 84 ± 2%, %; 7AAD− CD45+ CD3− CD56+ (NK cells): 7.1% ± 3% versus 6.8% ± 6% and CD3− CD56+CD16+ (CD16 positive cells within NK cells): 89% ± 3% & 82% ± 9%.