Anti mouse cd16 cd32 fc block

The immune cell samples were transferred on a V-bottom 96-well plate and blocked with Fc block (anti-mouse CD16/CD32, 1:100 dilution, Thermofisher Scientific, Belgium) for 30 min on ice in the dark. Afterward, the samples were washed with flow cytometry buffer and centrifuged at 1600 RPM at 4 °C for 3 min. The pellets were then stained with the fluorochrome-conjugated antibodies for 30 min on ice in the dark. Afterward, the samples were washed with flow cytometry buffer and centrifuged at 1600 RPM at 4 °C for 3 min. Finally, all the samples were resuspended in 200 µL of flow cytometry buffer and analyzed on a CANTO II flow cytometer (Becton-Dickinson Benelux, Belgium). All antibodies used in this procedure were used at 1:100 dilution/10⁶ cells stained and are listed in the Supplementary Information 3, Table S4.
Cells were gated on singlets and T lymphocyte (CD3⁺ cells) subpopulations were defined as follows: T cytotoxic (CD3⁺, CD8⁺, CD4⁻), T helper (CD3⁺, CD8⁻, CD4⁺), T helper 1 (CD3⁺, CD4⁺, CXCR3⁺), T helper 2 (CD3⁺, CD4⁺, CXCR3⁻, CCR6⁻, CCR4⁺, CCR8⁺), T helper 9 (CD3⁺, CD4⁺, CXCR3⁻, CCR6⁺, CCR4⁻), T helper 17 (CD3⁺, CD4⁺, CXCR3⁻, CCR6⁺, CCR4⁺, CCR10⁻), T helper 22 (CD3⁺, CD4⁺, CXCR3⁻, CCR6⁺, CCR4⁺, CCR10⁺). Data were analyzed using FCS Express 7 (DeNovo Software, California, USA) by a blinded observer (P.C.) and expressed as a percentage of CD3⁺ cells.

NPICCs were dissociated into single cells by treatment with TrypLE (Thermo Fisher Scientific, Germering, Germany) and washed with PBS + 10% foetal calf serum (FCS) and filtered through a 30-µm pre-separation filter (Miltenyi, Bergisch-Gladbach, Germany). Then cells were fixed/permeabilized using an intracellular staining buffer set and incubated with Fc-Block (anti-mouse CD16/CD32) for 10 min at room temperature (Thermo Fisher Scientific, Germering, Germany). Intracellular staining was performed using the following fluorochrome-labeled antibodies: mouse anti-insulin AF647 (clone T56-107), mouse anti-glucagon-PE (clone U16-850), mouse anti-somatostatin AF488 (clone U24-3545), mouse anti-Pdx-1-PE (clone 658A5), mouse anti-Pdx-1-AF488 (clone 658A5), mouse anti-Nkx6.1-AF647 and mouse anti Nkx6.1-PE (clone R11-560) (all from BD Biosciences, Heidelberg, Germany). All antibodies were pretested for appropriate dilution and for specificity using isotype control antibodies. Antibodies were incubated at 4 °C for 30 min followed by two washing steps with a permeabilization buffer. Flow cytometry data were acquired on a flow cytometer (BD Accuri™ C6 Plus Flow Cytometer, BD Biosciences, Heidelberg, Germany) and analyzed using FlowJo software version 10.4 (TreeStar, Ashland, USA).

All cells were stained with Live/Dead Fixable Aqua (1:2000; Invitrogen) at room temperature for 30 min in the dark. Following the Live/Dead viability stain, cells were incubated with anti-mouse CD16/CD32 (Fc block; 1:100, eBioscience), CD3 PE-eFluor610 (1:100; eBioscience), CD4 PE (1:100; eBioscience), CD8b APC-eFluor780 (1:100; eBioscience), CD11b PE-Cy7 (1:200; eBioscience), Ly6G Pacific Blue (1:100; BioLegend), CD45 PerCP-Cy5.5 (1:100; eBioscience), CD11c Alexa Fluor 700 (1:50; BioLegend) Ly6C FITC (1:200; eBioscience) in FACS buffer. Samples were run on an LSRII (BD Biosciences) flow cytometer and analyzed with FlowJo_V10 software.
Single cell lymphocytes were gated on forward scatter area (FSA; size) and side scatter area (SSA; granularity) and then by forward scatter height (FSH) and FSA. Live cells were selected as the negative Fixable Aqua population. CD45⁺ cells were selected and gated by CD3 (T cells) versus CD11b (myeloid cells). The CD3⁺ population was then gated on CD4 (helper T cells) and CD8 (cytotoxic T cells). CD11b⁺ cells were further gated on CD11c and Ly6G for the following populations: CD11c⁻Ly6G⁻ (microglia and macrophages), CD11c-Ly6G⁺ (neutrophils), and CD11c⁺Ly6G⁻ (dendritic cells). CD11c⁻Ly6G⁻ cells were gated for CD45 and low and high populations were further gated for MHCII and Ly6C to determine their activation state (Suppl. Fig. S1).

Blood was collected in heparinized tubes and before centrifugation, 10 μl of blood sample were taken for determining the absolute number of leukocytes using CD45-FITC antibody (BioLegend, Fell, Germany) and AccuCount particles (Spherotech Inc., Lake Forest, IL, USA). After centrifugation plasma was taken and the cell pellet was depleted of erythrocytes by two treatment steps with 50 ml erythrocyte lysis buffer (0.15 M NH₄Cl, 10 mM KHCO₃, 0.1 mM Na₂EDTA pH 7.3). After washing with PBS, cells were incubated for 10 min on ice with FACS buffer (1 % fetal bovine serum in PBS) containing 1 μg of purified anti-mouse CD16/CD32 Fc block (eBioscience, Frankfurt am Main, Germany) per 10⁶ cells. Cells were subsequently stained for 30 min at 4 °C in the dark with CD45-APC-Cy7, GR1-BV510 and Ly6C-PerCP-Cy5.5 (all from BioLegend) and then fixed for 10 min at room temperature with 1 % paraformaldehyde (PFA; Merck, Darmstadt, Germany) in PBS. Samples were analyzed using FACS Canto II flow cytometer and FACS Diva software (BD Bioscience, Heidelberg, Germany).