Cd19 percp

Intracellular ASC-speck formation in vivo was determined by flow cytometry on fresh whole blood samples according to previous studies (30 (link)–32 (link)). The gating strategy is shown in Supplementary Figure 2. The following antibodies were used: CD3-PerCP, CD19-PerCP, CD56-PerCP, CD66b-PerCP, ASC-PE, and HLA-DR PE-Cy7 are from BioLegend (San Diego, CA); CD14 APC-Cy7 and CD16 Krome Orange are from BD Bioscience (Heidelberg, Germany) and Beckman Coulter (Krefeld, Germany), respectively. Samples of sepsis patients and healthy controls were processed in exactly the same way. In brief, 300 µl of fresh whole blood was incubated with the antibodies (CD3, CD19, CD56, CD66b, HLA-DR, CD14, and CD16) for 15 min at room temperature (RT) in the dark. Erythrocytes were lysed using FACS lysing solution (BD Biosciences) and leukocytes were fixed simultaneously. Cells were washed and permeabilized using 0.1% saponin followed by incubation with anti-ASC monoclonal antibody for 40 min at 4°C in the dark. After staining, cells were washed and flow cytometry was performed on a Gallios Flow Cytometer (Beckman Coulter). Counting beads (Molecular Probes Invitrogen, Paisley, UK) were added into each sample before acquisition to study absolute numbers of different leukocytes subsets. Flowjo software (Version 10.0.7 for windows) was used for flow cytometric analysis.

To harvest peritoneal cells, 5 mL of Dulbecco’s Modified Eagle Medium (DMEM) containing 5% fetal bovine serum (FBS) was injected into the peritoneum of mice using a 27G needle. After injection, the DMEM was collected from the peritoneum using a 5ml syringe with a 25G needle. RBCs were lysed using ACK Lysing Buffer (Quality Biological). Peritoneal Cells were resuspended at 10⁶ cells/ml, blocked with Fc blocker and stained for the lineage markers CD19-Percp (Biolegend), B220-FITC (eBioscience), CD11c-PECy7 (Biolegend), B220-APC (eBioscience), CD3-FITC (BD Pharmingen), CD11b-PE (Biolegend).

Cells were incubated with 1% BSA in PBS for 10 min, centrifuged and stained for 15 min with the following antibodies: CD3-FITC (cat no 300406), CD4-APC/Cy7 (cat no 300518), CD8-PE/Cy7 (cat no 301012), CD19-PerCP (cat no 302228), CCR7-BV421 (cat no 353208), CD45RA-APC/Cy7 (cat no 304128), CD27-PE (cat no 302808), CD28-PE/Cy7 (cat no 302926), Tim-3-BV421 (cat no 345007), PD-1-PE (cat no 329906) all purchased from Biolegend, α-CAR-DyLight649 AffiniPure F(ab')₂ Fragment Goat Anti-Human IgG (H+L) purchased from Jackson ImmunoResearch Europe LTD (cat no 109-496-088), or negative isotype control antibodies (Biolegend). Cells were washed with PBS. Analyzed on BD Canto II (BD Biosciences, San Jose, CA) and evaluated with FlowJo (Treestar, Ashland, OR, USA). The transduction efficiency of 3G CAR was lower than that of 2G CAR. Therefore, a correction factor was calculated based on the CAR expression of the 2G and 3G T cells from the same donor at the same time point. This correction factor was then used to normalize the values of the functional assays.

Bone marrow myeloid cells were obtained by flushing tibiae and femurs with growth medium, and enriched for CD45 expression using CD45 MicroBeads (Miltenyi Biotec) and magnetic-activated cell sorting according to the manufacturer’s instructions. Cells were spun down and labelled with the following antibodies for 45 min at 4 °C: APC-CD11b (101211, BioLegend), FITC-Gr-1 (108405, BioLegend), FITC-F4/80 (123107, BioLegend), PE-CD115 (165203, BioLegend) and PerCP-CD19 (115531, BioLegend). The number of Gr-1⁺CD11b⁺ neutrophils, F4/80⁺CD11b⁺ macrophages, CD115⁺CD11b⁺ monocytes and CD19⁺ B cells was assessed by flow cytometry (BD FACSCanto, BD Biosciences) as described before^{49 (link)} and quantified using Kaluza software (Beckman Coulter).
Circulating myeloid cells in blood were analysed using the Sysmex XN1000 and XN2000 Hematology Analyzer (Sysmex Corporation; University Hospital Leuven).

Cells were washed and resuspended at a density of ≤10⁶ cells/25 µl flow buffer, and Fc receptors were blocked by adding 10 µg ml^–1 TruStain FcX™ blocking solution (BioLegend). Cells were then stained in a 50 µl final volume in 96-well round-bottom plates (Corning) covered for 30 min at 4°C and washed twice with flow buffer (1× PBS, 2% FBS, 1% sodium azide) before resuspension in 150 µl of BD™ Stabilizing Fixative (BD Biosciences) and transfer to polystyrene tubes (12×75 mm) (Becton Dickinson). A total of 0.5×10⁵ to 1×10⁵ events were acquired on a BD FACSCanto™ flow cytometer with FACSDiva™ software (BD Biosciences) and analyzed by FlowJo. Fluorophore-conjugated antibodies were APC-CD45 (BD Biosciences) and BV421-CD11c, BV421-CD8, FITC-Ly6C, PE-F4/80, PE-CD31, PerCP-CD3, PerCP-CD19, PE/Cy7-Ly6G, APC/Cy7-CD11b, and APC/Cy7-CD4 (BioLegend) (antibody details in Table S6).

Cell suspensions were stained with appropriate antibodies for 30 min on ice. The commercial antibodies used in this study included anti-mouse FITC-CD45 (BioLegend, CA, USA, #103108; 1 µg per 10⁶ cells), PE-CLEC2 (BioLegend, CA, USA, #146104; 1 µg per 10⁶ cells), BV421-F4/80 (BioLegend, CA, USA, #123137; 1 µg/10⁶ cells), Pe/Cy7-CD64 (BioLegend, CA, USA, #128016; 1 µg per 10⁶ cells), Percp/Cy5.5-CD11b (BioLegend, CA, USA, #101228; 1 µg per 10⁶ cells), APC/Cy7-PirB (R&D, CA, USA, #FAB2754S; 5 µg per 10⁶ cells), APC-Ly6C (BioLegend, CA, USA, #128016; 1 µg per 10⁶ cells), APC-Ly6G/Ly-6C (Gr-1) (BioLegend, CA, USA, #108411; 1 µg per 10⁶ cells), PerCP-CD19 (BioLegend, CA, USA, #115531; 1 µg per 10⁶ cells), APC-CD3 (BioLegend, CA, USA, #100235; 1 µg per 10⁶ cells), APC-CD206 (BioLegend, CA, USA, #141707; 2 µg per 10⁶ cells), and FITC-CD11c BioLegend, CA, USA, #117305; 1 µg per 10⁶ cells); and anti-human PE-CD14 (BD Biosciences Pharmingen, USA, #555398; 20 µl per 10⁶ cells), Mouse anti-human LILRB2 (R&D, R&D Systems, MN, USA, #MAB2078; 0.25 µg per 106 cells), and Goat Anti-Mouse FITC-IgG (Servicebio, Wuhan, China, #SF131; 1:200 dilution). All antibodies were diluted according to the manual from the manufacturer’s website. Dead cells and doublets were removed by dead-cell dye staining (Zombie Aqua Fixable Viability Kit, BioLegend, CA, USA, #B297827).