Fcr blocking reagent

For ABCs detecting, PBMCs/B cells to be detected were incubation with FcR blocking reagent (Miltenyi Biotec) for 10 minutes at 4 °C, then stained with BV421 anti-CD11c (BioLegend) and percp-Cy5.5 anti-CD19 (BioLegend) at 4 °C for 30 minutes. For intracellular staining, cells were surface stained, fixed using eBio fix/perm kit (eBioscience), washed and stained with PE-Cyanine7 anti-T-bet (BioLegend) in 1 × eBio fix/perm buffer for 1 hour at 4 °C. Mouse IgG1 kappa Isotype Control, PE-Cyanine7 (eBioscience) was used as isotype control for T-bet staining.
For RNASE2 detecting, PBMCs to be detected were incubation with FcR blocking reagent (Miltenyi Biotec) for 10 minutes at 4 °C, then stained with APC anti-CD14 and viability dye efour 506 (Invitrogen) at 4 °C for 30 minutes. For intracellular staining, cells were surface stained, fixed with fixation/permeabilization solution (Cytofix/Cytoperm kit, BD), washed and stained with RNASE2 antibody (Invitrogen) in 1× Perm/Wash buffer for 1 hour at 4 °C, then stained with AlexaFlour 488 Goat anti-rabbit IgG (FCMACS). Cells stained with CD14 and AlexaFlour 488 Goat anti-rabbit IgG were used as isotype control for RNASE2 staining.
Then Cells were analyzed on a BD FACSAria III flow cytometer (BD Biosciences) using FACSDiva software. Cells were analyzed on a BD FACSAria III flow cytometer (BD Biosciences) using Flowjo VX software.

Following FcR blocking reagent (Miltenyi) step, CD14+ and CD14- enriched mononuclear cells were stained with antibodies described in S1 Table. In vitro generated macrophages were blocked with FcR blocking reagent (Miltenyi) and stained with antibodies listed in S2 Table. Cells were analyzed on a BD™ LSR II flow cytometer (BD Biosciences). Final immunophenotyping results were generated using FlowJo software (FlowJo LLC, Ashland, OR) according to size, granularity, live/dead marker and surface expression of targeted molecules. Results are summarized in S1 Fig.

Approximately 1 × 10⁶ cells were incubated with FcR blocking reagents (Miltenyi Biotech) for 10 min before staining to block the unwanted binding of antibodies. Mouse flow cytometry antibodies: CD19, CD45, CD5 and IgM (Miltenyi Biotec) diluted to 1:50 in 100 μL FACS buffer were added to the cells. Samples were analyzed on a FACSCanto II. Data were analyzed by FlowJo™ Software v10.9. The IgM-producing B1b B cells were gated as ‘B220^mid/low CD19^highCD5⁻IgM⁺’, with B220^mid/low CD^19high gated as B1 B cells, CD5⁻ is B-1b B cells (a subtype of B1 B cells) and IgM⁺ means total IgM production by B-1b B cells. The final data were represented as the percentage of the total population.

SFMCs were cultured at a density of 1 × 10⁶ cells/ml in a complete RPMI-1640 (Welgene) medium containing Golgi stop (BD Bioscience). Cells were challenged with various stimuli, such as NETs, LPS (1 μg/ml), or MSU crystals (200 μg/ml) for 6 h at 37 °C. To exclude dead cells, SFMCs were incubated with Fixable Viability Dye eFluor® 506 (eBioscience) at 4 °C for 30 min. After incubation with FcR blocking reagents (Miltenyi Biotec), primary antibodies were used to detect surface markers. Synovial macrophages were identified by expression of CD14 in the absence of CD1c after exclusion of CD3⁺CD15⁺CD56⁺ cells (Supplementary Figure 1). For evaluating the expression of intracellular cytokines by staining, cells were fixed and permeabilized according to the manufacturer’s protocol (eBioscience). Data were acquired on a BD FACSCanto™ II flow cytometer (BD Biosciences) and were analyzed using FlowJo software (Tree Star, Ashland, OR).

HuT78 cells (1 × 10⁵ cells per well) and Raji cells (1 × 10⁵ cells per well) were cocultured in lymphocyte medium for 24 h with or without anti-CD19-anti-CD3 bispecific antibody (equivalent to blinatumomab) (10 ng/ml; BPS Bioscience) and anti-PD-L1-hIgG1 antibody (N298A; equivalent to atezolizumab) (Cat: hpdl1-mab12, 5 μg/ml; InvivoGen) or isotype control (Cat: bgal-mab12; InvivoGen). Monensin and brefeldin A (1:1,000 each; Cytek) were added for the last 6 h. Cells were stained with Zombie NIR Fixable Viability dye (1:1,000 in PBS; BioLegend) for 15 min at 4°C in the dark, washed with FACS buffer, and fixed and permeabilized with the Foxp3/Transcription Factor Staining Buffer Set (Cytek). Cells were then stained by incubation overnight at 4°C in the dark with the following reagents in permeabilization buffer: FcR blocking reagent (1:50; Miltenyi Biotec), anti-CD3-APC (Clone: UCHT1, 1:100; Cytek), anti-IFN-γ-PE-Dazzle 594 (Clone: 4S.B3, 1:500; BioLegend), and anti-TNF-BV711 (Clone: MAb11, 1:500; BioLegend) mAbs. The cells were washed with FACS buffer and acquired with an Attune NxT Flow Cytometer with the CytKick MAX Autosampler (Invitrogen). Data were analyzed with FlowJo and R software. The percentage of IFN-γ⁺ cells was used as a readout. The data were normalized against the mean for the blinatumomab plus atezolizumab group for each combination of HuT78 and Raji cells.