Anti cd25 3c7

Cells were isolated 72 hours after injection with LNPs unless otherwise noted. Mice were perfused with 20 mL of 1X PBS through the right atrium. Liver and lung tissues were finely minced, and then placed in a digestive enzyme solution with Collagenase Type I (Sigma Aldrich), Collagenase XI (Sigma Aldrich) and Hyaluronidase (Sigma Aldrich) at 37 ºC at 550 rpm for 45 minutes^4,5. The spleen, bone marrow and thymus tissues were placed in 1X PBS solution. Cell suspension was filtered through 70μm mesh and red blood cells were lysed. Cells were stained to identify specific cell populations and sorted using the BD FacsFusion and BD Facs Aria IIIu cell sorters in the Georgia Institute of Technology Cellular Analysis Core. The antibody clones used were the following: anti‐CD31 (390, BioLegend), anti‐CD45.2 (104, BioLegend), anti‐CD68 (FA11, Biolegend), anti‐CD3 (17A2, Biolegend), anti‐CD19 (6D5, Biolegend), anti‐CD11b (M1/70, Biolegend), anit‐CD11c (N418, Biolegend), anti‐CD4 (GK1.5, Biolegend), anti‐CD8a (53‐6.7, Biolegend), anti‐CD34 (SA376A4, Biolegend), anti‐CD25 (3C7, Biolegend), anti‐CD326 (G8.8, Biolegend), and PE anti‐mCD47 (miap301, BioLegend). Representative flow gates are located in Supplementary Figure 3. PBS‐injected Ai14 mice were used to gate tdTomato populations for intravenous administration.

The peritoneal macrophages treated as in the section, ‘In vitro stimulation of peritoneal macrophages’ were harvested from culture plates using a 5 mM cold PBS-EDTA mixture for 10 min, washed, and then re-suspended in an FACS buffer (BD Biosciences). To avoid unspecific unions with antibodies, anti-mouse CD16/32 (93, dil. 2:100) was added for 20 min at 4°C to block the Fc receptors on the cell surface of macrophages. Then cells were washed with FACS buffer (BD Biosciences) and stained as indicated, with antibodies against F4/80 (BM8, 1:100), MHC-II (M5/114.15.2, 1:100), CD206 (C068C2, 2:100), IL-4Rα (I015F8, 4:100), PD-L1 (B7-H1, 2:100), PD-L2 (TY-25, 2:100), CD80 (16-10A1, 4:100), and CD86 (GL-1, 2:100) in staining buffer at 4°C and used according to manufacturer’s instructions (Biolegend). The stained cells were analyzed on a FACsCalibur flow cytometer (BD Biosciences) using Cell Quest and FlowJo software. Parallel samples of the cells were stained with the corresponding isotype control. T CD4⁺ cells obtained from the section, ‘Co-culture of peritoneal macrophages with lymphocyte T cells’ were washed with the FACS buffer (BD Biosciences), marked with anti-CD4 (GK1.5, 1:100), anti-CD25 (3C7, 1:100) and anti-CD69 (H1.2F3, 1:100) antibodies (Biolegend), and analyzed on Attune NxT flow cytometer (Thermo Fisher) using FlowJo software.