Anti foxp3

PBMC or cultured T cells were stained for 30 min at 4°C with following fluorochrome-conjugated Abs: APC-Cy7-anti-CD3, v500-anti-CD4, PE-Cy5-anti-CD25, APC-anti-CD25, PE-Cy7-anti-CD45RA, BV421-anti-CD161, (six from BD Bioscience), FITC-anti-GARP (Enzo Life Science, Farmingdale, NY), PE-Cy7-anti-GITR, APC-Cy7-anti-CD73, BV421-anti-CD39, PerCP-Cy5.5-anti-CTLA-4, PE-Cy7-anti-CXCR3 (five from BioLegend, San Diego, CA), PE-anti-IL-1RI, APC-anti-IL-1RII, and FITC-anti-IL-1RII (three from R and D systems). For intracellular cytokine staining (ICS), cultured T cells were re-stimulated for 6 hr with PMA (50 ng/ml) and ionomycin (1 μg/ml) in the presence of BFA for last 4 hr, followed by staining with PE-anti-IL-1RI and FITC-anti-IL-1RII Abs. The stained cells were fixed and permeabilized using Fix/Perm buffer (BioLegend), followed by staining with anti-IL-17A, anti-IFN-γ (both from eBioscience), and anti-Foxp3 (Biolegend) Abs. The cells were acquired using a BD LSRFortessa (BD Bioscience) and analyzed using FlowJo software (Tree Star, Ashland, OR).

Different panels of antibody cocktails were generated from anti‐CD45 (BioLegend, 103116, 30‐F11), anti‐CD3 (BioLegend, 100218, 17A2), anti‐CD4 (BioLegend, 100421, GK1x.5), anti‐CD8a (BioLegend, 100711, 53‐6.7), anti‐NKp46 (BioLegend, 137606, 29A1.4), anti‐CD11c (BioLegend, 117307, N418), anti‐CD25 (Biolegend, 101908, 3C7), anti‐FOXP3 (Biolegend, 126404, MF‐14), anti‐granzyme B (BioLegend, 372208, QA16A02), anti‐IFNγ (BioLegend, 505849, XMG1.2), anti‐IFNγ (BioLegend, 505806, XMG1.2, anti‐CD62L (BioLegend, 104432, MEL‐14) and anti‐CD44 (BD Biosciences, 560568, IM7) antibodies, and the AmCyan Live/Dead Cell Staining Kit (Thermo Fisher Scientific). All antibodies were diluted at optimized dilutions before being used.
Female 7–8 weeks old C57BL/6 mice (n = 4–5 per group) were intravenously administered with the equivalent dose (3.8 × 10¹⁰ vg/mouse) of either free AAV‐fLuc or RBC‐AAV‐fLuc. Mice were then euthanized on day 31, and spleen and lungs were collected. Organ dissociation kits (Miltenyi Biotec) were used per manufacturer's instruction to generate a single‐cell suspension from excised organs, and cells were stained with the antibodies listed above and analyzed using flow cytometry (BD LSR II Analyser). Flow cytometry data analyses were performed using FlowJo 10 software.

Lactic acid (LA), glycolic acid (GA), stannous octoate, hydroxyl-terminated PEG, combretastatin A4 disodium phosphate (CA4P) and epirubicin (EPI) were purchased from Sigma–Aldrich (USA). Primary antibodies, including anti-PCNA, anti-CD34, anti-CD31, anti-MTA1, anti-TGF-β, anti-CRT and anti-HMGB1, were obtained from Abcam (Cambridge, UK). Antibodies for flow cytometric analysis, including anti-CD45, anti-CD11c, anti-CD86, anti-CD11b, anti-Gr-1, anti-CD3, anti-CD4, anti-CD8 and anti-FOXP3 were purchased from Biolegend. The 4′,6-diamidino-2-phenylindole (DAPI) and TUNEL assay kit were purchased from Thermo Fisher Scientific, Waltham, MA, USA.

We isolated T lymphocytes from spleens. After preparing a single cell suspension and counting the cells, we used magnetic beads (Miltenyi Biotec) to isolate CD4⁺ cells. Isolation was performed in the dark, and the total number of cells added to the LS column was restricted to 1 × 10⁸. Anti-CD4, anti-CD25, anti-IFN-γ, anti-IL-4, anti-IL-17A, and anti-Foxp3 antibodies were purchased from BioLegend. Intracellular straining for IFN-γ, IL-4, IL-17A and Foxp3 was performed using the Prem/Fix Buffer set, according to the manufacturer’s instructions [24 (link)]. After staining, cells were suspended in PBS + 1% FBS and analyzed using an Accuri C6 flow cytometer from BD Biosciences (San Jose, CA, USA). We also detected the serum IFN-γ, IL-4, and IL-17 levels using enzyme-linked immunosorbent assay (ELISA) kits, according to the manufacturer’s protocol (R&D Systems, Minneapolis, MN, USA).

Human peripheral blood mononuclear cells (PBMCs) were isolated using density-gradient centrifugation as previously described (Ahmad et al., 2017). Flow cytometric analysis was performed to assess Ki-67 production in CD3+, CD4+, CD8+, CXCR4+, CXCR7+, CD45R+, HLA-DR+, GATA3+, Helios+, and FOXP3+ cells. Briefly, PBMCs were stimulated for 4 h with PMA/ionomycin (Sigma-Aldrich, St. Louis, MO, USA) in the presence of GolgiStop ((BD Biosciences, San Jose, CA, USA)) as previously described [10 (link),29 (link)]. PBMCs were washed, and anti-CD3, anti-CD4, anti-CD8, anti-CXCR4, anti-CXCR7, anti-CD45R, and anti-HLA-DR (BioLegend, San Diego, CA, USA) staining was performed. Cells were fixed and permeabilized for staining with anti-Ki-67, anti-GATA3, anti-Helios, and anti-FOXP3 (BioLegend) antibodies. Forward scatter/side scatter and single-cell gating were used to exclude dead cells from all analyses. Data were acquired and analyzed with an FC 500 flow cytometer Beckman Coulter (Indianapolis, IN, USA) using CXP software (Beckman Coulter, USA).

Heart allografts and LNs were harvested at designated time points after transplantation. Heart allografts were fixed in formalin, embedded in a paraffin block, or preserved in optimal cutting temperature (OCT) compound (Tissue-Tek) and stored at –80°C. Samples were cut into 5-μm sections and stained with H&E. For immunofluorescent staining, sections were stained with conjugated or purified antibodies. Purified antibodies were detected using secondary antibodies. The antibodies included anti-CD11b (BioLegend, 101202), anti–collagen I (Abcam, ab34710), anti–Lyve-1 (Abcam, ab14917), anti-ERTR7 (Santa Cruz Biotechnology, sc73355), anti-fibronectin (Abcam, ab2413), anti-Foxp3 (BioLegend, 126401), and anti-CD3 (BioLegend, 100201). DAPI (VECTASHIELD, Vector Laboratories) was used to counterstain the cell nuclei. The stained tissue sections were visualized using an EVOS FL Auto 2 Imaging System (Thermo Fisher Scientific). Quantification was performed on 4 to 5 sections from at least 3 separate mice using Celleste (Invitrogen) and ImageJ (NIH, v1.8.0_112) image analysis software.

The cells were surface-stained with fluorochrome-conjugated Abs for 20 min on ice using the following Abs: anti-CD3, anti-CD4, anti-CD8, anti-NK1.1, anti-CD69 (eBioscience), and anti-Foxp3 (BioLegend, San Diego, CA, USA). To perform intracellular cytokine staining, IHLs were co-cultured with SL4 cells (1 × 10⁵ cells/well) for 4 h at 37°C in 96-well round-bottom plates containing 200 μL/well RPMI-1640 medium. Mouse recombinant IL-2 (50 units) and 0.2 μL of BD GolgiPlug protein transport inhibitor (BD Biosciences) were added to each well. After incubation, the cells were harvested, washed in PBS containing 1% FBS, and incubated for 10 min on ice with unlabeled anti-mouse CD16/32 Ab (BD Biosciences) to block FcγRII/III binding. The cells were then surface-stained for 20 min on ice with the indicated Abs. Following staining, the cells were washed to remove unbound Ab and fixed using a Cytofix/Cytoperm Kit (BD Biosciences). Cells were then subjected to secondary staining with reagents obtained from BioLegend except as noted, including the following: FITC-conjugated CD107a, PE-conjugated anti-interferon gamma, and anti-IL-10 (BD Biosciences). Data were acquired using a FACSCalibur flow cytometer and analyzed using CellQuest (BD Immunocytometry Systems, San Jose, CA, USA) and FlowJo software (Tree Star, San Carlos, CA, USA).