Mouse foxp3 buffer set

To examine the immunogenicity of the AAV2-hFIX gene delivery protocol, we assessed the T-cell, B-cell, and regulatory T-cell (Treg) population in experimental animals, 12 weeks after gene transfer. After red blood cell lysis, pelleted cells were incubated with FITC-labeled anti-CD3, PE-labeled anti-CD8, PerCP-labeled anti-CD4, and APC-labeled anti-CD19 antibodies for 30 min at room temperature. The percentage CD3+, CD4+, CD8+, and CD19+ cells were then assessed by flow cytometry (BD Accuri C6 Plus). These data were used to enumerate B-cells (CD19+) and the double positive markers among the CD3+ population including, CD4 helper cells (CD3+ CD4+), CD8 cytotoxic cells (CD3+CD8+) in each of the AAV2 vector or PBS-administered mice (Figure S1).
To estimate the percentage Treg population in mouse splenocytes, ~1 × 10⁶ cells were stained with PerCP-labeled anti-CD4 and APC-labeled anti-CD25 antibodies for 30 min. Subsequently, cells were washed, fixed, and permeabilized using the mouse Foxp3 buffer set (BD Pharminogen) and further stained with the PE-conjugated Foxp3 antibody for 30 min. Flow cytometry was performed to enumerate the Treg (CD4+ CD25+ Foxp3+) cells.

To determine the expression of T cell markers after pDom-M/F treatment in mouse models, tumor infiltrating lymphocytes (TILs) were isolated from mouse tumor samples. Briefly, 500mm³ tumors were harvested and minced into smaller pieces (<3mm) and digested in 5 ml digestion buffer [RPMI 1640 containing collagenase type I (200 U/ml; Gibco, US) and DNase I (10 U/ml; Invitrogen, US)] and incubated at 37°C for 20 min with agitation. After incubation, tumor suspension was pressed through a 40 µm cell strainer, and rinsed with cold MACS buffer (0.5% BSA+2mM EDTA in PBS). Cells were spun at 500g for 5 min and finally resuspended in PBS with purified rat anti-mouse CD16/CD32 (mouse Fc blocker) (BD Pharmingen US), and proceed to stain with viability dye (FVS780), anti-CD4, CD8 and PD-1 antibodies (all from BD Biosciences, US) for 30 min at 4°C. To determine the presence of MDSC and T-regs, additional tubes were stained with viability dye (FVS780), anti-CD4, CD45.2, CD11b, and GR1. Cells were subjected to fixation and permeabilization with Mouse FoxP3 Buffer Set (BD Biosciences, US), and stained with anti-FOXP3 (eBioscience, US). Details of all antibodies used in the flow cytometry staining is provided in Table S1. PD1-positive CD4+ or CD8+ T cells were quantitated based on the relative expression of PD1, gating strategy is detailed in Figure S3.

Tissues were harvested and processed as described previously (11 (link)). After incubation with anti-FcReceptor, cells were stained with CD44-FITC, CD8α-PerCP (eBioscience), CD69-APC-Cy7, CD19-BV510, and CD4-BV711 (BioLegend) for 20 min on ice. For intracellular staining of FoxP3-PE (eBioscience), cells were fixed and permeabilized with Mouse Foxp3 Buffer set (BD Biosciences) after surface staining, according to the manufacturer’s instructions. Samples were run on a BD LSRFortessa analyzer and the results evaluated in FlowJo. Data were plotted in GraphPad Prism for statistical analysis.

At day 45 p.t.i., five mice from PBS and LVR01x3 groups and five mice with residual lymphoma (partial response) from CHOPx2 and CHOPx2 + LVR01x3 were sacrificed and tumors were removed and prepared to obtain a single-cell suspension. 1 × 10⁵ cells per tumor were immunostained at 4°C in the dark for 30 min with the following antibodies panel: FITC-conjugated anti-CD49b, PECy7-conjugated anti-CD8, APC-conjugated anti-CD3, APCCy7-conjugated anti-CD4, PerCPCy5.5-conjugated anti-CD19, FITC-conjugated anti-CD4, PE-conjugated anti-FoxP3, PECy7-conjugated anti-CD3, APC-conjugated anti-CD25, FITC-conjugated anti-Ly6C, and PE-conjugated anti-Ly6G (all reagents from BD Pharmingen, San Diego, CA, USA). The optimal antibody concentration was defined by titration. For Treg cells analysis, cells were first stained with anti-CD4 and anti-CD25 antibodies, then fixed and permeabilized with a mouse FoxP3 buffer set (BD Pharmingen) and then washed twice with permeabilization buffer and incubated with anti-FoxP3 at 4°C for 30 min in the dark. Flow cytometry data were collected on a FACS Canto II Cytometer (Becton–Dickinson, Oxford, UK). For data acquisition and analysis, FACSDiva (Becton–Dickinson) and Infinicyt (Cytognos, Spain) software were used, respectively.

For surface markers, the cells were stained in PBS containing 1% BSA with indicated antibodies for 30 min on ice. For intracellular markers, the cells were first fixed with Fixation Buffer (420801; Biolegend, San Diego, CA, USA) at 4 °C for 30 min and then stained in Permeabilization Wash Buffer (421002; Biolegend, San Diego, CA, USA) with relevant antibodies at 4 °C for 30 min. Foxp3 staining was conducted according to the manufacturer’s instructions for the Mouse Foxp3 Buffer Set obtained from BD Bioscience (San Diego, CA, USA). The following antibodies were used for the studies: APC anti-mouse CD45 (103112), PE anti-mouse F4/80 (123110), PerCP/Cy5.5 anti-mouse F4/80 (123128), FITC anti-mouse CD11c (117306), APC anti-mouse CD206 (141708), APC anti-mouse/human CD45R/B220 (103211), PerCP/Cy5.5 anti-mouse Ly-6G/Ly-6C (108427), FITC anti-mouse CD4 (100406), PerCP anti-mouse CD8a (100732), AlexaFluor 647 anti-mouse/rat/human Foxp3 (320014), PE anti-mouse/human CD44 (103008), and APC anti-mouse CD62L (104412) from Biolegend (San Diego, CA, USA), and PE-Cy7 anti-mouse CD11b (552850) from BD Bioscience (San Diego, CA, USA). Flow cytometry data were acquired on MACSQuant^TM (Miltenyi Biotec, Auburn, CA, USA) and analyzed by FlowJo software (v10.5.3).