Cd3 pe

Flow assisted cell sorting (FACS) was performed as described elsewhere (16 (link)) with the following conjugated antibodies; Mac1 PE, Gr1 FITC, B220 APC, B220 FITC, CD19 PE, surface IgM PE, CD43 FITC, CD3 PE, CD4 APC, CD8 FITC, CD71 PE, Ter119 FITC, Sca-1 PE, cKit FITC, cKit APC-Cy7, IL7Ra PECy7, CD34 FITC, CD16/32 PE (eBiosciences or BD Biosciences). StemSep Mouse Hematopoietic Progenitor Kit Lineage Positive antibody cocktail (Stem Cell Technologies, Vancouver, Canada) was used to detect lineage positive bone marrow cells, stained as previously described (16 (link)). Apoptosis assays were conducted using AnnexinV-FITC Apoptosis Detection Kit I following the manufacturer’s recommended protocol. IHC and immunoblotting were performed as previously described (49 (link)). Formalin-fixed paraffin embedded sections were stained with hematoxylin and eosin (H&E), myeloperoxidase (MPO) (A0398, DAKO), CD3 (MCA1477, AbD Serotec), B220 (553086, BD), Ter119 (116201, BioLegend). Stained slides were scanned and imaged as described elsewhere (49 (link)). For immunoblots, primary antibodies used were anti-V5 (ab9116, Abcam or R960-25, Life Technologies), and beta-actin (Cell Signaling Technology). Protein was visualized using Pierce ECL Western Blotting Substrate (Thermo Scientific, Illinois, USA) and Amersham Hyperfilm ECL (GE Healthcare Ltd, Buckinghamshire, UK).

To evaluate PVL and CD4⁺ T-cell decline, peripheral blood was obtained on a weekly and bimonthly basis, respectively. Plasma RNA was extracted utilizing the E.Z.N.A. Viral RNA kit (Omega bio-tek, Norcross, CA) and the manufacturer’s instructions. Viral load was quantified using the iScript One-Step RT-PCR kit with SYBR Green (BioRad, Hercules, CA) per the manufacturer’s instructions as described previously.^{10 (link),14 (link)} CD4⁺ T-cell decline was determined by staining whole blood with fluorophore conjugated anti-human CD45-FitC (eBioscience), CD3-PE (eBioscience), and CD4-PE/Cy5 (BD Pharmigen, San Jose, CA) antibodies. BD Accuri C6 FACS Analyzer was used to determine cell counts as described previously.^{13 (link),14 (link)} The CD4⁺ T-cell decline between the infected and uninfected mice was assessed using a two-tailed Student’s t-test (p<0.001).

Cell surface and intracellular immunostaining analyses were performed using an Accuri C6 Flow Cytometer. NK cells and T cells were stained with the dye-conjugated mouse mAbs to human CD56-PE-Cy5 (Beckman Coulter), CD3-PE (eBioscience), CCR7-FITC (R&D Systems), granzyme B-PE (Invitrogen), and CD16-FITC, CD8-PE-Cy5, CD45RA-FITC, CD45RO-PE, and CD57-FITC (BD Biosciences). MDSCs were stained for CD11b-FITC, CD14-PE, CD33-APC, CD34-PE-Cy5, CD11c-PE, HLA-DR-PE, DC-SIGN-FITC, CD80-FITC, CD86-FITC, and CD83-PE (BD Biosciences and eBioscience), as well as IDO-A488 (R&D Systems), NOS2-PE (Santa Cruz Biotechnology), and COX1-FITC/COX2-PE (BD Biosciences). The corresponding mouse antibody isotype controls IgG1-FITC, IgG2b-FITC, IgG1-PE, IgG2a-PE, IgG1-PE-Cy5, IgG1-APC, and IgG1-A488 (BD Biosciences) were used, as appropriate. Before staining, the cells were treated for 20 min at 4°C in PBS buffer containing 2% human serum, 0.5% BSA, 0.1% NaN₃, and 1 μg/ml of mouse IgG (Sigma-Aldrich) to block non-specific binding. Cell permeabilization for intracellular staining was performed using the Foxp3 Fix/Perm Buffer Set (eBioscience), according to the manufacturer’s protocol. Cells were stained for 40 min at 4°C followed by washing with PBS buffer containing 0.5% BSA and 0.1% NaN₃, then fixed and stored in 4% paraformaldehyde until analysis.

Three-color immunostaining of cell surface and intracellular markers was performed utilizing the Accuri C6 flow cytometer. Isolated splenocytes were stained for CD11b-PE, Gr1-APC, and F480-FITC to evaluate MDSCs; CD3-PE, CD8-FITC, and Granzyme-B-PECy7 (intracellular) to evaluate CD8 cells; and CD4-APC, CD25-FITC, and FoxP3-PE (intracellular) to evaluate T_reg cells (eBio-science). Before intracellular staining, cells were fixed and permeabilized using the FoxP3 Fix/Perm Buffer Set solution (eBioscience). Tumors from mice were morselized into a single cell suspension to evaluate tumor-infiltrating lymphocytes and probed using APC-conjugated anti-mouse antibody directed against CD4 with isotype control (mouse IgG) and PE-conjugated anti-mouse mAb directed against CD8 with isotype control (mouse IgG1) (eBioscience).