Anti cd3e

Pooled cervical, axial, and inguinal lymph node cells (10⁶/well) from naïve A/J, BALB/c, and C57BL/6 mice were extracted. Cells obtained were exposed to IL-2 (100 UI/mL, Peprotech) alone or with the addition of plate-immobilized anti-CD3e (2 μg/mL, BD), anti-CD28 (2 μg/mL, BD), and TGF-β (2 ng/mL, Peprotech) for 72 h at 37°C and 5% CO₂ [25 (link)]. Cells were retrieved for immunophenotyping, and when relevant, supernatant was evaluated for detection of cytokine secretion, as described on the previous section.
mLN cells from OVA-challenged mice were exposed in vitro to IL-2 (100 UI/mL) alone or with the addition of OVA (0.5 mg/mL) with or without TGF-β (2 ng/mL) for 72 h at 37°C and 5% CO₂.

BAL fluid, lungs, and mediastinal LNs were analyzed by FACS (FACSCalibur BD Biosciences). The following mAbs were used for T cell analysis: rat anti-mouse CD4, rat anti-mouse CD8a, anti-CD3e, allophycocyanin-conjugated rat anti-mouse CD25, anti-CD62L, and anti-TCR Vβ5.1/5.2 (all from BD Pharmingen). Granulocytes in the BAL were detected using rat anti-mouse GR-1 (BD Pharmingen).

Cells were stained with saturating amounts of various fluorescent-labeled antibody combinations including anti-EpCAM (EBA-1 clone), anti-CD45 (HI30 clone), anti-CD3e (UCHT1 clone), anti-CD56 (B159 clone), anti-CD4 (SK3 clone), anti-CD8 (SK1 clone), anti-CD25 (M-A215 clone), anti-CD107a (H4A3 clone), anti-CD45RO (REA611 clone), anti-NKG2A (REA110 clone), anti-NKG2D (BAT221 clone), anti-CD137 (4B4–1 clone), anti-CD16 (3G8 clone), anti-HLA-E (3D12 clone), Annexin V and co-stained with DAPI (all from BD Biosciences or Miltenyi). Cells were analyzed with the Attune NxT flow cytometer (Life Technologies) and further analyses were performed with FlowJo software (Tree Star).

Isolated liver MNCs were resuspended in DPBS containing 0.5% BSA and 0.05% sodium azide. After washing, the cells were pre-incubated with anti-mouse CD16/32 Fc blocker (BD Pharmingen, USA) prior to treatment with antibodies to block non-specific reactions. The cells were stained with fluorescence-conjugated anti-CD45, anti-CD3e, anti-CD4, anti-CD8, anti-CD11b, anti-CD25, anti-CD44, anti-CD69, anti-Ly6C, anti-Ly6G, and anti-F4/80 antibodies (BD Pharmingen, USA). The stained cells were analyzed using a BD™ LSR II Flow Cytometer (BD Pharmingen, USA) and the FlowJo software (Tree Star, Ashland, OR, USA). PE-conjugated anti-IFN-γ and TNF-α antibodies (BD Bioscience, USA) were used for intracellular staining. For intracellular cytokine staining, the cells were re-stimulated with phorbol-myristate acetate/ionomycin for 1 hour in addition to brefeldin A for 5 hours and then the cells were fixed and permeabilized using the BD Cytofix/Cytoperm kit (BD Pharmingen, USA).

Lymphocytes isolated from lymph nodes of C57BL/6 mice were treated with anti-CD3e (BD Biosciences) or anti-CD3e+IFN-α (Pbl Assay Science, USA) or anti-CD3e+CD28 by plating 2x10⁶ cells in each well in 24 well plates. The plates were coated with various concentrations (from 0 – 20 μg/ml) of anti-CD3e in 200 μl of PBS overnight at 4° C. Plates were washed with PBS before lymphocytes were plated in Complete RPMI 1640. IFN-α (10 ng/ml) or CD28 (1 μg/ml) was added to wells as indicated in results section at the time of plating. With all treatments, lymphocytes were incubated at 37 °C and 5% CO₂ for 24 hrs before cells were collected for flow cytometry.

Frozen coronal sections (40 μm) were taken through the entire brain using a Microm HM 450 sliding microtome (Thermo Fisher Scientific, Walthman, MA). Primary antibodies included anti-B220 (biotinylated, 1:500; BD Biosciences, Franklin Lakes, NJ), anti-CD68 (1:1000; AbD Serotec, Raleigh, NC), and anti-CD3e (the ε chain of the T cell receptor associated CD3 complex, 1:500; BD Biosciences). Briefly, free floating sections were immunolabeled with antibodies in conjuction with ABC Vector Elite and 3,3’-diaminobenzidine kits for visualization. B220+ and CD3e + cells were counted using the NIH Image J (Media Cybernetics, Rockville, MD) cell count function in the stroke core in 2 sections (bregma −0.46 mm and −1.94 mm) per mouse, and 3 non-overlapping fields (112 μm × 85 μm using a 20× objective). Due to the denseness and intensity of CD68 immunoreactivity within each lesion, CD68 was quantified by measuring the percent area occupied by CD68 (n = 5–8 mice per group for CD3e-immunostaining, n = 4–12 mice per group for B220-immunostaining, and n = 4–7 mice per group for CD68-immunostaining).