Anti cd4 pacific blue

Responses of NK cells and T cells were assessed as described previously.15 (link) Briefly, cells were stained with fluorophore-labelled monoclonal antibodies to cell surface molecules, fixed, permeabilized and stained for intracellular molecules using a Cytofix/Cytoperm kit (BD Biosciences). Cells were analysed by flow cytometry on an LSR II (BD Biosciences). Samples with fewer than 100 NK cells in each subset were excluded. The following reagents were used: anti-CD56-phycoerythrin (PE) -Cy7, anti-CD16-allophycocyanin (APC) -H7, anti-CD4-Pacific Blue, anti-IFN-γ-e780, anti-IFN-γ-APC, anti-CD3-V500 and anti-CD69-phycoerythrin-cyanine5 (PE-Cy5) (all BD); anti-CD8-PE-Cy5, anti-CD25-PE, anti-IL-18Rα-PE, anti-CD62L-PE-Cy5, anti-CD57-e450 and anti-IL-2-APC (all e-Biosciences/Affimetrix, Hatfield, UK). Anti-IL-12Rβ2 monoclonal antibody was obtained from R&D Systems (Oxford, UK) and conjugated to PE-Cy5 using an Easylink PE/Cy5® Conjugation Kit (Abcam, Cambridge, UK).

Peripheral blood mononuclear cells (PBMCs) were isolated from whole-blood samples using Ficoll-Paque Plus™ (GE Healthcare) and cryopreserved in liquid nitrogen. PBMCs were depleted of CD8 cells using anti-CD8 Ab-coupled magnetic beads and LD columns (Miltenyi Biotec GmbH, Germany), as previously described (17 (link)). CD8-depleted PBMCs were incubated overnight at 37°C in 5% CO₂ in serum-free medium (AIM-V; Gibco) and resuspended at a final concentration of 2 × 10⁶ cells/ml in AIM-V. The purity of CD8-depleted PBMCs in each sample was assessed by flow cytometry using anti-CD8-APC, anti-CD3-PE, anti-CD4-PacificBlue™, and 7-aminoactinomycin D (all purchased from BD Bioscience), which showed that purity of CD8-depleted PBMCs was >99%.

The TCR α and β chain usage of dNPM1-specific clone 6F11 and EBV-specific clone 20–16 were determined as described previously [13 (link),19 (link)]. Codon-optimized variable α and β chain sequences were synthesized and cloned in the MP71-TCR-flex retroviral vector containing murine–TCR-constant regions [20 (link)] by BaseClear. The constructs were transfected in phoenix-AMPHO packaging cells and the supernatants were harvested and stored at −80 °C. For TCR gene transfer, PBMCs from healthy individuals were thawed, resuspended in TCM and stimulated with T-cell TransAct (Miltenyi Biotec). After 2 days, cells were transduced with TCRs using the transduction protocol as described for cell lines. On day 11, TCR-transduced CD8 and CD4 T-cells were sorted by FACS using anti-CD8 PE (catalog 555367), anti-CD4 Pacific Blue (catalog 558116) and anti-mouse TCR β chain APC (catalog 553174) antibodies (BD Biosciences) and stimulated with TCM containing irradiated allogeneic PBMCs at a T-cell:feeder cell ratio of 1:5 and 0.8 μg/mL PHA. Experiments with TCR-transduced T-cells were performed 10–13 days after the second stimulation.

Peripheral blood mononuclear cells (PBMCs) were separated from whole-blood samples using Ficoll-Paque Plus™ (GE Healthcare) and were cryopreserved in liquid nitrogen, as previously described (23 (link)). PBMCs were thawed and depleted of CD8-positive cells using magnetic beads coupled with anti-CD8 antibody and LD columns (Miltenyi Biotec GmbH, Germany), as previously described (24 (link)). The depleted PBMCs were incubated overnight in serum-free medium (AIM-V; Gibco) at 37°C in 5% CO_2. For use in ELISPOT assays, cells were resuspended at a final concentration of 2 × 10⁶ cells/ ml in AIM-V. The purity and viability of CD8-depleted PBMCs in each sample was assessed using anti-CD8-APC, anti-CD3-PE, anti-CD4-PacificBlue™, and 7-aminoactinomycin D (BD Bioscience) and flow cytometry (23 (link)). After depletion, CD8-positive cells were <1%. Plasma and serum samples were stored at −20°C.

The capacity of immature or mDC to stimulate an allogeneic T cells response was determined by mixed lymphocyte reaction assay. Briefly, immature or mDC derived in the presence or absence of hCPC, were co-cultured with allogeneic CFSE-labeled PBLs in 6-well plates (Falcon, Cat. 353046) for 5 days at 300,000 PBLs per 60,000 DC (ratio5:1). Proliferation of CD4⁺, CD8⁺ T cell subsets was monitored by flow cytometry using anti-CD3-APC, anti-CD4-Pacific blue, anti-CD8-APC-H7 (BD Biosciences), anti-CD25-PE (Miltenyi Biotec), and 7-AAD.