Anti cd8

FCM analysis was performed to analyze the expression of marker molecules, including CD11c, DEC205, CD4, CD8, CD45, CD25, Foxp3, and Gr-1, on the cell membranes, as well as intracellular cytokines, including IFN-γ, TNF-α, and IL-2.24 (link) Cells stained with fluorescent dyes, such as CFSE and DiI, were also analyzed by FCM. The cells isolated from various tissues were incubated with the corresponding fluorescent dyes or monoclonal antibodies conjugated to FITC, PE, PerCP-Cy5.5, or APC for 30 min at 4°C in 1:100–150 dilutions. The following monoclonal antibodies were purchased from eBioscience (San Diego, California, USA) or BD Biosciences: anti-DEC205, anti-CD11c, anti-CD45, anticalreticulin (anti-CRT), anti-CD8, anti-IFN-γ, anti-TNF-α, anti-CD4, anti-CD25, anti-FOXP3, anti-Gr-1, anti-CD206, and anti-F4/80. Before intracellular cytokine staining, 2×10⁶ splenocytes were stimulated with tumor lysates (5 µg/mL) in culture medium with 10% fetal calf serum and 2 µg/mL brefeldin A (BD Bioscience) for 6 hours at 37°C. The intracellular cytokines in splenocytes or TILs were stained using the Cytofix/Cytoperm kit (BD Bioscience) as per the manufacturer’s protocol. The stained cells were detected by FCM (CyFlow Cube 6; Sysmex, Japan), and the resultant data were analyzed to capture the FCM images with FlowJo software version 10 (Tree Star, Ashland, Oregon, USA).

Phenotypic characterization on resting T cell clones was performed on 10⁵ T cells labeled with MELOE-1 and Melan-A tetramers (10 μg/mL) (Recombinant protein facility, SFR Santé, Nantes, France), anti-CD8 (clone BW135/80, Miltenyi Biotec), anti-CD45RO (clone UCHL1, BD Biosciences), anti-CD27 (clone M-T271, BD Biosciences), anti-CD28 (clone CD28.2, BD Biosciences), anti-CD62L (clone DREG-56, BD Biosciences), anti-PD-1 (clone EH12, BD Biosciences), anti-CTLA-4 (clone BNI3, Miltenyi Biotec), anti-BTLA (clone J168–540, BD Biosciences), anti-Tim-3 (clone F38–2E2, eBiosciences) and anti-CD95 (clone DX2, BD Biosciences) specific antibodies. PD-1 expression (Clone EH12, BD Biosciences) was tested on specific T cell clones or sorted T cells at rest and after activation by quadruple labeling with specific tetramers, anti-CD8 and anti-CD25 (clone M-A251, BD Biosciences), as activation marker. All the antibodies were used at a concentration of 5 μg/mL. Vß diversity of sorted Melan-A-specific T cell lines was analyzed by labeling with 24 anti-Vß mAbs included in the IOTest Beta Mark TCR V Kit (Beckman-Coulter, IM3497). All the cytometric analyses were performed on a Facs Canto II (BD Biosciences).