Anti cd8α clone 53 6

The following fluorochrome-conjugated antibodies against surface and intracellular antigens were used at 0.25-1×10^–3 mg/ml: anti-Thy-1.1 (OX-7), anti-CD45.1 (A20), anti-KLRG1 (2F1), anti-CD25 (PC61), anti-CD62L (MEL-14), anti-IFN-γ (XMG1.2), anti-TNF (MP6-XT22), anti-CD44 (IM7), and anti-CD8α clone 53-6.7 (BD Biosciences). Cells were incubated with specific antibodies for 30 min on ice in the presence of 2.4G2 mAb to block FcγR binding. All samples were acquired with a FACS Canto II flow cytometer (Becton Dickinson) and analyzed using FlowJo software (TreeStar). To determine cytokine expression, cellular suspensions containing T cells were stimulated with phorbol 12-myristate 13-acetate (PMA), ionomycin and brefeldin-A (Leukocyte activation cocktail with Golgiplug; BD Biosciences) for 4 h. After stimulation, cells were stained an amine-reactive exclusion-based viability dye (Invitrogen) and antibodies against cell-surface antigens, fixed and permeabilized and intracellularly stained with specific anti-cytokine antibodies. Intranuclear staining for cytokines was carried out using the Foxp3 staining kit (eBioscience). Countbright beads (Invitrogen) were added for the flow cytometric quantification of absolute cell number.

Tetramers were generated for the following viral epitopes as previously described (12 (link), 32 (link)): HLA-B*0702/M_195-203 (M195) [APYAGLIMI], H2-D^b/F_528-536 (F528) [SGVTNNGFI], and H2-K^b/N_11-18 (N11) [LSYKHAIL]. Lymphocytes were isolated from spleens and lungs of infected animals and stained as previously described (12 (link)). Cells were stained with PE- or APC-labeled tetramers (0.1-1 μg/ml), anti-CD8α (clone 53-6.7, BD Biosciences), and anti-CD19 (clone 1D3, iCyt). In some experiments, cells were also stained for the inhibitory receptors PD-1 (clone RMP1-30), TIM-3 (clone RMT3-23), LAG-3 (clone C9B7W) and 2B4 (clone m2B4 (B6)458.1) or with appropriate isotype controls (all from Biolegend). For mixed bone marrow chimera experiments, cells were stained for Thy1.1 (clone OX-7, BD Biosciences) and Thy1.2 (clone 53-2.1, BD Biosciences). Surface/tetramer staining was performed for 1 hour at RT in PBS containing 1% FBS and 50nM dasatinib. Intracellular cytokine staining (ICS) was performed in parallel with tetramer staining as previously described (12 (link)). Flow cytometric data were collected using an LSRII or Fortessa (BD Biosciences) and analyzed with FlowJo software (Tree Star). Boolean gating in FlowJo was used to assess inhibitory receptor co-expression and patterns were visualized using the SPICE program (NIAID).