Thy1.1 clone ox 7

Tissues were homogenized by crushing between two frosted glass slides followed by straining through a 40uM nylon filter. Red cell lysis was followed by hypotonic disruption as described previously (22 (link)) and remaining cells were counted. Abs used for flow cytometric staining included CD4 (clone RM4-5), CD44 (clone IM7), PD-1 (clone J43), IgD (clone 11-26), IL-4 (clone 8D4-8) (all from eBioscience), B220 (clone RA3-6B2), Thy1.1 (clone OX-7), CXCR5 (2G8), CD62L (clone MEL-14), GL-7 (clone GL7), FITC CD95 (clone Jo2), PE-Cy7 (all from BD Biosciences) and IL-21R-FC (R&D). Anti-PSGL-1 (BD Biosciences) was directly conjugated to Alexa-647 as described previously (23 (link)). Staining for CXCR5 was performed at room temperature (25°C) with 30 min incubation. Intracellular staining for cytokines was performed using BD Cytofix/Cytoperm™ kits following the manufacturer's protocol. Stained and rinsed cells were analyzed using an LSRII Multilaser Cytometer (BD Biosciences). For certain experiments (Fig. 4), CD4 T cells were enriched using a biotin-based magnetic separation kit (EasySep™, StemCell Technologies) prior to cell surface staining, with specific populations sorted using a FACSAria™ (BD Bioscience). Biotin-based magnetic separation kits (EasySep™) were used to isolate T and B cells for cell transfer studies.

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).