CD69 Role on T Cell Priming Figure 6. CD69 targeting does not alter CD8+ T cell priming threshold at different peptide agonistic affinities. Whole LN and spleen OT-I RAG12/2 cells were stained with 2 mM CFSE and cultured at 106 cells per well with the indicated doses of SIINFEKL (A), SIIGFEKL (B), Catnb1 (C) and Catnb1 plus 1 mg/ml of LPS (D), and 10 mg/ml of anti-CD69 2.2 mAb (dashed line) or isotype control (solid line). Graphs showing the number of proliferated (left column) and of CD25+ (right column) CD8+ T cells per well. Results representative of two experiments. doi:10.1371/journal.pone.0048593.g006 respectively. Transferred recipients were anesthetized and received a subcutaneous injection in a posterior hind-footpad of the indicated OVA doses in a 10 ml volume using a Hamilton syringe (Hamilton Bonaduz, Bonaduz, Switzerland). At the indicated times, popliteal LN were collected, stained with anti-CD8+ or anti- CD4+ MAbs, and transferred cells were analyzed for CFSE dilution. Vaccinia virus infection and ex vivo intracytoplasmic staining (ICS) Infections and ICS were performed as described [37]. Briefly, mice were infected with VACV-WR [38] or VACV-OVA257–264 (encoding the miniprotein MSIINFEKL) [39] with an intraperi- toneal injection of 107 plate forming units. After 7 days, spleens were harvested. For VACV-WR-infected mice, 106 spleen cells were reestimulated with either RMA or RMA-S HLA-A*0201 cells infected with VACV-WR (at a multiplicity of infection of 10 plate forming units per cell for 2 h at 37uC, and washed), in the presence of brefeldin A (5 mg/ml). For VACV- OVA257–264- infected mice, 36106 spleen cells were reestimulated for 4h with SIINFEKL in the presence of brefeldin A (5 mg/ml). Cells were then surface-stained with anti-CD8 MAb (ProImmune, Oxford, United Kingdom), fixed with 4% paraformaldehide, washed, and incubated with anti-IFNc MAb (BD PharMingen, San Diego, CA) in the presence of 0.1% saponin for 20 min at 4uC. Events were acquired and analyzed by flow cytometry as described. Results CD69 is upregulated on DC upon activation CD69 upregulation on T cells upon specific Ag encounter has been well documented. Instead, CD69 expression by the other PLOS ONE | www.plosone.org 7 October 2012 | Volume 7 | Issue 10 | e48593