CD69 Role on T Cell Priming Figure 4. CD69 targeting on DC does not alter their costimulation and priming capacity. A. Flt3l BMDC were cultured with 50 nM CpG and anti-CD69 2.2 (solid line) or IgG1 control (dashed line) for 24 h. CD86 and CD40 expression levels were determined on pDC (CD11c+, CD45RA+, left) and cDC (CD11c+, CD45RA 2 , right). Result representative of two experiments. B. Purified DC were cultured with OT-II CD4+ T cells, in the presence of the indicated OVA doses, 0.03 mM CpG and 10 mg/ml anti-CD69 2.2 or IgG1 isotype control, in duplicate, for 3 days. C. Sorted pDC were cultured with the indicated OVA doses and 10 mg/ml anti-CD69 2.2 or IgG1 isotype control for 1 h. After wash, they were co-cultured with CD4+ OT-II T cells for 3 days. D. Sorted cDC were pulsed with the indicated OVA doses for 45 minutes and treated with 0.025 mM CpG and anti-CD69 2.2 or IgG1 control for 18 h. Then, they were cultured with OT-I CD8+ T cells for 2 days. In all cases, the number of divided cells within Va2+ CD8+ or Va2+ CD4+ live cells is represented. Bars represent SD of duplicate cultures. doi:10.1371/journal.pone.0048593.g004 mycin D (7AAD, BD Pharmingen) or Propidium Iodide (Molec- ular Probes, Eugene, OR, USA) were added in order to exclude dead cells. The following antibodies against mouse surface antigens were used: anti-CD4 (GK1.5), -CD8 (YTS 169.4), - CD11c (N418), -CD40 (FGK45.5), -B220/CD45RA (14.8), - CD69 (H1.2F3), -CD86 (PO3.1), and -Bst2 (120G8), all in-house produced; -CD4 (GK1.5), -CD8 (53–6.7), -CD25 (PC61.5), -Va2 TCR (B20.1), and -DO10.11 TCR (KJ1), from eBioscience (San Diego, CA, USA). Cells were analyzed on FACScan, FACScali- bur, FACScanto or LSRII flow cytometers (Becton Dickinson, Franklin Lakes, NJ USA), using CELLQuest or BD FacsDiva software (Becton Dickinson) and data was analyzed with FlowJo (Tree Star Inc., Ashland, OR, USA). In the experiments where the Mean Fluorescence Intensity (MFI) is represented, this parameter corresponds to the Geometric Mean. BMDC generation Bone marrows were lysed and cultured in complete medium. For GM-CSF-derived BMDC, cells were cultured at 0.56106/ml in 24 well plates in the presence of 20 ng/mL Granulocyte and Macrophage Colony Stimulating factor (GM-CSF) (Immunotools, Friesoythe, Germany) for 6 days, changing half of the media for fresh media every two days. For Fms-related tyrosine kinase 3 ligand (Flt3l)-derived BMDC, cells were cultured at 1.56106/ml in 24well plates supplemented with in-house produced Flt3l for 9 days. DC Isolation Splenic DC were isolated as described [36]. Briefly, C57BL/6 spleens were digested with DNase I (0.1%, Roche Molecular Biochemicals, Mannheim, Germany) and Collagenase (type II, Worthington Biochemical, Lakewood, NJ, USA), and centrifuged over Nycodenz medium (density 1.082 g/cm3, 17006 g for 10 PLOS ONE | www.plosone.org 5 October 2012 | Volume 7 | Issue 10 | e48593