CD69 Role on T Cell Priming results. D. and E. CD69+/+ or CD692/2 Flt3l-derived BMDC were treated with various CpG doses for 24h and stained for the different DC subset markers and CD69. D. Overlay of the CD69 histograms of 50nM CpG-treated CD692/2 (grey filled), and untreated (dashed line) or 50nM CpG-treated (black line) CD69+/+ pDC (CD11c+, CD45RA+, left) and cDC (CD11c+, CD45RA2, right). E. CD69+/+ and CD692/2 BMDC were cultured with growing doses of CpG for 24h. CD69 surface levels are expressed as the difference in MFI between CD69+/+ and CD692/2 BMDC. The results shown are of one out of two similar experiments. doi:10.1371/journal.pone.0048593.g002 tography (HPLC) using a Superdex 200 column (GE Healhcare,) and stored at 280uC. The IgG1 isotype control antibody was produced and purified likewise. The resulting antibody prepara- tions were tested on CD692/2 bone marrow-derived DC (BMDC) cultures at 10 mg/ml, and were unable to upregulate CD80 or CD86 expression levels on these cells. Phosphorothioated CpG oligodeoxynucleotide 1668 was from Geneworks (Hindmarsh, Australia). Ovalbumin (OVA) and E. coli Lipopolysaccharide (LPS) were from Sigma (St. Louis, MO, USA). The SIINFEKL was synthesized by the Proteomics facility of the Instituto de Salud Carlos III using a peptide synthesizer (model 433A; Applied Biosystems, Foster City, CA, USA) and purified by reverse-phase HPLC. The SIIGFEKL and the Catnb1 (b-catenin 329–336, RTYTYEKL) were purchased from Peptide2.0 Inc. (Chantilly, VA, USA). All cell line cultures and in vitro cultures were performed in complete medium (RPMI medium 1640 supplemented with 10% FCS, 50 mM 2-mercaptoethanol, 2 mM L-glutamine, 100 units/mL penicillin, and 100 mg/mL streptomycin) at 37uC. Flow cytometry LN and spleen cells were treated with anti-CD16/32 (Fc-block 2.4G2, BD Biosciences, Franklin Lakes, NJ), and 7-Aminoactino- Figure 3. CD69 deficient DC do not have altered priming capacity. A. CD69+/+ or CD692/2 Flt3l-derived BMDC were cultured with 0.5 mM CpG for 24 h. Overlays of CD69+/+ (solid line) and CD692/2 (dashed line) Flt3l-derived BMDC showing CD86 and CD40 expression on pDC (CD11c+, CD45RA+, left) and cDC (CD11c+, CD45RA2, right). B. and C. CD69+/+ or CD692/2 Flt3l-derived BMDC were pulsed with OVA at the indicated doses for 45 minutes, washed, and further cultured with OT-II (B.) or OT-I (C.) T cells for 3 or 2 days, respectively, in the presence of 0.5 mM CpG in duplicate. Percentage of proliferated Va2+ CD4+ or Va2+ CD8+ cells is depicted. Bars represent Standard Deviation (SD) of duplicate cultures. Experiments representative of two with similar results. doi:10.1371/journal.pone.0048593.g003 PLOS ONE | www.plosone.org 4 October 2012 | Volume 7 | Issue 10 | e48593