CD69 Role on T Cell Priming Figure 7. CD69 deficiency does not alter CD8+ T cell priming threshold at different peptide agonistic affinities. Spleen CD8+ T cells were purified from CD69+/+ (solid line) or CD692/2 (dashed line) OT-I mice, stained with 2 mM CFSE and cultured at 0.56106 at 10:1 with APC in the presence of the indicated doses of SIINFEKL (A), SIIGFEKL (B), and Catnb1 (C) peptides. Graphs showing the number of proliferated (left column) and of CD25+ (right column) CD8+ T cells per well. The results are representative of two similar experiments with similar results. doi:10.1371/journal.pone.0048593.g007 prototypic cell type implicated in T cell priming, the DC, has not been extensively studied. We have recently described certain constitutive CD69 expression by lymph node (LN) and spleen plasmacytoid DC (pDC) and by LN conventional DC (cDC) at steady state [34]. Here, we assess activation-induced CD69 upregulation on different DC subsets, activating them with Toll- like Receptors (TLR) ligands. CD69 was upregulated on both splenic pDC and cDC upon CpG olidodeoxynucleotide stimula- tion (Fig. 1A) in a dose-dependent manner (Fig. 1C), and it was prominent as soon as 3 hours after activation (Fig. 1B). CD69 upregulation was much higher in pDC than in the cDC subsets, which has been observed previously [40], and within the cDC subset, it was more prominent in CD8+ cDC than in CD82 cDC (Fig. 1C). After 12 hours culture in the absence of CpG, there was also a marked CD69 upregulation on splenic DC (Fig. 1C), consistent with the activation that ex vivo purified DC undergo in culture [41]. CD69 was not expressed by unstimulated Granulo- cyte-Macrophage Colony Stimulating Factor (GM-CSF) and Fms- related tyrosine kinase 3 ligand (Flt3l)-derived Bone Marrow DC (BMDC), but it was upregulated on GM-CSF BMDC when we treated them with Lipopolysaccharide (LPS) (Fig. 2A) and on Flt3l BMDC when we treated them with CpG (Fig. 2D), in both cases in a dose dependent manner (Fig. 2B, E). Therefore, CD69 is upregulated on endogenous DC upon activation with TLR ligands, and this induced expression can be reproduced in in vitro-generated BMDC models. CD69 deficiency or targeting on DC do not affect their costimulation or Ag-presentation capacity We next aimed at studying whether this CD69 expression by DC can have a role in T cell priming by putatively influencing costimulation or Ag presentation. To this end, we analyzed the effect of DC CD69 deficiency on the extent of the proliferation undergone by Ag-specific T cells, as well as on the expression of costimulatory molecules by DC. CD69 deficiency did not affect CD86 or CD40 expression on CpG-activated Flt3l-derived BMDC (Fig. 3A), and did not alter Ag-specific T cell proliferation in in vitro co-cultures of ovalbumin (OVA)-pulsed CD69+/+ or CD692/2 Flt3l-derived BMDC with OVA-specific, transgenic OT-I CD8+ or OT-II CD4+ T cells (in the presence of CpG to induce CD69 expression) (Fig. 3B, C). We also analyzed whether MAb-based engagement of CD69 expressed on DC could have an effect on their Ag-presenting capacity. CD69 targeting with the anti-CD69 2.2 MAb in in vitro co-cultures of purified DC and OT- PLOS ONE | www.plosone.org 8 October 2012 | Volume 7 | Issue 10 | e48593