CD69 Role on T Cell Priming Figure 1. CD69 is upregulated on endogenous DC upon activation. A.–C. DC were purified from spleens of C57BL/6 mice and cultured with CpG in various conditions. 10 cg/ml of anti-CD69 2.2 were added to control samples in order to block CD69 staining and provide a background staining control. 10 mg/ml of Isotype control Ab were added to test samples. All samples were stained for the different DC subsets markers and for CD69, and analyzed by flow cytometry. A. Overlay of CD69 expression between CD69 blocked control samples cultured with 0.03 mM CpG for 18h (grey filled), and unblocked samples, uncultured (dashed line) or cultured with 0.03 mM CpG for 18h (solid line), gated on pDC (CD11cint, CD45RA+), and cDC (CD11chi, CD45RA2). B. DC were cultured with 0.03 mM CpG during various time-spans and CD69 was assessed in pDC (CD11cint, CD45RA + ) and cDC (CD11chi, CD45RA2). C. pDC (CD11cint, CD45RA+), CD8+ cDC (CD11chi, CD45RA2, CD8+) and CD82 cDC (CD11chi , CD45RA2, CD82) were analyzed for CD69 expression after 12h culture with different doses of CpG or without having been cultured. In B and C, CD69 levels are expressed as the difference of CD69 MFI between the unblocked and blocked samples. Results representative of two similar experiments are shown. doi:10.1371/journal.pone.0048593.g001 In this study, we revisit the question of a role for CD69 on T cell costimulation and priming, which could contribute to the observed effects of CD69 on the different T cell-dependent immune responses. To this effect we use antibody-based targeting and gene knock-out approaches in in vitro and in vivo transgenic T cell mouse models as well as viral infection models. We expand the study upon a possible effect of CD69 on the extent of T cell priming, not only from its expression on T cells, but also from its expression on the other cell type participating in T cell priming, the dendritic cells. Our results point to that CD69 does not affect the extent of T cell priming, suggesting that it does not function as a costimulatory molecule, and that it does not affect Ag presentation. Materials and Methods Mice Balb/c, DO10.11 RAG22/2 Balb/c, C57BL/6 and OT-I C57BL/6 mice, all both CD69+/+ and CD692/2, and OT-I RAG12/2 C57BL/6, OT-II C57BL/6 and H-2 class I knockout HLA-A*0201-transgenic [35] mice were bred and housed under specific pathogen free conditions in the animal facilities of the Parc Cientific de Barcelona, Barcelona, Instituto de Salud Carlos III, Madrid, and the Walter and Eliza Hall Institute, Melbourne. CD692/2 mice had been backcrossed on the C57BL/6 and the Balb/c backgrounds at least nine times [27]. All procedures involving animals and their care were approved by the University of Barcelona and ISCIII Ethics Committees and were conducted according to institutional guidelines in compliance with local (Generalitat de Catalunya decree 214/1997, DOGC 2450) and international (Guide for the Care and Use of Laboratory Animals, NIH 85–23, 1985) laws and policies. Unless otherwise stated, C57BL/6 mice were used. Antibodies and immunological reagents The anti-CD69 2.2 mAb (IgG1 isotype) was generated in our laboratory [32] by the fusion of NS-1 myeloma cells with spleen cells from a CD692/2 mouse previously immunized three times with mouse 300–19 pre-B cells. The Ab was purified from concentrated hybridoma supernatants using a protein G column (GE Healhtcare, Piscataway, NJ, USA), dialyzed extensively against PBS, further purified by high-performance liquid chroma- PLOS ONE | www.plosone.org 2 October 2012 | Volume 7 | Issue 10 | e48593