that a role in costimulation would be better noticed using weaker agonist peptides, we used the SIIGFEKL peptide variant and the Catnb1 peptide (an endogenous peptide from the b-catenin protein mediating positive thymic selection of the OT-I clone). In both cases, anti-CD69 2.2 MAb addition neither affected the activation threshold, the extent of the proliferative response, nor CD25 expression (Fig. 6B, C). We have also recently shown that anti-CD69 2.2 induces bystander T cell proliferation in vivo and in vitro, dependent on IL-2 production by pDC and CD25 upregulation by T cells. In vitro, for CD8+ T cells, this bystander proliferation was observed when LPS was added to whole LN and spleen cultures. It is unclear whether bystander proliferation depends on the contact of the TCR with MHC molecules presenting endogenous weak agonist peptides. In an attempt to address this question, we added LPS to OT-I RAG12/2 cultures with Catnb1 peptide. If the anti-CD69 2.2-induced bystander proliferation was dependent on the recognition of endogenous weak agonist peptides, the difference in proliferation between the isotype control and the anti-CD69 2.2-treated samples would be higher at higher Catnb1 peptide occupancy of the MHC-I, that is, at higher Catnb1 peptide doses. However, this difference was already maximal in the absence of peptide, was maintained through a range of growing peptide doses, and disappeared at peptide doses giving maximal proliferation (Fig. 6D). This suggests that this bystander proliferation might be rather independent of TCR recognition of endogenous weak agonist ligands. In agreement with this hypothesis, anti-CD69 2.2 treatment induced bystander proliferation of DO10.11 CD4+ T cells when these were transferred into CIITA2/2 recipients, which have deficient MHC- II expression, to the same extent as when they were transferred into WT mice (our unpublished data). In Fig. 6D, as expected from previous results, in the presence of LPS and in the absence of peptide or at low peptide doses, the anti-CD69 2.2 induced CD25 upregulation on CD8+ T cells. Similarly to CD69 targeting, we did not find a significant effect of CD69 deficiency on the CD8+ T cell activation threshold, as measured by the peptide dose from which T cell proliferation and CD25 upregulation start to be apparent (even though the extent of proliferation and CD25 upregulation were slightly lower for CD692/2 OT-I cells than for CD69+/+ OT-I cells). This was true for the three peptides tested (Fig. 7). These results point to that CD69 does not function as a costimulatory molecule on T cells. Neither CD69 targeting nor CD69 deficiency affect the formation of virus-specific CD8+ T cell populations in the primary immune response to Vaccinia virus infection To check for a possible role of CD69 expressed by either DC or T cells on T cell priming in a physiological model of infection, in which more factors and possible indirect effects can also play a role, we used various experimental settings of the Vaccinia virus (VACV) infection model. CD69 deficiency of VACV-WR (wild type) (Fig. 8A) or VACV-OVA (Fig. 8B) -infected mice did not alter the formation of the VACV- or SIINFEKL-specific CD8+ T cell pools, respectively, as measured by the percentage of IFNc producing spleen CD8+ T cells in response to VACV or SIINFEKL reestimulation at the peak of the primary immune responses. To test for the effect of anti-CD69 2.2 treatment on the anti-Vaccinia virus CD8+ T cell response, we took advantage of the availability of the H-2 class I knockout HLA-A*0201- transgenic mouse model, whose surface MHC-I expression is only the one of HLA-A2. In these mice, the MAb treatment did not affect the percentage of CD8+ T cells responding to HLA-A2- presented Vaccinia virus antigens either (Fig. 8C). These results indicate that neither CD69 targeting nor CD69 deficiency affects CD69 Role on T Cell Priming the primary expansion and formation of Ag-specific CD8+ T cell pools, at least in the Vaccinia virus infection model. Discussion CD69 has been found to be rapidly upregulated on all the leukocyte lineages studied, upon activation with the corresponding stimuli. When testing whether this was also the case for DC, we found that CD69 is upregulated on this cell type as soon as 3 hours after addition of TLR ligands. Thus, the expression pattern of CD69 on both T cells and DC is reminiscent of the one of costimulatory molecules. Some C-type lectins are upregulated upon activation on T cells, and have a costimulatory or coinhibitory role on Ag-driven T cell activation, influencing, among other parameters, the proliferative response. Also, C-type lectins expressed on DC can initiate signaling or modulate TLR signaling, affecting DC maturation and thus, possibly altering their Ag presentation and costimulation abilities. In this work we have examined a possible role for CD69 in the mentioned processes through analyzing the effect of CD69 targeting and deficiency on the extent of T cell priming. In vitro, we did not observe any effect of CD69 deficiency or targeting of DC using different DC types, OT-I or OT-II responders and graded OVA doses. In addition, neither CD69 targeting nor its deficiency on T cells influenced the minimal peptide dose needed for a proliferative response of OT-I T cells to different peptides, not even to the weak agonist ones. In contrast to CD69 deficiency, the deficiency of costimulatory molecules such as CD28 has been observed to lead to the lack of T cell priming to weak agonist peptides, even at high doses [42]. Consistent with the in vitro results, in in vivo adoptive transfers of transgenic OT-I CD8+ or DO10.11 CD4+ T cells into recipient mice receiving OVA subcutaneously in the footpad, CD69 deficiency did not affect the extent of the Ag-driven T cell proliferation in the Ag draining LNs. By parallel experimental approaches, we have previously reported that CD69 deficiency [10] or anti-CD69 2.2 treatment [34] of the recipient mice do not affect T cell cross-priming. All these data point to that CD69 does not quantitatively affect the priming of Ag-specific T cells. In a recent work we have shown that, in the absence of specific Ag, CD69 targeting with the anti-CD69 2.2 MAb induces bystander T cell proliferation dependent on IL-2 and on CD25 upregulation on T cells (in vitro, for CD8+ T cells, it needed the addition of LPS). This might seem contradictory with the results shown in the present work, and also with the results in that same work showing that anti-CD69 2.2 in vivo treatment did not affect Ag-specific T cell proliferation, since one might expect that the increased IL-2 and CD25 expression would lead to increased Ag- specific proliferation. However it is possible that in the presence of specific Ag, the TCR signaling itself already induces the production of such IL-2 and CD25 amounts that overrule the ones induced by CD69 targeting. Alternatively, in the presence of TCR ligation, CD69 could be uncoupled from the downstream signaling events leading to the bystander proliferative effect. These hypotheses are consistent with the data shown in Fig. 5D, in which the addition of LPS allows anti-CD69 2.2-induced bystander T cell proliferation in the absence of Ag. In this experiment, the difference of proliferation versus the wells with isotype control is already maximal in the absence of peptide, is maintained during a range of growing specific peptide doses, suggesting that the bystander proliferation is added on top of the Ag-specific proliferation, and disappears at the dose that gives a maximal response. PLOS ONE | www.plosone.org 10 October 2012 | Volume 7 | Issue 10 | e48593