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.
, 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 
. 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 
or anti-CD69 2.2 treatment 
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 , 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.
Our results are in contrast with the initial in vitro
data showing that anti-CD3 or PMA-activated human T cells were further induced to proliferate by CD69 targeting, but are in agreement with a posterior observation indicating that Ag-specific T cell proliferation was unaffected in CD69−/−
mouse T cells in vivo
. Of notice, not all the anti-CD69 MAbs tested in initial works were reported to have this proliferation-enhancing effect on human T cells 
. Altogether, more physiological in vitro
and in vivo
data argue against the initially proposed role for CD69 as a costimulatory molecule.
When studying the influence of CD69 in a physiological setting in which the priming occurs to endogenous naïve T cell pools of various frequencies and TCR affinities, such as the Vaccinia virus infection, we observed that CD69 targeting or deficiency did not alter the size of the VACV-specific T cell population at the peak of the primary response. This is in support of our previous hypothesis that, if we found a slightly smaller population of Lm-specific T cells in Lm-infected CD69−/−
, it was not due to an intrinsic defect in Ag-specific T cell priming. Instead, it could be a collateral effect of the Lm-induced, type I IFN-mediated massive lymphocyte apoptosis 
affecting also primed Lm-specific T cells. Of notice, the defect in the control of Lm infection in CD69−/−
mice was noticeable as soon as day 1 post-infection (and thus, induced by differences in the innate immune response), was associated to increased type I IFN levels and increased spleen cell death, and was not observed in lymphocyte deficient mice. In contrast to Lm infection, Vaccinia virus infection does not lead to massive lymphocyte apoptosis, and this might be the reason for it not inducing this innate immunity-based difference in the size of specific T cell populations.
Altogether, the in vivo
and in vitro
data point to that targeting of CD69 expressed or up-regulated on DC does not affect their Ag processing, Ag presentation or costimulation capacity, and that CD69 does not function as a costimulatory molecule on DC or on T cells. These results do not rule out, though, that CD69 could affect other important aspects influencing or being determined by DC-T cell interaction, such as DC polarization or T cell polarization and programming. In this regard, CD69 has recently been reported to inhibit Th17 differentiation in Ag primed CD4+
T cells 
. Taking this into account, it could be hypothesized that the exacerbated forms of Ag-specific T cell-dependent diseases reported in CD69−/−
and anti-CD69 2.2-treated mice are not due to differential priming of Ag-specific T cells but might rather be owed to skewed polarization of primed T cells.
On the whole, this work contributes to resolving a previous controversy and to shift the focus on CD69 towards its effect on T cell polarization, as it is starting to be apparent, rather than on the extent of T cell priming and T cell costimulation.