We followed the occurrence and consequences of T cell–DC interactions subsequent to the initial activation events. In general, T cells detached from DCs before cell division but often reengaged a different DC in an Ag-dependent manner. This seemed to happen at various stages of the activation process, including after commitment to division. Importantly, our results provide in vivo evidence that signals that are delivered through different DC encounters can be integrated by T cells and reflected in their differentiation program. Our approach took extensive advantage of the 2-d delay that is required to observe T cell proliferation after DC injection. By performing two DC injections that were spaced so interactions with DCs from the first injection were necessary to observe proliferation, we could determine the history of Ag recognition of some activated T cells, and assess the functional consequences of DC reencounter after the initial activation events.
Most undivided and divided T cell blasts did not seem to interact with male DCs at 48 h, which supported the idea that T cell–APC contact falls apart before T cell division. This observation differs from a previous study that concluded that most T cells proliferated while clustering with DCs (14
). However, it is in agreement with a two–photon imaging study that visualized T cells undergoing cell division without contacting any Ag-bearing APCs (5
). Differences in the overall avidity of T cell–APC interactions might explain these discrepancies. The remaining 20% of undivided blasts and divided T cells contacted male DCs in an Ag-dependent fashion. Experiments that aimed at tracking the history of Ag recognition events for these T cells revealed that the DC that contacted activated/divided T cells usually was different from the one that delivered the initial signal that was required for proliferation. The frequency of recently activated T cells reencountering DCs is likely to be >20%, because our analyses provided a snapshot image of contacts at a fixed time point. Whereas high DC numbers likely favors DC reengagement by T lymphocytes, the high motility of T cells may suffice to promote multiple T cell–DC contacts, even when a relatively low number of Ag-bearing DCs is present in the lymph node (1
). This view is supported by an in silico model of T cell activation in the lymph node, which predicted that divided T cells have the opportunity to reencounter rare Ag-bearing DCs (16
). Because the lifespan of activated DCs is ~3 d (17
), T cells have the opportunity to contact multiple DCs at the early stages of activation, and at later time points, including after commitment to cell division.
Previous studies analyzed the dynamics of T cell–DC contacts in intact lymph nodes at various time points (1
). During the first hours, transient contacts (<10 min) dominate; these are followed by long-lived interactions and then by the reacquisition of a motile behavior as T cells are blasting. In good agreement with these reports, we found that at 20 h, long-lasting interactions were dominating, and that most T cell blasts were crawling freely at 48 h. However, our experimental strategy enabled us to identify a subset of T cell blasts that makes prolonged interactions (>30 min) with DCs that are encountered in the late phase of priming (DCs from the second injection). These results suggest that opportunities for T cell blasts to receive signals at late time points are not limited to brief contacts with DCs, but include the establishment of relatively long-lived interactions. Based on our observations and the work of others, we propose that Ag recognition for an individual T cell could involve a series of short-lived contacts with DCs, as well as several relatively prolonged (>30 min) interactions.
The strength of the signal that is received by CD4 or CD8 T cells dictates their differentiation programs (19
). In particular, short in vitro stimulations induce an abortive clonal expansion that is associated with low CD25 expression, whereas longer stimulations promote sustained CD25 expression and optimal T cell expansion (20
). Prolonged TCR stimulation also is required for CD4 T cells to acquire the ability to produce IFN-γ (22
). We found that increasing the probability of DC reencounter in vivo promoted expression of CD25 and IFN-γ production. Our findings provide in vivo evidence that the overall signal that is received by a T cell integrates stimuli that are delivered by multiple DCs before and after commitment to cell division. They also indicate that, under certain circumstances, signals that promote cell cycle and CD25 expression can be delivered by different Ag-bearing DCs. A recent study by Jenkins et al. demonstrated that distinct populations of DCs affect T cell activation programs differently after immunization with a soluble Ag (23
). Together with our results that showed that T cells can accumulate sequential signals in vivo, this suggests that the T cell activation program integrates the type and the number of DCs that are encountered. Conversely, there may be an upper limit to the number of DC encounters that promote optimal CD4 T cell activation. A recent report found that CD4 T cells that were stimulated by a high number of DCs for 5 d in a row displayed reduced protective function (24
Although our results do not exclude that a single T cell–DC interaction may be sufficient to trigger an optimal activation program under optimal conditions of Ag presentation, they do suggest that T cells that received suboptimal activation signals (e.g., because of low Ag amount or low TCR affinity) can be rescued by additional DC encounters. Such an additional effect might explain why some CD4 T cell responses are dependent on the presence of Ag for several days (25
), and why the life span of DCs can influence the extent of T cell activation (27
). In this respect, it is tempting to speculate that the 3–4-d period during which activated T cells are sequestered in the lymph node (30
) may increase the number of subsequent T cell–DC contacts. Although imaging experiments that were performed in the present study were focused on interactions between CD4 T cells and injected DCs, interactions with endogenous DCs that have engulfed dead DCs can be an additional source of Ag reencounter for activated T cells. In an infectious context, the capacity for T cells to integrate signals from multiple APC encounters may adapt the strength of the adaptive response to the extent of Ag dissemination.
In summary, we documented that recently activated CD4 T cells can reinteract with Ag-bearing DCs in vivo, and subsequently, integrate these late signals in their differentiation program. Thus, the number of Ag-bearing DCs that reaches the draining lymph node may act as an important parameter by dictating the number of Ag-specific T cells that is recruited into the immune response, and by qualitatively modulating the activation program of T cells through APC reencounter.