Our results show that OVA-specific T cells slowed down and exhibited confined motion upon encountering antigen, many of them arresting in the vicinity of HEVs. Meanwhile, the non-specific cells in the same field maintained their baseline speed and directionality. The early arrest near HEVs was identical in conditions of tolerance and priming, but antigen-specific T cells regained mobility more rapidly under conditions of tolerance than priming.
Intravital imaging of lymph nodes in mice injected with mature antigen-loaded DCs, showed that naïve T cells interacted with DCs in three stages: during the first 8 h, or phase 1, T cells interact with antigen-bearing DCs dynamically with no prolonged stopping, T cell immobility was seen between 8–20 h, or phase 2, and motility recovered thereafter in phase 37
. Similar kinetics were observed in explanted lymph nodes when antigen was delivered by migrating mature DCs5
. It was suggested that transient T-DC interactions in phase 1 changed the T cell activation state so that it could undergo stable interactions in phase 27,36
. Although our finding that T cells stop shortly after leaving the HEVs was surprising in this light, it was independently supported by histological evidence showing preferential retention of antigen-specific T cells around HEVs, which indicated constrained movement. Our results agree with previous work showing histologically that immunization with complete Freund’s adjuvant resulted in early antigen-dependent retention of T cells near HEVs37
. Furthermore, in vitro
experiments show that T cells stop migrating immediately upon exposure to their cognate antigen38,39
. Steady-state lymph node DCs express high amounts of MHC class II and intermediate amounts of CD80 and CD8610,40
. Therefore, the rapid arrest we observe under tolerogenic conditions in vivo
is not inconsistent with in vitro
observations of poor immunological synapse formation in the absence of costimulation41,42
. We conclude that a prolonged period of transient interactions is not necessary to form stable conjugates between T cells and DCs. Potential explanations for the differences in the length of phase 1 between ours and previous studies include their use of exogenously administered DCs that must migrate to the lymph node, and enter the T cell area before presenting antigen7
. Another possibility is that T cells integrate signals during phase 1 encounters with DC. In this model, antigen presented by many DCs after DEC-205 targeting43,44
might produce an integrated signal of the same magnitude as that obtained by T cells interacting with much less frequent immigrant DCs for 8 h. While further experimentation is needed to test these possibilities, it is likely a priori
that the number of antigen-positive DCs will vary depending upon how antigen reaches the lymph node. For example, soluble antigen rapidly drains to the lymph nodes and is widely accessible to resident lymph node DCs45
. In addition, self-antigens from dead cells and serum proteins are continually picked up, processed and presented by large numbers of resident DCs in the steady state. Thus, it seems likely that under conditions of immunity or tolerance, T cells can encounter varying numbers of antigen-bearing DCs, and that the kinetics of the initial T cell-DC interaction will depend on the relative frequency of antigen-positive DCs.
Imaging of T cell priming in explanted lymph nodes showed antigen-specific T cell clustering around DCs in the deep paracortex3,6
. We rarely observed formation of discrete T-DC clusters but this was expected, as large numbers of DEC-205+
DCs in the lymph node present antigen after targeting with the α-DEC-OVA antibody23,30
, and the number of T cells in each cluster is inversely proportional to the number of antigen presenting DCs3.
In our experiments, arrested antigen-specific T cells were not always in clear contact with CD11chi
DCs, which is consistent with the partial overlap between EYFP and DEC-205 expression in CD11c-EYFP Tg mice10
We found that the early stages of tolerance and immunity were similar in terms of T cell arrest near HEVs at 1–5 h followed by resumed movement and dispersion in the deep paracortex by 18 and 24 h, respectively. The only difference observed was that return to rapid migration was slower under conditions of priming than tolerance. Our imaging experiments are consistent with our finding that T cell activation markers are identical and proliferation and responses to antigen in vitro
are similar in the first 3 days of priming and tolerance induction. These experiments are also in agreement with physiologic studies showing that T cells activated by soluble peptide in vivo
are not committed to tolerance after 3 days31–33
. Our results differ from an earlier two-photon study of tolerogenic T-DC interactions which used the same antigen delivery method (α–DEC-205-OVA)6
. This study reported that CD8+
T cell arrest exclusively in priming and not in tolerance. However, these experiments were performed in explanted lymph nodes where DCs were labelled by direct lymph node injection of anti-CD11c, and non-specific T cells were not simultaneously imaged6
. Importantly, our use of intravital microscopy preserves blood and lymph flow whereas these physiological processes were lacking in the explanted lymph nodes used previously6
What is the significance of the prolonged T cell-DC interactions early in immune responses? Our experiments suggest that these conjugates are antigen-specific, but not associated with commitment to immunity or tolerance and independent of the state of DC maturation. We speculate that stable contacts between DCs and T cells are an integral part of initial T cell activation in vivo, and that commitment to immunity or tolerance occurs later as T cells dynamically contact DCs.