Cells were transferred, imaged, and tracked as above, and the median velocity of the non-specific cells was determined for each imaging field; fields with fewer than three non-specific cells were excluded. imaged by intravital microscopy differed from your explanted preparations in preserving blood and lymph circulation. Mature DCs have been visualized by labeling with exogenous fluorescent dyes and were found to crawl slowly in random directions, rapidly extending and retracting long dendritic processes1,3C5,7. We have examined the behavior of constant state DCs in live anesthetized mice by intravital 2-photon TAPI-0 microscopy CD11c-EYFP transgenic mice that express high amounts of enhanced yellow fluorescent protein (EYFP) in CD11c+ DCs10. DCs in these mice are indistinguishable from DCs of wild-type mice based on immunohistochemistry and circulation cytometry. In the constant state, DCs actively probed passing T cells with dendritic processes, but showed little translational movement and were found in extensive interdigitating networks within the T cell zones. These DCs networks were present throughout the T cell area, including around High endothelial venules (HEVs), such that T cells exiting HEVs would encounter the DC network rapidly. In addition, we noted that transferred mature DCs displayed translational movement as they dispersed throughout the networks of endogenous DCs10. The outcome of antigen presentation by DCs depends on the state of DC differentiation or maturation11C16. In the constant state, immature DCs capture, process, and present to T cells a variety of self and environmental antigens including proteins from serum, commensal microbes, and lifeless cells17C22. Presentation of agonistic peptides on major histocompatibility complex (MHC) by constant state DCs tolerizes T cells and prevents autoreactivity when the same antigens are subsequently offered under immunizing conditions16,23C27. In contrast, DCs activated to mature by inflammatory stimuli, including contamination, Toll-like receptor ligands, and CD40 ligation, induce strong effector T cell responses to the same antigens11C16,23,24,28C30. Intravital two-photon microscopy TAPI-0 was used to image T cell priming in lymph nodes7, and showed that T cells interact with fully mature, antigen-loaded, DCs in 3 phases. For the first 8 h, T cells transiently contacted DCs, between 8C20 h they established stable contacts, and finally resumed dynamic movement. In contrast, when tolerance was TAPI-0 examined in explanted lymph nodes6, T cells did not establish prolonged contacts with antigen-loaded TAPI-0 DCs. These differences in early T cell migration suggests that the stability of T cell-DC interactions determines commitment to tolerance or immunity6. However, non-specific control T cells were not simultaneously visualized, and experiments based on classical immunological methods indicate that the early stages of T cell-DC interactions are comparable in tolerance and priming23,24,27,31,32 and do not commit T cells to either 33. To examine tolerance and immunity in live mice, Goat polyclonal to IgG (H+L)(HRPO) we developed a method for visualizing endogenous DCs simultaneously interacting with antigen-specific and non-specific CD4+ T cells. This allowed us to control for the variability in T cell movement within different sub-compartments of the lymph node. Here we statement a novel antigen-specific stop transmission that acts within an hour of T cells leaving the HEVs in both immunity and tolerance. RESULTS Antigen delivery by -DEC-205 We sought to determine how widely divergent endpoints of tolerance and immunity relate to the physical conversation between DCs and CD4+ T cells during early stages of antigen acknowledgement. To deliver antigen to DCs and therefore tolerant (Fig. 1d). When -CD40 was given in conjunction with CDEC-OVA, comparable expression of CD69 and CD62L was found and comparable amounts of OT-II cell division were seen at day 3 (Fig. 1a). However, OT-II cells responding to antigen in the presence of -CD40 adjuvant persisted at day 7 and showed enhanced proliferation in response to antigen by day 3 and are deleted in the absence of -CD40 by day 7. (a) Comparable activation of OT-II T cells after delivery of -DEC-OVA with and without -CD40. Histograms show TAPI-0 surface expression of the indicated marker.