Distinct T cell dynamics in lymph nodes during the induction of tolerance and immunity Ste ´phanie Hugues 1,6 , Luc Fetler 2,6 , Laura Bonifaz 3,5 , Julie Helft 4 , Franc ¸ois Amblard 2 & Sebastian Amigorena 1 Induction of immunity and peripheral tolerance requires contacts between antigen-bearing dendritic cells (DCs) and cognate T cells. Using real-time two-photon microscopy, we have analyzed the dynamics of CD8 + T cells in lymph nodes during the induction of antigen-specific immunity or tolerance. At 15–20 h after the induction of immunity, T cells stopped moving and established prolonged interactions with DCs. In tolerogenic conditions, despite effective initial T cell activation and proliferation, naive T cells remained motile and established serial brief contacts with multiple DCs. Thus, stable DC–T cell interactions occur during the induction of priming, whereas brief contacts may contribute to the induction of T cell tolerance. Dendritic cells (DCs) are essential both in the induction of antigen- specific immune responses and in the maintenance of peripheral tolerance 1 . Inflammation controls the balance between induction of immunity and tolerance. In the steady state, antigen targeting to DCs, both biochemically 2 and genetically 3 , induces transient activation and then rapid death of antigen-specific T cells. In the same antigen- targeting conditions, inflammation causes effective T cell proliferation and priming. Therefore, the maturation state of DCs, which depends on inflammation, determines the outcome of an immune response. Both the induction of antigen-specific immunity and tolerance rely on the direct interaction of DCs with naive T cells. These interactions occur in the T cell zone of lymph nodes, in the vicinity of high endothelial venules 4 . In the context of priming, DC–T cell interactions have been analyzed in vivo and ex vivo by two-photon microscopy of intact lymph nodes 5–10 . Contacts between adoptively transferred DCs and antigen-specific T cells are extremely dynamic, on the order of a few minutes, during both the first 2–8 h and 1–2 d after immuniza- tion. Between these two periods, DC–T cell interactions become more stable, lasting for at least 1 h. The factors determining the duration of the contacts as well as the influence of contact duration on T cell activation, however, are still unclear. In vitro, the duration of the contacts between DCs and T cells depends on the substrate (for example, interactions in collagen matrix are shorter than on a coated surface 11,12 ) and on the maturation state of DCs (immature DCs establish shorter contacts than do mature DCs 12 ). It is unclear, however, if priming of naive T cells requires a prolonged contact with a single DC or whether multiple brief contacts with different DCs may suffice. Here we have examined the interactions between resident DCs and naive CD8 + T cells, as reflected by the dynamics of T cell activity, in intact lymph nodes during the induction of immunity or tolerance. During priming, DC–T cell dynamics followed the same three phases described before 9 : we noted dynamic interactions (corresponding to T cell arrests lasting a few minutes) both 8–12 h and 30–48 h after immunization, whereas after 15–20 h, most of the T cells stopped migration. This phase of stable contacts was ‘dictated’ by the activation state of DCs, rather than by T cell activation. During the induction of antigen-specific tolerance, we noted no ‘stop signal’. T cell arrests were transient at all times after initiation of the T cell response. Thus, stable T cell contacts are required for priming, whereas continuous brief DC– T cell interactions are characteristic of induction of T cell tolerance. RESULTS Induction of CD8 + T cell priming or tolerance We achieved antigen presentation to naive T cells by lymph node– resident DCs using an antibody to an endocytic receptor (DEC205) with high expression at the DC surface, conjugated to ovalbumin (OVA; aDEC205-OVA) 2,13 . We adoptively transferred OVA-specific CD8 + OT-1 T cells labeled with CFSE (5-(and-6)-carboxyfluorescein diacetate succinimidyl ester) into C57BL/6 host mice. The injection of aDEC205-OVA conjugates into these mice in the absence or in the presence of lipopolysaccharide (LPS) or antibody to CD40 (anti- CD40), induced similar T cell proliferation in the first 3 d after stimulation (Fig. 1a). To examine the subsequent immunological outcome (priming or tolerance) of this initial T cell activation with or without adjuvant, we © 2004 Nature Publishing Group http://www.nature.com/natureimmunology Published online 31 October 2004; doi:10.1038/ni1134 1 Institut National de la Sante ´ et de la Recherche Me ´dicale U365, Immunite ´ et Cancer, Institut Curie, 26 rue d’Ulm, F-75248 Paris Cedex 05, France. 2 Centre National de la Recherche Scientifique UMR 168, Laboratoire de Physico-Chimie Curie, Institut Curie, 26 rue d’Ulm, F-75248 Paris Cedex 05, France. 3 Laboratory of Cellular Physiology and Immunology, Chris Browne Center for Immunology and Immune Diseases, The Rockefeller University, New York, New York 10021, USA. 4 Institut National de la Sante ´ et de la Recherche Me ´dicale U520, Biologie Cellulaire de l’Immunite ´ Tumorale, Institut Curie, 26 rue d’Ulm, F-75248 Paris Cedex 05, France. 5 Present address: Research Unit on Autoimmune Diseases, Centro Medico Nacional Siglo XXI, Instituto Mexicano del Seguro Social, Mexico City, Mexico. 6 These authors contributed equally to this work. Correspondence should be addressed to S.A. (sebastian.amigorena@curie.fr). NATURE IMMUNOLOGY ADVANCE ONLINE PUBLICATION 1 ARTICLES