itchy mice are less capable than wild-type T cells of becoming quiescent in response to sustained calcium signaling in vitro, which indicates that Itch is an important factor in calcium-induced ‘anergy’11. Thus, Itch defi-ciency may sensitize antigen receptor sig-nal transduction in T cells and, as a second function, may provide epidermal cells with constitutive NF-κB activity after TNF stimu-lation. Itch- and A20-dependent ubiquitin editing could also affect additional substrates in antigen receptor or costimulatory recep-tor signal transduction. In support of that idea, Shembade et al. show Itch- and A20-dependent deubiquitination of K63-linked chains on TRAF6 (ref. 3), and K63 ubiqui-tination has been found to be important for

signaling by the MALT1 adaptor protein12. Finally, in certain environments, Itch- deficient T cells may develop constitutive Jnk and IKK activation due to defective termi-nation of cytokine signaling. This basal sig-naling may coincide with recognition of self antigen in some T cells. Self recognition in the absence of costimulation would normally only activate NFAT and induce anergy13 but may in this situation be supplemented by constitutive activation of NF-κB and tran-scription factor AP-1 and lead to produc-tive T cell stimulation. A self-reactive T cell could therefore, instead of entering a state of hyporesponsiveness, become fully activated and autoaggressive. Future studies should delineate the actual contributions of these

signal-transduction pathways to the autoim-munity of itchy mice.

1. Fang, D. et al. Nat. Immunol. 3, 281–287 (2002).2. Perry, W.L. et al. Nat. Genet. 18, 143–146 (1998).3. Shembade, N. et al. Nat. Immunol. 9, 254–262

(2008).4. Lin, A.E. & Mak, T.W. Curr. Opin. Immunol. 19,

665–673 (2007).5. Adhikari, A., Xu, M. & Chen, Z.J. Oncogene 26, 3214–

3226 (2007).6. Wertz, I.E. et al. Nature 430, 694–699 (2004).7. Shembade, N., Harhaj, N.S., Liebl, D.J. & Harhaj, E.W.

EMBO J. 26, 3910–3922 (2007).8. Lee, E.G. et al. Science 289, 2350–2354 (2000).9. Zenz, R. et al. Nature 437, 369–375 (2005).10. Heyninck, K. & Beyaert, R. Trends Biochem. Sci. 30,

1–4 (2005).11. Heissmeyer, V. et al. Nat. Immunol. 5, 255–265

(2004).12. Oeckinghaus, A. et al. EMBO J. 26, 4634–4645

(2007).13. Macian, F. et al. Cell 109, 719–731 (2002).

How T cells ‘find’ the right dendritic cellAndrey S Shaw

Naive T cells can spend hours ‘sampling’ dendritic cells before making a stable conjugate with a single dendritic cell. It is the antigen ‘dose’ that determines how long this process takes.

Andrey S. Shaw is in the Department of Pathology

and Immunology, Howard Hughes Medical

Institute, Washington University, St. Louis, Missouri

63110, USA.

e-mail: ashaw@wustl.edu

Imaging studies show that after entering a lymph node, naive T cells may spend several

hours ‘scanning’ and touching many antigen-bearing dendritic cells (DCs) before devel-oping a long-term contact with a single DC. Why they do this has perplexed immunolo-gists for a long time. In this issue of Nature Immunology, von Andrian and colleagues use intravital microscopic approaches to evaluate the influence of the antigen dose presented by individual DCs on the length of time T cells ‘scan’ DCs before making stable, productive contacts with them1.

Given the large number of distinct T cells in an animal, each specific for a limited num-ber of antigens, a principal challenge for the immune system is to ensure that all T cells have an opportunity to find and interact with a DC bearing foreign antigens. The system does this by directing both the naive T cell and the antigen-bearing DC to travel to the lymph node. Because the probability that a T cell will encounter its antigen is low, T cells spend their entire life moving from lymph node to lymph node in search of their antigenic ligand2. In

vitro experiments suggest that when a specific T cell finally encounters its antigenic ligand in the lymph node, its response is to immediately stop and become activated so that it can pro-liferate and differentiate3.

The use of imaging techniques that allow the activity of T cells in the lymph node to be monitored suggests that this process is not so simple. Naive T cells that first enter the lymph node seem to be highly migratory, making many contacts with both antigen-bearing and non-antigen-bearing DCs in the lymph node4–6. This first period, called ‘phase I’, can last up to 8 h and is thought to be productive because T cells begin to express activation markers such as CD69 and CD44 during this time (Fig. 1). This is followed by a second period lasting 8–20 h, ‘phase II’, during which T cells form stable contacts with a single antigen- bearing DC. During this time, T cells upregu-late CD25, the receptor for interleukin 2, and begin to secrete interleukin 2 and interferon-γ and commit to enter the cell cycle. During the final period, ‘phase III’, T cells regain their motility and begin to proliferate.

Although phase I, during which naive T cells explore which DCs are available to them before ‘choosing’ a partner, is perhaps not unlike the behavior of humans in a singles’ bar, it was unexpected to immunologists because some of the initial studies were con-sistent with the idea that naive T cells quickly

stop and make stable contacts7–9. One poten-tial explanation for an extended period of phase I is that it allows T cells to assess the abundance of antigen present, perhaps allow-ing for some competition between T cells with varying avidity for a specific antigen. In this model, the prolonged migratory activity is a fixed ‘stereotypical’ activity of the naive T cell. A less exciting explanation is that given the wide diversity of peptides present on any DC, the probability of a productive, antigen-specific encounter between a naive T cell and a DC might be low and phase I simply reflects the requirement for many interactions before a productive interaction occurs.

In their paper, von Andrian and colleagues test the hypothesis that frequent interactions are due to the low probability of a productive interaction1. If this were true, they reason, the length of phase I would be influenced by the amount of antigen present. In a clever and elegant series of experiments, they find that both the antigen density on a DC and the fre-quency of DCs are inversely correlated with the length of phase I. In other words, more antigen decreases the length of phase I.

In their experiments, they compare two peptides that differ in only one of the major histocompatibility complex (MHC) class I anchor residues. They find that both pep-tide-MHC complexes have similar affinities for the P14 T cell antigen receptor. Because of

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the differences in the MHC anchor residues, the two peptides have very different half-lives bound to MHC, with one peptide having a half-life of about 6 h and the second peptide having a half-life of only about 2.5 h. In vitro, this difference ‘translates’ into a difference of about 50-fold in the stimulatory capacity of the two peptides. In vivo, because the experi-ments occur over a much longer time scale, the difference is much greater. In this experi-mental design, T cells do not encounter DCs in the lymph node until 18 h after DCs are first injected. Then, phases I and II take place over the ensuing 18–24 h.

With the assumption that both peptides are loaded to the same extent at ‘time zero’, these differences would result in large differences in the amount of peptide present at 18 and 36 h. For example, if every MHC molecule were loaded with the peptide (a gross over-estimation), after 18 h a maximum of only 127 peptides would be present on the DC loaded with the peptide with a short half-life, compared with 1,568 for the peptide with a longer half-life, which represents a difference of 12-fold. Over the ensuing 18 h, while T cells are transiting through phases I and II, the number of peptides should reach zero for the peptide with a short half-life, whereas hundreds of the peptide with a longer half-life should remain.

By varying the concentration of peptide loaded, these authors find that the threshold amount of peptide required to activate T cells in vivo varies by about 30,000, with 200 pM of the peptide with a longer half-life being sufficient versus 6 µM of the peptide with a short half-life. At these threshold doses for each peptide, phase I is long. But increasing the dose of either peptide above the threshold concentration results in a much shorter dura-tion of phase I. Thus, the length of phase I is related to the amount of antigen present.

What, then, is happening during phase I? The simplest model is that productive inter-actions are of low probability, which necessi-tates multiple interactions before a productive interaction can occur. This is consistent with the finding that the length of phase I is related to antigen quantity. A related model is that T cells retain a ‘memory’ of each interaction and accumulate signals across multiple inter-actions, which lowers the threshold for T cell activation after each successive interaction. To distinguish between those two possibili-ties, von Andrian and colleagues differentially label DCs loaded with 200 pM or 10 µM pep-tide and deliver them together to a lymph node. By itself, the 200-pM dose results in a long phase I, whereas the 10-µM dose results in a short phase I. By focusing their imaging

analysis only on T cells interacting with DCs loaded with 200 pM, they find that the pres-ence of DCs loaded with 10 µM in the same lymph node results in a more rapid transi-tion to phase II with DCs loaded with 200 pM. They interpret this to mean that prior interac-tions with DCs loaded with 10 µM result in a lowering of the threshold, which enhances the probability that a T cell will have a productive interaction with a DC loaded with 200 pM.

Examination of the raw data, however, demonstrates that even at early time points with the lower (threshold) 200-pM dose of peptide, there are detectable numbers of T cells that already show stable contacts with the DC. Although this does not invalidate the idea that T cells’ previous encounters with the DC can lower the threshold, it does suggest that many of the interactions (at the threshold dose) are nonproductive and that only a few productive encounters might be sufficient to induce the transition to phase II. To clarify this issue, it will be necessary to develop methods to detect T cell anti-gen receptor signaling in real time so that nonproductive and productive encounters with a DC can be distinguished. In addition, mathematical modeling may help to clarify the details10.

Like most important studies, this work raises more questions than it answers. The present dogma is that naive T cells require prolonged interactions with antigen-present-ing cells (between 4 and 16 h) before they are committed to enter the cell cycle11,12. Is the in vivo correlate for this phase I, phase II or both? If T cells can be activated during phase I, before forming stable contacts, what

is the function of the immunological syn-apse? As stable synapses by definition form only during phase II, is their main function perhaps to sustain signaling rather than initi-ate it? What is the function of T cell antigen receptor microclusters13, and do they act in phase I or in phase II? Finally, it is notable that half-life estimates suggest that very little if any of a peptide with an MHC-binding half-life of less than 3 h would be present by the time phase II begins. This suggests that the presence of antigenic peptide may not be required for phase II. Given the importance of the question, newer technologies, includ-ing computational studies, combined with clever experiments should continue to ‘tease out’ the mysteries of naive T cell activation.

1. Henrickson, S.E. et al. Nat. Immunol. 9, 282–291 (2008).

2. von Andrian, U.H. & Mempel, T.R. Nat. Rev. Immunol. 3, 867–878 (2003).

3. Dustin, M.L., Bromley, S.K., Kan, Z., Peterson, D.A. & Unanue, E.R. Proc. Natl. Acad. Sci. USA 94, 3909–3913 (1997).

4. Mempel, T.R., Henrickson, S.E. & Von Andrian, U.H. Nature 427, 154–159 (2004).

5. Miller, M.J., Safrina, O., Parker, I. & Cahalan, M.D. J. Exp. Med. 200, 847–856 (2004).

6. Miller, M.J., Wei, S.H., Parker, I. & Cahalan, M.D. Science 296, 1869–1873 (2002).

7. Bousso, P. & Robey, E. Nat. Immunol. 4, 579–585 (2003).

8. Hugues, S. et al. Nat. Immunol. 8, 921–930 (2007).

9. Shakhar, G. et al. Nat. Immunol. 6, 707–714 (2005).

10. Chakraborty, A.K., Dustin, M.L. & Shaw, A.S. Nat. Immunol. 4, 933–936 (2003).

11. Huppa, J.B., Gleimer, M., Sumen, C. & Davis, M.M. Nat. Immunol. 4, 749–755 (2003).

12. Iezzi, G., Karjalainen, K. & Lanzavecchia, A. Immunity 8, 89–95 (1998).

13. Yokosuka, T. et al. Nat. Immunol. 6, 1253–1262 (2005).

Figure 1 A period of T cell motility precedes a period of stable interactions with DCs. During phase I (left), naive T cells enter the lymph node and interact with many DCs for up to 8 h. During this time, they upregulate CD44 and CD69, which prevents their exit from the lymph node. During phase II (middle), T cells interact with a single DC for between 8 and 20 h. During this time, T cells begin expressing cytokines and upregulate the high-affinity interleukin 2 (IL-2) receptor CD25. During phase III (right), T cells break their contacts with the DCs and begin to move. During this period, they enter the cell cycle and begin to proliferate. IFN, interferon.

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