Imaging the function of regulatory T cells in vivo Qizhi Tang 1,2 and Matthew F Krummel 2 Despite extensive research on regulatory T cells (Tregs) since their rebirth more than twenty years ago, the cellular and molecular mechanisms by which they act to suppress immune responses remain largely elusive. In vitro suppression assays are instrumental in the functional identification of these cells. However, suppressive mechanisms defined in in vitro assays might not be relevant to situations in vivo. Advances in live tissue and intravital imaging technologies combined with the ability to grow large numbers of Tregs for in vivo experimentation have created an opportunity to analyze Treg function in vivo in their native environment in real-time. Two- photon laser-scanning microscopic studies of Treg control of lymph node priming suggest that Tregs exert their function by limiting T helper (Th) cell access to dendritic cells (DCs). In the absence of Tregs, Th cells initially form transient interactions with DCs that lead to arrest of the Th cells and to the formation of stable conjugates between Th cells and DCs. In the presence of increasing number of Tregs, Th cell arrest and their prolonged contacts with DCs are progressively inhibited. The reduced DC contacts in the presence of Tregs are associated with suppressed proliferation and differentiation of Th cells. Expansion of such analysis to peripheral tissues together with the development of functional reporter mice will help to further elucidate the mode of operation of Tregs in vivo. Addresses 1 University of California, San Francisco (UCSF) Diabetes Center, Department of Medicine, University of California, 513 Parnassus Avenue, San Francisco, CA 94143-0511, USA 2 UCSF Diabetes Center Department of Pathology, University of California, 513 Parnassus Avenue, San Francisco, CA 94143, USA Corresponding author: Krummel, Matthew F (matthew.krummel@ucsf.edu) Current Opinion in Immunology 2006, 18:496–502 This review comes from a themed issue on Immunological techniques Edited by Sebastian Amigorena Available online 12th June 2006 0952-7915/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. DOI 10.1016/j.coi.2006.05.007 Introduction Regulatory T cells (Tregs) exert a powerful inhibitory effect on immune responses to autoantigens, tissue trans- plants, tumors, allergens and microbial pathogens (reviewed in [1]). Despite extensive research on Tregs since the landmark study by Sakaguchi et al. [2], pub- lished more than 20 years ago, how these cells control immune responses is still poorly understood. Although most in vitro analyses provide strong evidence for contact- dependent suppression of T cell proliferation through inhibition of interleukin (IL)-2 production, results from in vivo experiments are much less clear. Although only some suggest that Tregs suppress clonal expansion of other T cells, most find marked inhibition of T cell differentiation in the presence of Tregs. In fact, in many cases, Tregs can inhibit an ongoing immune response and can reverse autoimmune diseases even after their onset. How do Tregs exert broad effects on an immune response in vivo? What cell types are directly targeted by Tregs, and how are these cells altered functionally and at a molecular level? In this review, we will summarize novel imaging approaches that have already begun to shed light on Treg function in vivo in the lymph node (LN), and will clarify the mechanisms of regulation in peripheral tissues. In vitro studies of Treg-mediated suppression The development of in vitro assays for Treg function provided a rapid and convenient way to phenotype these cells [3,4]. This coculture-based assay has also been used widely to decipher the mechanism of Treg suppression. Results obtained to date uniformly support the notion that Tregs suppress effector T cell proliferation through inhibition of IL-2 transcription, although often permitting other aspects of proximal signaling such as CD69 upre- gulation [5]. Several in vitro studies have suggested that Tregs function in a cytokine-independent, cell–cell con- tact-dependent manner and, under certain conditions, regulation of T effectors appears to depend upon direct contact with Tregs themselves [5]. Whereas some studies suggest that in vitro suppression can be mediated by transforming growth factor b (TGFb) and/or IL-10, other mechanisms of in vitro suppression that have been pro- posed, including cell–cell signaling via surface-bound TGFb and even direct killing of responder T cells by Tregs (reviewed in [6]). However, suppressive mechan- isms in vivo are not well defined, and the majority of models provide strong evidence for dependence on IL-10 or TGFb leading to bystander suppression (for review, see [6]). Little is known regarding whether cell–cell contacts between Tregs and other T cells actually take place in vivo. Imaging of the cell–cell contacts that occur in an in vitro suppression assay is technically straightforward and indeed supports the contention that initial signaling occurs when Tregs and T helper (Th) cells meet at an antigen presenting cell (APC; Figure 1). However, the in Current Opinion in Immunology 2006, 18:496–502 www.sciencedirect.com