The CC chemokine receptor CCR7 has been identified as a key regulator of homeostatic B and T cell trafficking to secondary lymphoid organs. Data presented here demonstrate that CCR7 is also an essential mediator for entry of both dermal and epidermal dendritic cells (DC) into the lymphatic vessels within the dermis while this receptor is dispensable for the mobilization of Langerhans cells from the epidermis to the dermis. Moreover, a distinct population of CD11c(+)MHCII(high) DC showing low expression of the costimulatory molecules CD40, CD80, and CD86 in wild-type animals was virtually absent in skin-draining lymph nodes of CCR7-deficient mice under steady-state conditions. We provide evidence that these cells represent a semimature population of DC that is capable of initiating T cell proliferation under conditions known to induce tolerance. Thus, our data identify CCR7 as a key regulator that governs trafficking of skin DC under both inflammatory and steady-state conditions.

Sphingosine-1-phosphate (S1P) represents a potent modulator of diverse cellular activities, including lymphocyte trafficking and maintenance of lymphocyte homeostasis. The five known receptors for S1P (S1P1–5) belong to the family of G protein-coupled receptors. Upon binding S1P, they act downstream via heterotrimeric G proteins on members of the small GTPase family (Cdc42/Rac/Rho), evoking a S1P receptor-dependent activation pattern of Cdc42, Rac, and Rho, respectively. This, in turn, triggers cytoskeletal rearrangements determining cellular morphology and movement. In this study we investigated the effects of S1P on murine dendritic cells (DC). Mature DC, but not immature in vitro differentiated DC, were found to migrate to S1P, a phenomenon that correlated to the up-regulation of S1P1 and S1P3 in maturing DC. The same pattern of S1P receptor regulation could be observed in vivo on skin DC after their activation and migration into the lymph node. The migration-inducing effect of S1P could be severely hampered by application of the S1P analogon FTY720 in vitro and in vivo. A similar, yet more pronounced, block was observed upon preventing Cdc42/Rac and/or Rho activation by specific inhibitors. These results suggest that S1P-mediated signaling plays a pivotal role in the life cycle of DC.

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Induced antigen-specific Foxp3 1 T cells (iTreg) are being discussed as a promising alternative to polyclonal natural Foxp3 1 T cells (nTreg) for cell-based therapies, particularly to achieve transplantation tolerance. Using Foxp3eGFP-reporter mice, we here establish an efficient protocol to induce and expand alloantigen-specific iTreg from Foxp3 À CD4 1 T cells with cluster-disrupted DC. These iTreg were mainly CD62L 1 and showed efficient suppressive activity in vitro. However, in contrast to nTreg, adoptively transferred iTreg entirely failed to prevent lethal graft versus host disease (GVHD). Within irradiated recipients, the majority of adoptively transferred Foxp3 1 iTreg, but not Foxp3 1 nTreg quickly reverted to Foxp3 À CD4 1 T cells. We therefore suggest that therapeutic approaches to treat GVHD should rely on nTreg, whereas the use of de novo alloantigen-induced iTreg should be handled with caution since the stability of the regulatory phenotype of the iTreg could be of major concern.Key words: Animal models . Dendritic cells . Graft rejection . Regulatory T cells See accompanying article by commentary by Edinger IntroductionRecent advances have demonstrated that adoptively transferred exogenous Treg can inhibit graft versus host disease (GVHD) [1][2][3]. However, the availability of sufficient numbers of donor Treg for cell-based therapies remains limited [4]. Besides the in vitro expansion of nTreg [5], an obvious approach to solve this problem would be the de novo induction and expansion of Foxp3 1 Treg from abundant naïve CD4 1 T cells with recipient alloantigens [6,7]. Instead of mediating unspecific suppression, such alloantigen-induced Treg potentially could provide the advantage of antigen-specific regulation, thereby reducing the risk of disease relapse and infections [8]. Here, we present an efficient protocol to induce Foxp3 1 Treg by the use of clusterdisrupted allogeneic DC. Such allo DC-induced iTreg were functionally active in vitro and displayed a stable regulatory phenotype upon adoptive transfer into untreated syngeneic recipients. However, when used in experimental acute GVHD, these cells quickly lost Foxp3 expression and, in contrast to nTreg, did not show any protective effect. SHORT COMMUNICATION Results and discussionTo define conditions suited for the de novo induction of alloantigen-specific iTreg, we isolated Foxp3 À CD4 1 T cells from C57BL/6 Foxp3EGFP mice and co-cultured them with BM-derived allogeneic BALB/c DC that were either matured by application of LPS or via ''cluster-disruption'' (CD-DC). Disruption of the Ecadherin-mediated cell-cell contacts induces DC maturation through mechanisms distinct from TLR signaling [9]. Since antigen-loaded CD-DC have been shown to induce tolerance in mice after i.v. injection [9] we established a protocol that allowed the conversion of naïve to Foxp3 expressing cells in vitro in the presence of allogeneic but not syngeneic CD-DC. When compared with LPS-matured DC, CD-DC showed equally high expression of MHC class II, but diminished up...

Adhesion and motility of mammalian leukocytes are essential requirements for innate and adaptive immune defense mechanisms. We show here that the guanine nucleotide exchange factor cytohesin-1, which had previously been demonstrated to be an important component of beta-2 integrin activation in lymphocytes, regulates the activation of the small GTPase RhoA in primary dendritic IntroductionIntegrin-mediated adhesion is an essential function of most metazoan cells, and plays a key role in, for example, organ development, the maintenance of tissue architecture, blood clotting, and many aspects of leukocyte biology. The mammalian integrin family consists of 24 heterodimeric cell surface receptors that bear distinct functional binding specificities for extracellular matrix proteins and plasma membrane counterreceptors. 1 Immune cells have highly specific requirements for timing and location of cell-cell and -matrix contacts, which need to be finely tuned to variable conditions of homeostasis and inflammation.An important hallmark of integrin function in the immune system is the rapid adhesion of leukocytes to activated endothelia, a process that is essential for immune cell egress from the vasculature into either lymphoid organs or into infected tissues. To perform such functions, integrins on leukocytes have to be activated by a process that has been termed "inside-out" signaling. It is now generally believed that integrins on circulating, resting leukocytes are conformationally restricted, so that they normally cannot bind to their protein ligands, such as ICAM-1 or VCAM-1. However, if a leukocyte becomes tethered to an endothelial cell, for example, by selectin-mediated adhesive "rolling," immune cell chemokine receptors such as CCR7 or CXCR4 may be activated by chemokines present on the activated vessel wall. 2,3 This, in turn, results in intracellular, heterotrimeric G-protein-coupled signal transduction, which enables very fast conformational changes and ligand binding of cell surface integrins, resulting in a robust firm adhesion, the so-called integrin-mediated leukocyte "arrest." 3 Shear forces imposed by the blood flow apparently play an important role in the regulation of this process. 4 The mode of activation of immune cell integrins is incompletely understood. Several intracellular proteins that directly or indirectly interact with the cytoplasmic domains of the leukocyte-specific beta-2 integrins, such as LFA-1, have been implicated in this function. These include the actin-and integrinbinding protein talin, which is important for the general coupling of integrin-mediated cell adhesion to the actin cytoskeleton, 5-7 the GTPase Rap1 7-9 and its effector proteins RAPL and RIAM, 9-11 the ADAP/Vav/SLP76 complex in T cells, 12-14 the Rho GTPases RhoA 15 and CdC42, 16 and, recently, the so-called kindlin proteins. 17,18 Our laboratory has identified the guanine nucleotide exchange factor (GEF) for ADP-ribosylation factor (ARF) GTPases, cytohesin-1, as an LFA-1 interacting protein. Cytohesin-1 binds specifical...