transported from intracellular vesicles to the cell membrane where it is expressed in a polarized manner at the immunological synapse [17]. The surface expression is mainly induced by TCR signaling strength, but can be enhanced by soluble factors [18,19]. Costimulation is an important control point to ensure that professional APCs define initiation, differentiation, and persistence of T cell responses. It has recently been proposed that tissue specific APCs direct and reprogram T cell migration into specific tissues [20,21]. Given the central role of costimulation in differentiation and function of T cells, we postulate that costimulatory molecules participate in the localization of adaptive immune responses by licensing T lymphocytes for chemotaxis. In the present study, we investigated the functional role of CD152 in migration of Th1 cells. We show that CD152 surface expression is induced in Th1 cells stimulated primarily by mature DCs when compared with other APCs. Surprisingly, CD152 engagement induced activated Th1 cell migration towards CXCL12, CCL19, and CCL4, the ligands for CXCR4, CCR7, and CCR5, respectively, whereas inhibition of CD152 abrogated migration. Importantly, CXCR4 and CCR5 and their ligands are associated with inflammatory diseases [22,23] and CCL19 as well as CXCL12 are also important for lymphocyte homing to lymph nodes and Peyer’s patches [24–26]. Moreover, we show that CD152-mediated signals endow effector T cells with the capacity to migrate to sites of inflammation and lymph nodes. This novel function of CD152 could explain the retention and directed migration of effector cells to secondary lymphoid organs and sites of antigenic-challenge and might be important for T cell homeostasis as well as Th1-dominated diseases. Results Serological and genetic inactivation of CD152 reduces chemotaxis of Th1 cells to CXCL12 and CCL4 To examine the role of CD152 in T cell migration, we stimulated CD152-deficient (CD1522/2) monoclonal OVA-spe- cific TCRtg CD4+ T cells from OTII mice (TCRtgCD1522/2) and monoclonal OVA-specific TCRtg CD4+ T cells from CD152+/+ OTII mice (TCRtgCD152+/+) with cognate antigen in vitro and tested chemotaxis to the CXCR4 ligand CXCL12, the CCR7 ligand CCL19, and the CCR5 ligand CCL4 in a Transwell chemotaxis assay (Fig. 1A, and data not shown). Similar baseline migration, which was routinely 5–10% in our Transwell migration assays (Figs. 1–5), was detectable for TCRtgCD152+/+ T cells and TCRtgCD1522/2 T cells. Under these conditions, no migration towards the CCR5 ligand CCL4 was seen in either population (data not shown). In contrast, TCRtgCD152+/+ T cells migrated 2- fold better in response to CXCL12 than did TCRtgCD1522/2 T cells (Fig. 1A). Similarly, migration towards the CCR7 ligand CCL19 was diminished by 70% in TCRtgCD1522/2 T cells compared with TCRtgCD152+/+ cells. To examine a role for CD152 in the contribution to the migratory properties of pro-inflammatory T lymphocytes upon primary stimulation, CD1522/2 and CD152+/+ TCRtg CD4+CD62L+ T lymphocytes were stimulated with antigen under Th1 conditions and migration was evaluated in Transwell chemotaxis assays on day 5 (data not shown) or day 6 after stimulation (Fig. 1B); both assays gave similar results. Cells capable of receiving a CD152 signal showed a significantly higher migratory response toward the inflammatory chemokine CCL4 (Fig. 1B). In contrast, for the mainly homeostatic chemokines CXCL12 and CCL19, no significant difference in migration was observed when comparing monoclonal CD4 cells from TCRtgCD152+/+ and TCRtgCD1522/2 mice. CD152 Promotes Migration CD152 functions are more pronounced upon activation of antigen-experienced T cells than upon primary stimulation of na¨ıve T cells [27]. Therefore, we inactivated CD152 on antigen- experienced Th1 cells by adding neutralizing anti-CD152 Fab fragments to Th1 cells during primary stimulation and recall response, and monitored migration on day 6 of recall response (Fig. 1C). Polarization of primary Th1 cells resulted in .98% pure effector populations, as confirmed by a CD44high IFN-c+ IL-42 phenotype (data not shown). Following recall responses of antigen- experienced Th1 cells, cells that received a CD152 signal migrated two-fold better towards CCL4 than Th1 cells that were activated under a serological blockade of CD152 (Fig. 1C). Similarly, blockade of CD152 by the addition of anti-CD152 Fab fragments also resulted in reduced frequencies of cells migrating towards CXCL12. To substantiate a role for CD152 in mediating the migratory capacity of antigen-experienced T lymphocytes, we used T cells from CD1522/2 and CD152+/+ mice. On day 4 after a polyclonally induced recall response of splenocytes with anti- CD3 and APCs, substantially more CD152+/+ T cells migrated compared with CD1522/2 T cells (Fig. 1D): wild-type cells migrated three- and two-times better than CD1522/2cells toward CCL4 and CXCL12, respectively. Intriguingly, using 10 mg/ml instead of 1 mg/ml OVA during stimulation enhanced the migration for TCRtg CD152+/+ T cells by 2-fold (Fig. 1E). In contrast, CD1522/2 T cells did not migrate significantly better with higher peptide concentrations (Fig. 1E). Thus, the enhanced migration of CD152+/+ T cells towards CCL4 and CXCL12 was dependent on the concentration of antigen during TCR stimulation (duration and strength of signal). Interestingly, this chemotaxis enhancing property was specific to CCL4 and CXCL12, because no difference in migration towards CXCL9 occurred, even when high OVA concentrations were used for stimulation. Migration towards CCL19 was slightly enhanced by altering the signaling strength during stimulation, when Th1 cells received a CD152 signal (Fig. 1E). Most strikingly, CD152+/+ but not CD1522/2 antigen-experienced Th1 cells migrated to the homeostatic chemokine CCL19. In summary, the presence of functional CD152 enhanced the chemotaxis of T cells, especially that of repeatedly activated Th1 cells. CD152 enhances chemokine-driven transendothelial migration Recruitment of lymphocytes to the site of infection involves a sequential, multi-step process leading to the active transmigration of lymphocytes across the blood vasculature endothelium [28]. As part of the process, lymphocytes migrate through endothelial cell junctions, a step referred to as diapedesis. Using mlEND endothelial cells grown confluently on a membrane (Fig. 2 inset), transmigration of Th1 cells towards CCL4, CXCL12 and CCL19 across an endothelial layer was compared to transmigration across a membrane only (Fig. 2). Migration of CD152+/+ Th1 cells towards indicated chemokine gradients of CCL4, CXCL12 and CCL19 was enhanced at least two-fold as compared with CD1522/2 Th1 cells, no matter whether transmigration was performed across a membrane only or across an endothelial layer. Thus, we conclude that CD152 engagement enhances transen- dothelial migration of T cells (Fig. 2). CD152-induced migration of pre-activated Th1 cells depends on Gai signaling and is independent of IFN-c Chemokine receptors are known to signal through G proteins [29]. By blocking Gai-linked chemokine receptor signaling with PLoS ONE | www.plosone.org 2 May 2009 | Volume 4 | Issue 5 | e5702