chemotaxis. Mainly DC-stimulated effector T cells up-regulated surface CD152 and as a consequence migrated in response to CCL4 and CXCL12. Importantly, CD152 signals also enhanced Th1 cell migration into lymph nodes and a site of cutaneous inflammation in a short term in vivo homing assay. Thus, CD152 ligation determines the positioning of inflammatory T cells at the site of inflammation in vivo. Expression of CD152 on the surface of primary T cells is difficult to detect using conventional cytometric and microscopic techniques [16,18,35,36]. Consequently, most studies on CD152 expression have used intracellular staining methods. In contrast, the sensitive liposome-based staining technique used in this study allowed the detection of as few as 100 to 200 surface molecules per cell [37]. Using this method, we showed that the type of activating APC determines the frequency of surface CD152 expressing T cells. Interestingly, mature DCs, but not activated B cells, were potent in up-regulating surface CD152 on T cells. The fact that differentially activated (i.e. DC vs. activated B cell) T cells differentially express CD152 during an immune response raised the possibility that surface CD152+ and surface CD152no/low T cells could also be heterogeneous in their migratory potentials. Indeed, many more DC-activated T cells expressed surface CD152 and were able to chemotax than were B-cell activated T cells (Fig. 5). Thus, different classes of APC induce distinct T cell migration patterns via the differential induction of CD152 on T cells. CD152 and CD28 share common B7 ligands. However, B7 expression is not limiting on APCs during an optimal immune response, enabling saturated engagement of both CD28 and CD152 [14], making a simple competition of CD152 over CD28 unlikely and dispensable in our experiments. Additionally, there is accumulating evidence that CD152 directly modulates proximal TCR signaling [38]. It is therefore unlikely that enhanced CD28 signaling occurs during serological or genetic blockade of CD152. Therefore, we conclude that CD152-enhanced migration of Th cells is not simply a result of impaired CD28 signaling. Most studies showed that CD152 signaling down-regulates expression of molecules and, ultimately, the function of T cells. In addition, CD152 signaling can up-regulate the expression and activation of molecules such as TGFb, Bcl-2, pFKHRL1, LFA1, and PI3’K [11,39,40]. Functionally, this means that cells expressing CD152 on the cell surface and receiving a CD152 signal are resistant against activation-induced cell death [11]. Additionally, CD152 signals in activated T cells lead to reduced adhesion and increased motility that result in shortened T cell- APC interactions within lymph nodes [38,41]. This finding was interpreted as a mechanism to down-regulate the immune response [42]. Thus, our data add an important new piece to the puzzle by showing that CD152 signals license T cells for chemotaxis and migration into lymph nodes and inflammatory sites. In conclusion, CD152-signaling does not simply silence T cells, but rather enables them to actively contribute to the immune response by enhancing effector T cell migration in vivo. We have now demonstrated that CD152 signaling enhances the cell surface expression of CCR5 and CCR7 on T cells. It is likely that up-regulation of CCR5 and CCR7 supports the CD152- mediated enhanced migration towards of the cognate ligands CCL4 and CCL19, respectively. However, this may not be the only mechanism. For example, we were not able to detect CXCR4 up-regulation in response to CD152 signaling, even though we detected enhanced migration to its ligand CXCL12 (Fig. 1A–E). Chemotaxis is not always accompanied by enhanced chemokine receptor expression on leukocytes because factors that influence chemokine receptor signaling additionally control the migratory capacity in response to chemokines [43–46]. Thus, CD152 could CD152 Promotes Migration regulate T cell chemotaxis alternatively or additionally to the induction of chemokine receptors on T cells by controlling downstream signaling events. Our study is the first report demonstrating that CD152 ligation as well as genetic or serological ablation of CD152 signaling affects chemotaxis of primary T cells. At first view, our data seems to be inconsistent with the observation that CD1522/2 mice show dramatic lymphocyte infiltration of tissues [14,28,35,47]. Howev- er, in these studies a massive expansion of activated lymphocytes is seen; and the cells might just overflow throughout the body in shortage of space. Therefore, it is likely that lymphocytic infiltration of tissues is not only a consequence of directed migration. Alternatively, migration of the expanded cell popula- tions into tissues might be fairly random, but as CD1522/2 cells express CCR7 only at low frequencies (Fig. 6B), and CCR7 expression has been shown recently to be a prerequisite for T cells to exit extralymphoid tissues [48,49], they could be retained in peripheral tissues. In this study, we have shown that enhanced avidity of the TCR determines migration of CD152+/+ T cells along chemokine gradients. This suggests that cells receiving the most optimal signals during stimulation show enhanced CD152 expression and are licensed to migrate. These data are consistent with work of Allison and colleagues, showing that T cells receiving a strong signal up-regulate CD152 [15,18]. However, our results demon- strate that CD152 expressing T cells are not silenced by CD152- signaling but, on the contrary, are directed for migration, thereby driving and preserving the immune response. Taking into account that CD152 induces resistance against activation-induced cell death, together with our new results that CD152 enhances chemotaxis of already activated, effector- cytokine producing T cells, we hypothesize that CD152 surface expression and signaling marks the ‘‘fittest’’ T cells out of a heterogeneous population of stimulated T cells [50]. Depending on the environment, the CD152-marked cells would home to secondary lymphatic organs or to the site of inflammation, probably depending on the stimulation history and the quality of the chemotactic gradient [51]. These cells will be allowed, via CD152 signaling to drive the immune response to fight pathogens immediately and to drive progressive T cell differentiation of TCM cells. Thus, CD152 might actively determine the fate of an effector T cell. Importantly, consistent with our data showing that CD152 up- regulates expression and function of CCR7, a receptor crucial for entry of resting T cells and Th1 cells from the bloodstream into lymph nodes [24,26,52], we found that migration of T cells into lymph nodes was enhanced by CD152 (Fig. 7A). Moreover, we found that CD152 signals promoted accumulation of Th1 cells at a site of cutaneous inflammation (Fig. 7B), which was most likely the consequence of the CD152-enhanced responsiveness to CCL4 and CXCL12 (Fig. 1E, 2) as both ligands are widely associated with inflammation [22,23]. However, CCR7 ligands can also be up- regulated in chronic inflammation [53,54] and others have shown a role for CCR7 ligands in T cell migration into inflamed non- lymphoid tissues [55]. Consequently, it is possible that CD152- regulated CCR7 expression contributes to effector T cell migration into both lymphoid organs and inflamed tissues. Our data suggest that upon activation, T cells will not migrate efficiently into lymph nodes and inflamed tissues unless they up- regulate surface CD152. The migratory properties of the cell population are important to mount an adaptive immune response in the periphery. Thus, CD152 might be one master switch to determine that lymphocytes are in the right time at the right place. PLoS ONE | www.plosone.org 8 May 2009 | Volume 4 | Issue 5 | e5702