expression on CD4 cells. 20% more CD152+/+ than CD1522/2 CD4 T cells expressed CCR5 on their cell surface (Fig. 6C). In addition, agonistic signaling of CD152 in CD152+/+ T lymphocytes using anti-CD3/anti-CD28/anti-CD152 cross-linking under Th1 conditions up-regulated CCR5 as compared to T cell activation with only anti-CD3/anti-CD28 cross-linking (Fig. 6D). Thus, CD152 regulates expression of the chemokine receptors CCR5 and CCR7 correlating with the observed CD152-enhanced migration to the corresponding ligands CCL4 and CCL19. Differential migration pattern of CD152+/+ and CD1522/2 Th1 cells in vivo To test the in vivo significance of the enhanced in vitro migration towards inflammatory and lymphoid chemokines due to CD152 signaling, we analyzed the in vivo homing capacity of CD152+/+ and CD1522/2 TCRtg Th1 cells. Recipient mice were either injected i.v. with radioactively labeled CD152+/+ TCRtg Th1 or CD1522/2 TCRtgTh1 cells. 24 h after cell transfer, mice were injected s.c. into one footpad with OVA-peptide in IFA and in a control footpad with PBS in IFA only. 24 h later, radioactivity in different organs, the inflamed and accordingly control footpad was measured (Fig. 7A). Both transferred cell populations accumulated with no difference in non-inflamed organs (including gut, skin, lung, spleen, liver, rest of the body). Strikingly, CD152+/+ Th1 cells migrated enhanced into resting and inflammation draining lymph nodes compared with CD1522/2 Th1 cells (Fig. 7A). CD152 Promotes Migration Importantly, CD152+/+ Th1 cells migrate 2-fold better into the site of inflammation (footpad) than did CD1522/2 Th1 cells (Fig. 7B). In independent experiments testing migration in the absence of inflammation, CD152+/+ Th1 cells also homed better than their CD1522/2 counterparts into lymph nodes (p| = |0.0006; data not shown). The detected differences in migration were not due to differences in polarization or activation status of CD1522/2 versus CD152+/+ T cells since both subsets expressed similar levels of the activation markers CD69, CD44, CD25, CD62L, had an identical capacity to secrete IFN-c before stimulation (Fig. 7C, upper panel) and on day 5 after induction of recall response (Fig. 7C, lower panel) and showed no IL4-, IL10- or IL17-producing T cells at the time point of analysis in chemotaxis assays (data not shown). Consistent with their in vitro migration to inflammation (CCL4, CXCL12) and lymph node expressed chemokines (CCL19, CXCL12) CD152 competent T cells are licensed for migration into both, sites of acute inflammation and lymph nodes. Discussion Our results show that CD152 expression and signaling in T cells is a major control point for their migration both in vitro and in vivo. The heterogeneity of CD152 expression in individual primary T cells stimulated by different APCs has not been analyzed previously and our study reveals that T cells stimulated by different APCs also differ in CD152-mediated enhanced in vitro Figure 7. Th1 cell homing to the site of inflammation and lymph nodes is enhanced by CD152. (A) Distribution of TCRtg CD1522/2 and CD152+/+ Th1 cells 48 hours after transfer. In vitro induced Th1 cells from a recall response were radioactively labeled and injected i.v. (56106 cells per mouse). After 24 hours OVA-peptide (250 ng) in IFA was administered s.c. into one footpad (DTH); PBS/IFA-injection in the other footpad served as control. Different organs were analyzed after 48 hours for radioactivity. Recovered radioactivity in organs was calculated as percentage of total amount of radioactivity per mouse (7 mice per group). (LN: mesenteric and axillary lymph nodes; drLN: draining (popliteal and inguinal) lymph nodes; PBL: peripheral blood lymphocytes). Results from one out of two similar experiments are shown. (B) Specific migration of transferred CD1522/2 and CD152+/+ Th1 cells to the site of inflammation. Radioactively labeled CD1522/2 and CD152+/+ Th1 cells were transferred into mice and DTH was induced in footpads as described in 7A. Shown is the antigen-dependent accumulation of Th1 cells to the site of inflammation indicating the ratio of recovered radioactivity in the inflamed footpad versus the PBS injected footpad. (C) Similar expression of activation induced markers and similar amount of IFN-c-producers. TCRtg CD1522/2 and CD152+/+ CD4+ T cells were either left unstimulated or were in vitro stimulated under Th1 conditions and a recall response was performed. Unstimulated cells were stained on day 3 (upper panel) and stimulated cells on day 5 of recall response for activation induced molecules (lower panel). Intracellular staining for IFN-c of fixed cells was performed on day 3 of recall response after restimulation with PMA/Ionomycin for 6 hours; Brefeldin A was added for the last 2 hours of incubation. One out of two similar experiments is shown. doi:10.1371/journal.pone.0005702.g007 PLoS ONE | www.plosone.org 7 May 2009 | Volume 4 | Issue 5 | e5702