CD152 Promotes Migration Figure 2. Only CD152+/+ but not CD1522/2 cells show efficient transendothelial migration. Primary stimulation and recall response of CD4+ OVA-specific TCRtg T cells from CD1522/2 and CD152+/+ mice were performed using Th1 conditions with 10 mg/ml OVA-peptide and T cell-depleted APCs. On day 6 of recall response migration of CD4+ cells through membranes coated with or without endothelial mono- layer was analyzed after 90 min. incubation at 37uC in Transwell chemotaxis assay. CD152 mediated migration of Th1 cells is G-Protein dependent: CD4+ OVA-specific TCRtg CD1522/2 and CD152+/+ Th1 cells were incubated for 2 hours at 37uC in the presence of 100 ng/ml Pertussis toxin prior to examination in chemotaxis assay. (Inset) Endothelial cells (mlEND) were grown to a confluent monolayer for 48 h on Transwell membranes and confluency was controlled by microscopy. Shown data represent one out of 2 similar experiments. doi:10.1371/journal.pone.0005702.g002 Similar IFN-c-production was detected in TCRtgCD1522/2 and TCRtgCD152+/+ CD4+ T cells under Th1 conditions and with strong antigenic stimulus (10 mg/ml OVA-peptide; data not shown). Furthermore we inhibited IFN-c by adding neutralizing anti-IFN-c to the cultures during the antigenic recall response. Adding anti- IFN-c led to an overall reduction in migration, however, the reduced migration of TCRtgCD1522/2 Th1 cells could not be reversed by blocking IFN-c (Fig. 3). Furthermore, addition of exogenous IFN-c to the cultures did not reduce migration of CD152+/+ Th1 cells, indicating that CD152-modulated migration of Th1 cells was not due to effects of different IFN-c concentrations between cultures of TCRtgCD1522/2 Th1 cells and TCRtgCD152+/+ Th1 cells. CD152 engagement induces chemokine responsiveness of recently activated T lymphocytes CD152 exerts its main function during its peak expression in a T cell response, which is typically 48 h following activation [11,16]. To monitor CD152-mediated effects on T cell migration, a recall response of Th1 cells was induced for 48 h with congenic APCs and OVA. Additionally, antibody-coupled microspheres were used to cross-link CD152 in combination with CD3 and CD28 coligation. The stimulation with anti-CD3/anti-CD28- coupled microspheres allowed for direct crosslinking of CD152 and also kept the CD28-signal constant in all populations. On day 3 of activation, migration of activated CD152-engaged Th1 cells towards CXCL12 and CCL19 was considerably enhanced compared with Th1 cells engaged only with CD3 and CD28 Figure 3. CD152-enhanced migration is IFNc independent. CD4+ OVA-specific TCRtg splenocytes from CD1522/2 and CD152+/+ were stimulated under Th1 conditions (as described in Fig. 2) in the presence of 20 ng/ml recombinant IFN-c or 10 mg/ml blocking anti-IFN-c - antibodies. On day 6 after inducing a recall response cells were analyzed for migration towards CCL4 in chemotaxis assays. The data represent one out of 3 similar experiments. doi:10.1371/journal.pone.0005702.g003 (Fig. 4A). Strikingly, 4-times more Th1 cells migrated towards CCL4 after stimulation with anti-CD3/anti-CD28/anti-CD152 compared with anti-CD3/anti-CD28-stimulated T cells. Thus, CD152 engagement in concordance with CD3 and CD28 of Th1 cells triggered chemokine responsiveness in the absence of additional signals provided by APCs. To confirm that enhanced migration of Th1 cells is regulated by CD152 and is not merely the result of an early differentiation event, we retrovirally transduced CD1522/2 cells during in vitro stimulation with the cDNA of full length CD152 (pMSCV- CD152). Upon a recall response, retroviral transduction with pMSCV-CD152, but not with the control vector, restored migration towards CCL4 in CD1522/2 cells (Fig. 4B). Thus, CD152 expression mediates enhanced migration even in highly activated T cells and the detected lack of migration of CD1522/2 T cells is due to their lack of CD152 and not to developmental alterations. Next, we determined whether the CD152-controlled migration towards the inflammatory chemokine CCL4 and the homoeostatic chemokine CCL19 reflects heterogeneity of the T cell population. When migration towards a single chemokine gradient of CCL4 or CCL19 was tested, TCRtgCD152+/+ Th1 cells showed enhanced migration compared with TCRtgCD1522/2 Th1 cells after antigen-specific recall responses (Fig. 4C). Migration to a combined chemokine gradient of CCL4 and CCL19 was similar to migration towards a single CCL4 gradient, suggesting that the same cell population responds upon CD152-signaling towards CCL4 and CCL19. PLoS ONE | www.plosone.org 4 May 2009 | Volume 4 | Issue 5 | e5702