CD152 Promotes Migration Figure 4. CD152-signaling mediates migration of recently activated Th1 cells. (A) CD4+CD62L+ TCRtg T cells were stimulated with 1 mg/ml OVA peptide and T cell-depleted APCs under Th1 conditions. Recall response of Th1 cells was induced by platebound anti-CD3/anti-CD28 for 48 hours. Cells were washed, incubated with microspheres coated with anti-CD3, anti-CD28 and anti-CD152 (0.15 mg/ml, 0.4 mg/ml, 4.5 mg/ml respectively) or anti-CD3, anti-CD28 and hamIgG for additional 24 hours and examined in chemotaxis assay. (B) Restored specific migration of CD1522/2 Th1 cells by CD152 expression. CD1522/2 splenocytes were activated with 2 mg/ml anti-CD3 under Th1 conditions and on day 2 were retrovirally transduced with cDNA of full length CD152 (pMSCV-CD152) or with empty vector pMSCV. Chemotactic capacity of transduced CD4+ cells was determined in chemotaxis assays. (C) Migration towards CCL19 plus CCL4. CD4+ TCRtg T cells from CD152+/+ and CD1522/2 mice were stimulated under Th1 conditions as described in figure 2. On day 6 of recall response viable cells were analyzed in chemotaxis assays for migration capacity towards CCL4 and CCL19 alone or in combination. One out of at least 3 experiments with similar results is shown. doi:10.1371/journal.pone.0005702.g004 CD152 is predominantly expressed on the surface of T cells stimulated by DCs CD152 is not expressed on the surface of resting T cells and is difficult to detect even after stimulation of T cells [18,31,32]. In order to analyze CD152 expression on individual T cells activated by different APCs we stimulated na¨ıve OVA-specific CD4+ T lymphocytes with the peptide OVA323–339 loaded on either activated B lymphocytes or DCs. To unambiguously identify T cells expressing surface CD152, we used an enhanced staining technique [33] based on CD152-specific immunofluorescent liposomes, which increased the detection sensitivity at least 1000-fold [11,16]. Employing this method, expression of surface CD152 was evaluated at various time points after T cell activation (Fig. 5A, C). B cells and DCs stimulated T cells equally well as determined by the induction of similar T cell proliferation rates of 85–86% (Fig. 5B) on day 3 after onset of stimulation. Specific surface expression of CD152 was detected with low frequencies on day 1 in primary T cell cultures activated with either activated B cells or DCs. Following primary stimulation, CD152 expression increased and peaked after 48 h. Thereafter, the percentage of surface CD152-expressing cells declined in both cultures and these cells were infrequent (,3%) after 3 days of stimulation. At the peak of surface CD152 expression, T cell populations stimulated by DCs displayed enhanced expression of surface CD152. Whereas more than 23% of the T cells stimulated by DCs expressed surface CD152, only ,3% of the T cells displayed CD152 on their surface 48 h after stimulation with activated B cells (Fig. 5A, C). The number of surface CD152-expressing cells could not be attributed to contaminating Th2 cells, as no IL-4 producers were present in the cultures (data not shown). Thus, mainly T cells stimulated by DCs are able to express surface CD152, showing that the activation history and activating cell type (DC versus B cell) regulate CD152 expression on T cells. Next, we asked whether the differential potential of DCs versus activated B cells to induce CD152 on T cells correlated with a differential potential to license T cells for chemotaxis. Correlating with the induction potential of CD152 on T cells, T cells stimulated by DCs migrated significantly better in response to CCL4 or CXCL12 than did T cells activated by B cells (Fig. 5D). Thus, the data suggest that chemokine-driven migration of T cells is controlled by mature DCs and correlates tightly with the up- regulation of surface CD152 on T cells. CD152 induces expression of chemokine receptors CCR5 and CCR7 Many reports show that CD152 down-regulates expression of effector molecules such as IL-2 and cyclin-dependent kinases [9,34]. We have shown recently that CD152 can indeed up- regulate the expression and activate molecules such as Bcl-2, pFKHRL1, and PI3’K [11]. To determine the effects of CD152 on the expression of chemokine receptors, cells from CD1522/2 and CD152+/+ mice were used. After polyclonal stimulation of splenocytes from CD1522/2 and CD152+/+ mice with anti-CD3 and induction of recall response with syngeneic APCs and anti- CD3, we detected no difference in CXCR4 expression (Fig. 6A), but we found enhanced expression of CCR7 by CD152+/+ cells compared with CD1522/2 cells (Fig. 6B). To further elucidate the role of CD152 in the induction of CCR5, splenocytes of CD1522/2 and CD152+/+ mice were activated with anti-CD3 under Th1 polarizing conditions, and upon recall response, analyzed for CCR5 PLoS ONE | www.plosone.org 5 May 2009 | Volume 4 | Issue 5 | e5702