CD152 Promotes Migration Figure 5. CD152-enhanced migration is primarily mediated by DCs. (A) Surface expression of CD152 on CD4+ cells is up-regulated by stimulation with DCs. Nave CD4+CD62L+ TCRtg T cells were stimulated with 1 mg/ml OVA-peptide presented by matured DCs or by LPS-activated B cells. After 48 hours surface expression of CD152 was detected by liposome staining technique and subsequent FACS analysis. Left panels show CD152 staining, right panels show blocking controls. Numbers indicate the percentage of CD152+ CD4+ based on total CD4+ cells. (B) Equal proliferation of T cells stimulated with activated B cells or matured DCs. Nave CD4+CD62L+ TCRtg T cells were labeled with CFSE and antigen- specifically stimulated with activated B cells or matured DCs. Proliferation of T cells was determined by flow cytometry 48 hours (filled curve) and 72 hours later (black line). T cells cultured with different APCs but without antigenic stimulation are shown as heavy grey line. Numbers indicate the percentage of divided T cells 72 hours after stimulation relating to unstimulated controls (after 48 h 48% of Dc-stimulated T cells and 46% of Bc- stimulated T cells proliferated). (C) Kinetics of CD152 surface expression on CD4+ T cells. Indicated time after onset of stimulation cells cultured as described in 5A were stained with liposome staining technique for CD152. Percentages of surface expressing CD152 cells of total CD4+ cells are shown. (D) Chemotactic index of Th1 cells stimulated with different APCs. CD4+CD62L+ TCRtg T cells were stimulated under Th1 conditions with 1 mg/ ml OVA peptide presented by activated B-cells or bone marrow derived DCs. On day 6 of primary stimulation, CD4+ cells were analyzed for migration capacity in chemotaxis assay. All data show representative data from at least two experiments. doi:10.1371/journal.pone.0005702.g005 Figure 6. CD152 mediates up-regulation of chemokine receptors CCR5 and CCR7. Recall response of splenocytes from CD152+/+ and CD1522/2 mice was induced polyclonally under Th1 conditions. On day 3 of recall response, cells were stained for surface markers and analyzed by flow cytometry. The histograms indicate surface expression on CD4+ lymphocytes of (A) CXCR4, (B) CCR7 and (C) CCR5; (M1: 48% CD152+/+; 29% CD1522/2). (D) After performing recall response of splenocytes from CD152+/+ mice with microspheres (as described in Fig. 4A) CCR5 was detected on CD4+ cells. Representative results from one out of 3–5 experiments are shown. doi:10.1371/journal.pone.0005702.g006 PLoS ONE | www.plosone.org 6 May 2009 | Volume 4 | Issue 5 | e5702