Materials and Methods Mice Transgenic (tg) mice for OVA-specific TCRtg/tg on BALB/c background (Do11.10, gift from Dennis Y. Loh, Washington University School of Medicine, St. Louis, MO) or C57/B6 background (OTII), CD152 knock-out mice on C57/B6 back- ground (gift from James P. Allison, University of California at Berkeley, CA). All mice were bred under specific pathogen-free conditions at the Bundesinstitut f u¨r Risikobewertung (Berlin) and were used at the age of 5 to 10 weeks except for non-transgenic CD1522/2 mice, which were used at the age of 3 weeks. Animal studies were reviewed and approved by Landesamt fu¨ r Gesundheit und Soziales, Berlin (G0075/02). Antibodies, cytokines and reagents The following antibodies were used: anti-CD152 (UC10-4F10-11), anti-CD25 (7D4), anti-CD69 (H1.2F3), anti-IL4 (11B11), anti-CCR5 (C34-3448), anti-CXCR4 (2B11/CXCR4), ratIgG1 (R3-34), ha- mIgG2 (KLH, HA4/8), ratIgG2c, ratIgG2b (A95-1) (all from BD Biosciences), anti-CCR7 (4B12, eBioscience) ratIgG2a (eBioscience) coupled to FITC, PE or Cy5-conjugates. Anti-CD28 (37.51), anti- CD3 (145-2C11), anti-IL4 (11B11), anti-CD4 (GK-1.5/4), anti- CD62L (MEL-14), anti-CD44 (IM7), anti-TCRtg (KJ1-26.1), anti- IFN-c (AN18.17.24), anti-CD11c (N418), anti-CD152 (UC10-4F10- 11) and Syrian hamster control antibody (560-31.1B9; kindly provided by J.P. Allison) were purified from hybridoma supernatants. Anti-CD152 Fab fragments were prepared with the Immunopure Fab preparation kit (Pierce) and used at 200 mg/ml. Recombinant chemokines and IL-12 were from R&D Systems, pertussis toxin from Sigma; magnetic microbeads (anti-CD4, anti-CD19, anti-CD43, anti-CD62L, anti-CD90, anti-FITC multisort) were from Miltenyi- Biotech; and sulfate polystyrene latex microspheres of 5 mm60.1 mm mean diameters from Interfacial Dynamics. Cell isolation and stimulation Isolation of na¨ıve CD62LhighCD4+ T cells was performed using magnetic cell separation (MACSTM) to a purity greater than 98%. Isolated TCRtg cells were stimulated with 1 or 10 mg/ml OVA323– 339 peptide (provided by Schneider-Mergener, Charite´) and T cell- depleted splenocytes as APCs. On day 6 after onset of stimulation, recall response was performed by adding freshly isolated APCs and OVA-peptide to density-gradient-purified cells. CD1522/2 or CD152+/+ TCRwt splenocytes were stimulated with 1.5 mg/ml ConA and recall response was induced on day 5 with 2 mg/ml anti- CD3 and CD902 APCs from syngeneic C57/B6 mice. CD432 B cells were activated with 20 mg/ml LPS for 24 hours. DCs were generated from bone marrow of syngeneic mice by culturing for 8 days with 10% GM-CSF and subsequent maturation for 24 hours with TNFa (10 ng/ml). For Th1 polarization recombinant IL-12 (10 ng/ml) and anti-IL-4 (6 mg/ml) were added to the cultures. Crosslinking of CD152 on CD4 cells was performed using latex microspheres coated with antibodies against CD3 (0.15 mg/ml), CD28 (0.4 mg/ml), CD152 or a hamster control antibody (N418, 4.5 mg/ml) as described [16,32]. CD4 T cells (16106/ml) were stimulated at a ratio of one bead per cell with antibody-coupled microspheres. Cell cycle progression was measured by labeling of T cells with CFSE (5 mM in PBS, Molecular Probes) for 5 min at room temperature. The reaction was stopped by washing and resuspending the cells in RPMI-1640 medium. CD152 Promotes Migration ml hamster CD152 antibody of for 15 min at 4uC. Cells were washed and incubated with Cy5 dye-filled liposomes, conjugated to hamster Fab fragments, for 30 min at 4uC. Unbound liposomes were removed by washing. The specificity of CD152 staining was controlled by isotype control antibody conjugated with Cy5-filled liposomes as well as by incubation of cells with Cy5-filled liposomes only. Retroviral transduction 293T fibroblasts were transfected with calciumphosphate-pre- cipitated pECO, pCGP, pMSCV-GFP (empty vector) or pMSCV- GFP-CD152 (containing full length CD152 cDNA) respectively. After 4 hours medium was exchanged and fibroblasts were cultured for additional 48 hours in DMEM containing 10% FCS, 2 mM glutamine and 50 mM 2-mercaptoethanol. CD1522/2 splenocytes, activated with 2 mg/ml anti-CD3 using Th1 conditions for 1–2 days, were resuspended in virus supernatant and retrovirally transduced by centrifugation for 90 minutes at 25uC. Cells were resuspended in conditioned medium, recall response was performed on day 4 and cells were analyzed in chemotaxis assays 3 days later. Chemotaxis assay Th1 cells were collected on day 4–6 of recall response. After density centrifugation on Histopaque 1083 (Sigma-Aldrich), chemotaxis assays were performed using 5-mm pore polycarbonate Transwell filters (Corning Costar). 56105 cells in 100 ml assay medium were added on fibronectin-coated filters (10 mg/ml, Invitrogen), chemokine dilutions (10 nM CCL4, 10 nM CCL19, 20 nM CXCL12, 100 nM CXCL9) or medium were given to lower chambers. For transmigration through endothelium, 56104 mlEND1 cells (gift from Prof. Dr. Rupert Hallmann, University Hospital, Lund) in medium were seeded on fibronectin-coated filters and incubated for 48 hours before adding Th1 cells. Migrated cells were recovered after 90 min of incubation at 37uC and frequency of migrated cells was determined by staining with anti-CD4 (L3T4, BD Biosciences) and comparing numbers to a fixed amount of fluorescent beads (Fluoresbrite Microparticles 20 mm, Polysciences Inc.) by flow cytometry [52]. All determinations were performed in triplicate. Chemotactic index indicates the ratio of cells migrated to chemokine versus medium. Confluence of the endothelial mono- layer was checked by microscopic analysis. To do so, cells were washed in PBS, fixed with paraformaldehyde, stained with GIEMSA-solution for 30 min. and dried over night. Membranes with cells were removed from the inserts and embedded in Entellan. Migration of lymphocytes in vivo In vitro cultured Th1 cells from OVA-TCRtg CD1522/2 and CD152+/+ were collected on day 5 of recall response. Cells were radioactively labeled with 20 mCi/ml sodium (51Cr) chromate (Hartmann Analytic) for 1 hour at 37uC. After washing and dead cell removal, 56106 cells in 200 ml HBSS per mouse were injected i.v. into tail veins in seven C57/B6 mice per group. 24 hours later, delayed type hypersensitivity (DTH) response was initiated by administration of OVA-peptide (250 ng) in IFA (Pierce) s.c. into one footpad. Injection of PBS/IFA in the other footpad served as control. 48 hours after cell transfer mice were sacrificed and organs were analyzed by Beckmann c-Counter for recovered radioactivity. Radioactivity in different organs was calculated as percentage of total amount of radioactivity per mouse. Flowcytometric analysis of CD152 surface expression Surface expression of CD152 was detected using magneto- fluorescent liposomes [16,33]. T cells were incubated with 1 mg/ Statistical Analysis Statistical analysis was performed using the two-tailed unpaired Student’s t-Test (Figure 1–5) or Mann-Whitney Test (Figure 7A, PLoS ONE | www.plosone.org 9 May 2009 | Volume 4 | Issue 5 | e5702