Eur. J. Immunol. 2011. 41: 2997–3005 Immunomodulation 3003 Cytokine treatment Corneal transplantation Recombinant murine pro-inﬂammatory cytokines TNF-a, IL-1a and IFN-g (PeproTech EC, London, UK) were added directly to cell cultures at ﬁnal concentrations of between 10 and 100 ng/mL and cultured for 48 h. In vitro T-cell proliferation assays Spleen cells from C57BL/6 mice were treated with a mixture of anti-CD45R/B220, anti-CD8 and anti-MHC class II supernatants (RA3-3A1, M5/114, 53.6.7 and 2.4G2) for 30 min. After antibody treatment, the cells were washed and incubated with goat anti- mouse IgG and goat anti-rat IgG-coated beads (Dynal, Brombor- ough, UK) for 30 min, followed by removal of bound cells with a magnet [39]. Responder CD41 T cells (1 105 cells/well) were stimulated with beads coated with anti-mouse CD3 and CD28. Tissues were ground using the beads. After centrifugation, 10 mL of supernatant from the homogenized tissues was mixed with MnCl2 and added to the T cells in the presence or absence of 10 mM competitive arginase inhibitors L-2-Amino-(4-(20-hydro- xyguanidino)butyric Acid (nor-NOHA) or BEC, or 0.1 mM L-arginine in 96-well plates. Proliferation was measured by a 16-h pulse with 3H thymidine (10 mL, 5 mCi/mL, Amersham Pharmacia Biotech). Orthotopic corneal transplantation was performed using a modiﬁ- cation of a technique described previously [44, 45]. Brieﬂy, donor C57BL/6 (H2b), (B6) corneas were excised using a 2.0-mm trephine (Duckworth & Kent, England). The right corneas of recipients were excised with Vannas scissors (Duckworth & Kent). B6 corneas were then applied to BALB/c corneal beds, secured with 8–9 continuous 11/0 nylon sutures. Sutures were removed on day 7, at which time any grafts showing technical failures were excluded from the study [44]. The speciﬁc arginase inhibitor N-hydroxy-nor-Arg (Bachem, Germany) was injected intraperitoneally (i.p.) at 100 mg/kg/day for the treatment group (Control animals received the same volume of PBS). All animals were obtained from Harlan Olac (Bicester, UK) and treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and United Kingdom Home Ofﬁce Guidance. Skin transplantation Skin grafting was conducted as described by Billingham and Medawar [46] using tail skin grafted onto the lateral thorax. Plasters were removed after 8 days and grafts were examined every 2 days until rejection (o10% of the original graft remains viable). Measurement of apoptosis by Annexin V staining Statistical analysis This has been done according to manufacture’s protocol (BD PharmingenTM, 556421). Nitric oxide colorimetric assay The NO level was measured using the Nitric Oxide Colorimetric Assay Kit (BioVision, K262-200) according to the manufacturer’s protocol. In vivo T-cell proliferation assays Spleen cells from Thy1.11 Rag2 / Marilyn mice (TCR trans- genic with speciﬁcity for HY/Dby peptide [43], provided by Dr. O. Lantz, Paris, France) were stained with CFSE (4 mM) and adoptively transferred into B6 females (Thy1.21) by i.v. injection (2 106 cells/mouse). On the same day (day 0), all recipients were immunized with male splenocytes. From day 1 to day 7, B6 female mice received intraperitoneal injections of either nor- NOHA at 100 mg/kg/day or PBS. At day 5 and day 10, three mice from each group were killed and splenocytes were stained for Thy1.1 marker. Cells were gated on Thy1.1 (eBioscience) positive population. Proliferation was measured by CFSE (Invitrogen) dilution. & 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Mean values with SD are shown where appropriate. Statistical signiﬁcance was determined using Student’s t-test. Differences in graft survival were analysed using log rank test and actuarial survival plotted according to the Kaplan–Meyer method. A value of po0.05 was regarded as statistically signiﬁcant. Acknowledgements: This work was funded by project grant (078732/Z/05/Z) from the Wellcome Trust. Additional support was from Roche Organ Transplantation Research Foundation (A. K.), Fight for Sight (S. Z.), Cancer Research UK (D. C. and J. G. C.). Conﬂict of interest: The authors declare no ﬁnancial or commercial conﬂict of interest. References 1 Williams, K. A. and Coster, D. J., The immunobiology of corneal transplantation. Transplantation 2007. 84: 806–813. 2 Fu, H., Larkin, D. F. and George, A. J., Immune modulation in corneal transplantation. Transplant. Rev. (Orlando) 2008. 22: 105–115. www.eji-journal.eu