Eur. J. Immunol. 2011. 41: 2997–3005 Immunomodulation 2999 Figure 1. Expression of arginase I by murine cornea. (A) Real-time PCR analysis of arginase I (top) and arginase II (bottom) in murine cornea and other tissues. Total RNA was extracted from cornea and other tissues, and arginase mRNA was quantiﬁed by real-time PCR. Arginase mRNA levels were normalized to HPRT. (B) Western blotting for arginase protein. Tissues were homogenized and the supernatants were run on SDS-PAGE and probed for both arginase I and II. GAPDH was used as a loading control. (C) Arginase activity was measured in tissue extracts. Tissues were homogenized and the supernatants were taken to measure arginase activity by conversion of L-Arg into urea. Urea concentrations were assessed using a colorimetric assay, using the conversion of a-isonitrosopropiophenone; 1 unit of enzyme arginase activity catalyses the formation of 1 mmol urea/min. Data are represented as the mean1SD. (D) Effect of cytokine stimulation on arginase I mRNA expression in cornea. Arginase I mRNA levels were normalized to HPRT. (E) Immunohistochemical staining of the cornea with antibodies against Arginase I or II and isotype controls show expression in both the endothelium and epithelium. Magniﬁcation: 20 . (F) Epithelium and endothelium of cornea were separated surgically. Total RNA was extracted and arginase I mRNA expression was analysed by RT-PCR. Data are representative of three independent experiments, and in each group (A, C and D) triplicates were performed. Inhibition of arginase has no effect on skin allograft survival Inhibition of arginase has no effect on systemic T-cell proliferation Inhibition of arginase might shorten corneal allograft survival either by local action on arginase activity within the transplant graft or by systemic modulation of the allograft response [20]. To determine which is the case, we performed B6 male to female skin transplants. This donor–recipient combination was chosen because it has similar rejection kinetics as the B6 to BALB/c corneal transplantation model. RT-PCR analysis revealed that neither donor tail skin nor shank skin (site of the graft bed) express arginase I and minimal arginase II (Fig. 3B). Recipients were randomized to receive either nor-NOHA at 100 mg/kg/day or control PBS from day 1 to day 21 post-transplantation. Figure 3C shows that the rejection of skin grafts in the nor-NOHA treatment group is similar to that of the control group (p 5 0.6). & 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim To further investigate the effect of arginase inhibition on systemic T-cell response, we tested the ability of T cells to proliferate after administration of the arginase inhibitor, nor-NOHA. Splenocytes from Thy1.11 Rag2 / Marilyn mice were stained with CFSE and adoptively transferred into B6 females (Thy1.21) by i.v. injection. On the same day (day 0), all recipients were immunized with male splenocytes. From day 1 to day 7, the animals received 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. Figure 4 shows a similar degree of proliferation of male-speciﬁc T cells in animals receiving nor- NOHA or PBS, suggesting that inhibition of arginase has little systemic effect on T-cell proliferation. www.eji-journal.eu