Human T-cell proliferation is suppressed by human PMN arginase. (A) Purified human T cells were stimulated with crosslinked anti-CD3 antibody at various concentrations, and proliferation was assessed by [³H]thymidine incorporation after 48 hours in triplicate wells. The cells were either stimulated in the presence (Arg(+) medium) or absence (Arg(-) medium) of 150 μM l-arginine. Alternatively, Arg(+) medium was preincubated for 12 hours with an aliquot of human PMN sonicate (PMN-S), corresponding to a final arginase activity of 300 mU/mL, in the presence or absence of the specific arginase inhibitor nor-NOHA, and T cells were then stimulated in these different media. Data are representative of 38 different experiments. (B) T-cell proliferation was analyzed as in panel A, but nor-NOHA and/or l-arginine (1 mM each) were added to arginine-depleted medium (Arg(+) medium + PMN-S) at the same time that the T cells were added. Results are expressed as mean cpm of triplicate cultures ± SD. (C) Human T-cell proliferation was assessed by cell division-mediated loss of intracellular CFSE fluorescence. T cells were labeled with CFSE and stimulated for 96 hours with antihuman CD3- and antihuman CD28-coupled microbeads in different cell culture media as described for panel A. Additionally, arginine was depleted in Arg(+) medium by 12 hours of preincubation with recombinant human arginase I (activity 300 mU/mL). Cell division (ie, loss of FL-1 fluorescence) was analyzed by flow cytometry. Data are representative of 4 different experiments.