Human PMN arginase-mediated arginine depletion induces down-regulation of CD3ζ in T cells. Purified human T cells were stimulated with antihuman CD3- and antihuman CD28-coupled paramagnetic microbeads. After 48 hours, cells were stained with FITC-labeled antihuman CD3, fixed, permeabilized, and stained with PE-labeled antihuman CD3ζ. Intracellular expression of CD3ζ was analyzed by flow cytometry. As control, CD3ζ expression in Arg(+) cell culture medium (150 μM l-arginine) is shown. The dashed lines represent stainings for the respective isotype control antibodies. The experimental conditions were as follows: (A) cell culture medium without arginine, (B) Arg(+) medium preincubated for 12 hours with human PMN-S (final arginase activity 300 mU/mL), and (C) as in panel B with or without inhibition of arginase by nor-NOHA (1 mM) from the start of the preincubation. (D) MFIs of the CD3ζ chain from 8 independent experiments are demonstrated as mean ± SD. CD3ζ expression upon stimulation in Arg(+) medium was set as 100% to allow for comparison between individual experiments. Data were analyzed with the paired Student t test. ^*P < .05 when comparing with T cells in Arg(+) medium; ^**P < .001 when comparing Arg(+) medium + PMN-S with or without nor-NOHA. (E) Purified human T cells were stimulated with different concentrations of PMA and ionomycin and in different cell culture conditions as indicated (see Figure 1 for explanation). After 48 hours, T-cell proliferation was assessed by [³H]thymidine incorporation in triplicate wells. Data are from 1 representative experiment (total, 4).