Human PMN arginase-mediated arginine depletion does not increase T-cell apoptosis or cell death but specifically alters T-cell activation. (A) Purified human T cells were stimulated with platebound crosslinked antihuman CD3 antibody for 48 hours in different cell culture conditions (nomenclature as in Figure 1). After double staining with annexin V-FITC and PI, cells were analyzed by flow cytometry. The percentages of T cells undergoing apoptosis (annexin V-FITC positive, PI negative) and of dead T cells (annexin V-FITC positive, PI positive) within the whole population are depicted within the respective quadrants. Data are representative of 3 individual experiments. (B) Human T cells were stimulated as in panel A and double stained with anti-CD3 and either anti-CD25, anti-HLA-DR, or anti-CD69. Analysis of the expression level of the different T-cell activation markers was done by flow cytometry after gating on CD3⁺ cells. To allow for comparison between 5 separate experiments, control MFI (activated human T cells in Arg(+) medium) was normalized to 100%. Data are shown as mean ± SD and were analyzed with the paired Student t test. ^*P < .05 and ^**P < .005. Arg(-) medium and Arg(+) medium + PMN-S are compared with Arg(+) medium. Arg(+) medium + PMN-S is also compared with Arg(+) medium + PMN-S + nor-NOHA, as indicated by bars. (C) Human PMN arginase suppresses T-cell IFN-γ synthesis by a posttranscriptional mechanism. Purified human T cells were stimulated with platebound crosslinked anti-CD3 antibody at the indicated concentrations. Cell culture media were as described in Figure 2. Supernatant was harvested after 48 hours, and IFN-γ was measured by ELISA. (D) A total of 3 × 10⁶ human T cells were stimulated as in panel C. At the indicated time points (3 hours, 9 hours, and 24 hours) mRNA was prepared and reverse transcribed. The frequency of IFN-γ transcripts was quantified by LightCycler Technology.