Figure 7.

Hyperactivation of T cell cytotoxicity is induced by polymorphonuclear neutrophil granulocytes supernatants (PMN-SN) and further increased by arginase inhibition. Human T cells and PMN were isolated from whole blood of healthy donors. Human T cells were retrovirally transduced with an HLA-A2 restricted p53(264-272)-specific T cell receptor and expanded by weekly restimulation with anti-CD3/anti-CD28-tagged beads and later via p53 peptide-loaded K562-A2.1 cells. PMN were incubated in cell culture medium for 72 h in the presence or absence of the arginase inhibitor nor-NOHA (1 mM). T cells were stimulated with anti-CD3/anti-CD28-tagged beads and cultured in the PMN-SN for 48 h. SAOS-2 cells, pulsed with p53(264-272) peptide as indicated, were cultured for 3 h with T cells (A) in the absence or (B) in the presence of PMN-SN at the indicated conditions. SAOS-2 cells were then stained with crystal violet to detect viable cells. (C) Quantification of viable SAOS-2 cells (n=3 independent experiments): OD values of control SAOS-2 cells (see Supplementary Figure S2 ) were set as 100% and OD values of the different experimental conditions were normalized to these controls. Statistical analysis: if not otherwise indicated, black asterisks refer to the control condition of SAOS-2 cells without p53 peptide (leftmost column), while blue asterisks refer to the control condition of SAOS-2 cells pulsed with p53 peptide and incubated with T cells in the absence of PMN-SN (leftmost blue column). Statistical calculations were performed with one-way ANOVA and Tukey´s post hoc test (***p < 0.001).