Abstract
Proteinase 3 (P3), a serine protease found in primary granules in granulocytes, is the target of T cell- and B cell-mediated autoimmunity in Wegener’s granulomatosis (WG) and of anti-leukemia immunity mediated by PR1 (VLQELNVTV)-specific cytotoxic T lymphocytes (PR1-CTL). Although aberrant P3 and neutrophil elasase (NE) expression in leukemia increases susceptibility to PR1-CTL-mediated killing, overexpression of P3 also induces apoptosis of the high affinity PR1-CTL leading to deletional tolerance and leukemia outgrowth. Because expression of P3 and NE in sera from leukemia patients is increased by 5-fold compared to healthy controls, we sought to determine whether such overexpression of P3 or NE impairs PR1-CTL immunity to leukemia by a direct affect on T lymphocytes. To study this, T cells from healthy donors were activated by anti-CD3 and anti-CD28 and exposed to increasing concentration of P3 or NE over one to five days, and the percentage of apoptotic cells and cell proliferation were determined by flow cytometry using PI, anti-Ki-67, and CFSE. P3, but not NE, induced dose-dependent apoptosis of up to 30% of T cells, and in the non-apoptotic cells, a 50% inhibition of CD4 and CD8 T cell proliferation at 1 μg/ml and 100% at 10 μg/ml compared to untreated cells. This effect was not enzyme-mediated since prior exposure of P3 to 56°C or co-incubation with the serine protease inhibitors Elafin and alpha-1 antitrypsin showed no affect on apoptosis or cell proliferation. P3 induced a cell cycle arrest at the G0/G1 interface, determined with PI and Ki-67 staining of healthy donor T cells that were exposed to P3 for up to 3 days. In contrast, at protein concentrations up to 25 μg/ml, NE showed no such inhibitory effect on apoptosis or cell proliferation. In addition to its role as a leukemia-associated antigen, P3 is also targeted by the cANCA antibody in patients with WG and the serum titer correlates with disease activity. Therefore, we hypothesized that the effect of P3 on T cell proliferation might also be affected by humoral immunity during circumstances of systemic autoimmunity. Co-incubation of P3 with a molar excess of cANCA reversed P3- mediated inhibition of both CD4 and CD8 T cells, consistent with a role of this antibody and the P3 target antigen in controlling T cell autoreactivity. Taken together, this data shows a new role for P3 in regulating T cell proliferation, which occurs only at high P3 concentration, similar to P3 in sera from leukemia patients, which is not enzymedependent. This supports a direct role for P3 in regulating both anti-leukemia immunity and autoimmunity. This data will need to be considered for effective immunotherapy targeting P3 in leukemia patients and these inhibitory effects also suggest a role for P3 in regulating autoimmunity at sites of inflammation, such as in patients with WG.
Proliferating Cell Nuclear Antigen as the Cell Cycle Sensor for an HLA-Derived Peptide Blocking T Cell Proliferation
