Proteinase 3 Mediates Enzyme-Independent Reversible Inhibition of CD4 and CD8 T Cell Proliferation by Blocking G1-S Transition
Authors: Yang Tian-Hui , Alatrash Gheath , Clise-Dwyer Karen , Quintanilla Kathryn , Lu Sijie , Molldrem Jeffrey J. 
Published: October 2019

Abstract
Abstract 2099
Proteinase 3 (P3), a serine protease found in primary granules in neutrophils, 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 elastase (NE) expression by leukemia targets increases their 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 serum level of P3 in leukemia patients is increased by 5-fold compared to healthy controls, we sought to determine whether such overexpression impairs PR1-CTL immunity to leukemia by a direct effect 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 soluble P3 or NE over one to five days, and the percentage of apoptotic cells and cell proliferation were determined by flow cytometry. P3, but not NE, induced dose-dependent T-cell apoptosis and inhibition of proliferation. Specifically, after 24 hours of incubation at a high concentration of P3 (’10 μg/ml), apoptosis of CD4+ and CD8+ T-cells was increased by 50% and 70%, respectively, compared to <10% in controls (p<0.05). No apoptosis of CD4+ or CD8+ T-cells was observed at a P3 concentration ≤ 1 μg/ml. Surprisingly, however, 14% inhibition of proliferation was observed at 0.5 μg/ml of P3, which increased to >90% inhibition at 5 μg/ml and was completely abrogated at 10 μg/ml in both CD4+ and CD8+ T-cells. Inhibition was reversible as T cells proliferated once again after removing P3 from media. These effects on apoptosis or cell proliferation persist after heating of P3 to 95°C or co-incubation with the serine protease inhibitors Elafin or alpha-1 antitrypsin, suggesting enzyme-independent. However, co-incubation of P3 with a molar excess of anti-P3 monoclonal antibody (mAb) reversed P3-mediated inhibition of both CD4+ and CD8+ T cells. Furthermore, P3 mediated cell cycle arrest by maintaining T cells primarily in the G0 phase of the cell cycle and thus blocking the G1-S transition, determined with Propidium Iodide (PI) and Ki-67 labeling of anti-CD3/anti-CD28 stimulated T cells. In contrast, at protein concentrations up to 25 μg/ml, NE showed no effect on apoptosis or cell proliferation. The inhibition is also independent of the tetrapeptide IVGG that suppresses granulopoietic progenitors. Currently, we are analyzing various forms of recombinant P3 to define the structural determinants of P3 responsible for the inhibition of T-cell proliferation. Neutrophils from patients with P3 anti-neutrophil cytoplasmic antibody (ANCA) associated vasculitis and some other chronic inflammatory diseases show higher levels of membrane P3 (mP3) expression than neutrophils from healthy controls. The presence of a high proportion of mP3 expressing neutrophils is associated with more frequent relapses of WG. Co-incubation of mP3 expressing neutrophils with T cells at a ratio of 5 to 1 significantly inhibited proliferation of both CD4+ and CD8+ T cells. The inhibition of T cell proliferation by mP3 expressing neutrophils was completely abrogated when anti-P3 mAb was added. Taken together, this data supports a new enzyme-independent function of soluble and membrane-bound P3 expressed on innate immune cells in regulating adaptive T cell immunity. These findings suggest P3 that is observed in myeloid leukemia patients may down-regulate anti-leukemia immunity and may also modulate autoimmunity and inflammation that is common to many cancers. The data will be useful in understanding the biology of immune regulation in autoimmune disease and leukemia, and may be useful in therapy planning and patient monitoring. Additionally, new approaches for targeting serum P3 may provide novel methods for the treatment of leukemia and autoimmune disease.

Disclosures:
No relevant conflicts of interest to declare.