Membrane-Associated Proteinase 3 on Granulocytes and Myeloid Leukemia Mediates Reversible Inhibition of T Cell Proliferation

Abstract 1916

Proteinase 3 (P3), a serine protease constitutively expressed in primary granules and on the membrane of some resting granulocytes, is the target of T cell-mediated autoimmunity in Wegener's granulomatosis (WG) and of anti-leukemia immunity mediated by PR1 (VLQELNVTV)-specific cytotoxic T lymphocytes (PR1-CTL). We have shown that soluble P3 is increased by 5-fold in sera from acute myeloid leukemia (AML) patients compared to healthy controls, and soluble P3 mediates enzyme-independent inhibition of T-cell proliferation. Moreover, tumor-associated neutrophils (TANs) are associated with a poor prognosis in a number of cancers including renal cell carcinoma and lung cancer, and P3 is also over-expressed in a variety of AML and chronic myeloid leukemia (CML). Therefore, we hypothesized that membrane-bound P3 (mP3) may similarly regulate adaptive immunity by suppressing T-cell proliferation. To study this, T cells from healthy donors were labeled with the membrane dye CFSE and stimulated with anti-CD3 and anti-CD28 in the presence or absence of mP3-expressing PMNs for five days. The percentage of proliferating cells was determined by flow cytometry. Proliferation of autologous CD8⁺ and CD4⁺ T cells was significantly inhibited by 75% and 72%, respectively, when PMNs were co-incubated with lymphocytes at a ratio of 3:1, and by > 90% at 5:1. This cell contact-dependent inhibitory effect was limited to PMN since PBMCs added to lymphocytes in place of PMNs at 5:1 had no effect on T cell proliferation. To determine whether the inhibitory effect was specifically mediated by mP3, we FAC-sorted CD177⁺PMNs and CD177⁻PMNs to obtain highly purified (>98%) mP3⁺ and mP3⁻ PMNs, respectively, because CD177 and mP3 are co-expressed on same subset of resting PMNs. At a ratio of 3:1 (CD177⁺PMNs or CD177⁻PMNs to lymphocytes), mP3⁺PMNs mediated > 75% growth inhibition of both CD8⁺ and CD4⁺ T cells compared to < 55% inhibition by mP3⁻PMNs (p<0.05). Furthermore, the inhibitory effect of mP3⁺PMNs on T cell proliferation was blocked (< 10% inhibition of proliferation) by anti-P3 but not by isotype control mAb. The inhibitory effect of mP3 was enzyme-independent because Elafin or α1-anti-trypsin did not affect inhibition by mP3⁺PMNs. In addition, mP3-mediated inhibition was fully reversible as T cells proliferated normally with anti-CD3/anti-CD28 stimulation after PMNs were removed from co-culture. Similarly, mP3⁺AML blasts inhibited autologous CD8⁺ and CD4⁺ T cell proliferation by 50% and 30%, respectively, at a 2:1 ratio of AML blasts: lymphocytes. Interestingly, bone marrow myeloid derived suppressor cells (MDSC) from leukemia patients express significantly higher mP3 (79.4±5.23% (mean±SEM, n=7)), compared to 22.4±11.55% mP3 on MDSC from healthy donors (p= 0.0007, n=3). Taken together, these data support an important new function of membrane-bound P3 on innate immune cells and leukemia in controlling adaptive T cell immunity. These findings suggest a novel mechanism whereby neutrophils could promote tumor growth in vivo through contact-mediated suppression of tumor-infiltrating lymphocytes by mP3 or by soluble P3 secreted by activated TANs in cancer and myeloid leukemia. Thus, targeting P3 with anti-P3 antibodies may be explored as a novel therapeutic strategy for leukemia and other cancers.