Immune escape in chronic leukemia

Reactive oxygen species (ROS) are produced by myeloid cells as a mechanism of defense against infection, but also to resolve inflammation, as ROS can induce cell death in T cells and NK cells. ROS production may also be deployed as a mechanism by which myeloid cells suppress anti-leukemic lymphocytes to promote malignant progression. The aim of this thesis was to define the role of myeloid cell-derived ROS in chronic leukemias as a putative target of immunotherapy. In paper I, the transductional pathways leading to ROS-induced lymphocyte death were investigated and found to involve the ERK1/2 mitogen-activated protein kinase (MAPK). These results challenge the view of ROS-induced cell death being a direct consequence of ROS-inflicted DNA damage. Papers II and III demonstrate that anti-CD20 monoclonal antibodies (mAbs) triggered ROS production by monocytes and neutrophils, which translated into reduced NK cell-mediated antibody-dependent cytotoxicity (ADCC) towards autologous leukemic cells derived from patients with chronic lymphocytic leukemia (CLL). The anti-oxidative agent histamine dihydrochloride (HDC) was found to restore ADCC by preventing ROS formation from adjacent monocytes, suggesting that anti-oxidative therapy might increase the efficacy of therapeutic mAbs. In paper IV, monocytic leukemic cells obtained from patients with chronic myelomonocytic leukemia (CMML) were shown to suppress T cells and NK cells by producing ROS. HDC counter-acted the suppression of lymphocytes by preventing ROS formation, and augmented the anti-leukemic activity of NK cells. Collectively, these results suggest that myeloid cell-derived ROS may be operational in CLL and in CMML as a mechanism of immune escape and that immunotherapy by anti-oxidative intervention should be further investigated in these forms of chronic leukemia

NOX2-dependent immunosuppression in chronic myelomonocytic leukemia

Chronic myelomonocytic leukemia (CMML) is a myeloproliferative and myelodysplastic neoplasm with few treatment options and dismal prognosis. The role of natural killer (NK) cells and other antileukemic lymphocytes in CMML is largely unknown. We aimed to provide insight into the mechanisms of immune evasion in CMML with a focus on immunosuppressive reactive oxygen species (ROS) formed by the myeloid cell NADPH oxidase-2 (NOX2). The dominant population of primary human CMML cells was found to express membrane-bound NOX2 and to release ROS, which, in turn, triggered extensive PARP-1-dependent cell death in cocultured NK cells, CD8(+) T effector memory cells, and CD8(+) T effector cells. Inhibitors of ROS formation and scavengers of extracellular ROS prevented CMML cell-induced lymphocyte death and facilitated NK cell degranulation toward Ab-coated, primary CMML cells. In patients with CMML, elevation of immature cell counts (CD34(+)) in blood was associated with reduced expression of several NK cell-activating receptors. We propose that CMML cells may use extracellular ROS as a targetable mechanism of immune escape

Role of the ERK Pathway for Oxidant-Induced Parthanatos in Human Lymphocytes

<div><p>Reactive oxygen species (ROS) are formed by myeloid cells as a defense strategy against microorganisms. ROS however also trigger poly(ADP-ribose) polymerase 1- (PARP-1) dependent cell death (parthanatos) in adjacent lymphocytes, which has been forwarded as a mechanism of immune escape in several forms of cancer. The present study assessed the role of mitogen-activated protein kinases (MAPKs), in particular the extracellular signal-regulated kinase (ERK), in ROS-induced signal transduction leading to lymphocyte parthanatos. We report that inhibitors of ERK1/2 phosphorylation upheld natural killer (NK) cell-mediated cytotoxicity under conditions of oxidative stress and rescued NK cells and CD8⁺ T lymphocytes from cell death induced by ROS-producing monocytes. ERK1/2 phosphorylation inhibition also protected lymphocytes from cell death induced by exogenous hydrogen peroxide (H₂O₂) and from ROS generated by xanthine oxidase or glucose oxidase. Phosphorylation of ERK1/2 was observed in lymphocytes shortly after exposure to ROS. ROS-generating myeloid cells and exogenous H₂O₂ triggered PARP 1-dependent accumulation of poly ADP-ribose (PAR), which was prevented by ERK pathway inhibitors. ERK1/2 phosphorylation was induced by ROS independently of PARP-1. Our findings are suggestive of a role for ERK1/2 in ROS-induced lymphocyte parthanatos, and that the ERK axis may provide a therapeutic target for the protection of lymphocytes against oxidative stress.</p></div

Inhibition of ERK phosphorylation in lymphocytes exposed to oxygen radicals by ERK pathway inhibitor but not by PARP inhibitor.

<p>PBMCs or NK cells were preincubated in presence or absence of ERK1/2 inhibitor PD98059 (25 µM) or PARP-1 inhibitor PJ34 (2 µM) for 1 h at 37°C. (<b>A</b>) A Representative dot plots of pERK⁺ NK cells after 10 min exposure to PMA-stimulated monocytes_.is shown. (<b>B</b>) Mean ± SEM of pERK positive cells in gated lymphocytes after 10 min exposure to H₂O₂. (<b>C</b>) Mean ± SEM of pERK positive NK cells after 10 min exposure to PMA-stimulated monocytes. <b>B–C</b>: mean ± SEM of 4–6 experiments. *P<0.05, **P<0.01 and ***P<0.001.</p

Retained cytotoxicity of NK cells rescued from ROS induced cell death by an ERK1/2 inhibitor.

<p>NK cells were co-incubated with monocytes at Mo:NK ratios of 0.25∶1, 0.5∶1 and 1∶1 overnight in the presence or absence of the ERK1/2 inhibitor PD98059 (25 μM). Anti-CD20 (rituximab) and CFSE-labeled 221 target cells were subsequently added at an effector to target cell ratio of 1∶1 as per the NK cell count prior to incubation. Lysis of 221 cells by ADCC was estimated in a 4 hour assay using the Live/Dead Fixable Far Red Dead Cell Stain kit and flow cytometry. Panel (<b>A</b>) shows contour density plots from a representative experiment displaying an inverse relationship between NK cell and target cell viability. Panel (<b>B</b>) shows NK cell cytotoxicity (mean ± SEM) of seven donors using the lowest Mo:NK cell ratio in which PD98059 rescued NK cells from apoptosis. *P<0.05, **P<0.01 and ***P<0.001.</p

Protection of lymphocytes from ROS-induced apoptosis by an ERK pathway inhibitor.

<p>MACS-purified human CD8⁺ T cells or NK cells were preincubated with the ERK1/2 inhibitor PD98059 (25 µM) (filled triangle) for 1 h at 37°C. The T cells and NK cells were then incubated overnight in the presence of PD98059 with H₂O₂ at indicated concentrations (<b>A–B</b>) or with ROS-producing monocytes (MØ) at indicated MØ:NK ratios (<b>C–D</b>). Lymphocyte viability was assessed using the Live/Dead Fixable Violet Dead Cell Stain kit. ERK inhibitor-equivalent concentrations of DMSO were used as control (open square). Results obtained using DMSO did not significantly differ from PBS. Data are the mean ± SEM of results obtained using blood from 3–7 donors. *P<0.05, **P<0.01 and ***P<0.001.</p

Oxidant-induced poly ADP-ribose accumulation: role of the ERK pathway.

<p>(<b>A–B</b>) Lymphocytes were preincubated with or without the ERK1/2 inhibitor PD98059 (25 µM) or the PARP-1 inhibitor PJ34 (2 µM) for 1 hr at 37°C before exposure to 500 µM H₂O₂ for 20 min. PAR accumulation was analyzed in whole cell lysates. (<b>A</b>) Representative Western blot and (<b>B</b>) mean ± SEM of results from 4 donors (O.D., optical density). β-tubulin was utilized as loading control and H₂O₂-treated HELA cells served as positive controls. (<b>C</b>) Flow cytometry analysis of PAR accumulation following exposure to H₂O₂ (500 µM, filled circle) or PBS (open square). (<b>D</b>) Inhibition of PAR formation in lymphocytes after preincubation with PD98059 (25 µM) or PJ34 (2 µM). PAR accumulation was measured after 20 min exposure to 500 µM H₂O₂ by flow cytometry. Data are the MFI of PAR in gated lymphocytes (mean ± SEM of results obtained in 4–6 donors) *P<0.05, **P<0.01 and ***P<0.001).</p