The recent contribution by Michelis et al. convincingly shows that an early recovery of lymphocytes in blood following allogeneic hematopoietic cell transplantation heralds a reduced risk of relapse for patients with acute myeloid leukemia (AML) (1). In a multicenter phase III trial (n=320), post-consolidation therapy with histamine dihydrochloride and low-dose interleukin-2 (HDC/IL-2) was found to significantly prevent relapse of AML, as accounted for in detail elsewhere (2). Immunotherapy with HDC/IL-2 aims to mimic the graft-versus-leukemia reaction achieved by allogeneic transplantation by optimally expanding and activating natural killer cells and T cells (3). This article is protected by copyright. All rights reserved.
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.
Despite that the initial phases of chemotherapy induce disappearance of leukemic cells in many patients with acute myeloid leukemia (AML), the prevention of life-threatening relapses in the post-remission phase remains a significant clinical challenge. Allogeneic bone marrow transplantation, which is available for a minority of patients, efficiently prevents recurrences of leukemia by inducing immune-mediated elimination of leukemic cells, and over the past decades, numerous immunotherapeutic protocols have been developed aiming to mimic the graft-versus-leukemia reaction for the prevention of relapse. Here we review past and present strategies for relapse control with focus on overcoming leukemia-related immunosuppression in AML. We envisage future treatment protocols, in which systemic immune activators, such as vaccines, dendritic cell-based therapies, engineered variants of IL-2, or IL-15, are combined with agents that counter immunosuppression mediated by, e.g., the PD/PDL interaction, CTLA-4, CD200, reactive oxygen species, IDO expression, CXCR4, or the KIR/class I interaction, based on characteristics of the prevailing malignant clone. This combinatorial approach may pave the way for individualized immunotherapy in AML.
The study of human leukocytes is almost exclusively conducted using cells isolated from peripheral blood. This is especially true for neutrophils, despite the fact that these cells are of main (pathological) importance in extravascular tissues upon e.g., infection and/or tissue damage. The journey from circulation to tissue is typically associated with a number of cellular changes, making the cells primed, or hyper-responsive, and in many aspects distinct from the cells present in circulation. Models to obtain in vivo transmigrated leukocytes from human tissue are available, but not widely used. We describe here an easy-to-use model for the study of local inflammation, stemming from limited tissue damage, which can be used to isolate viable and functional leukocytes. The model is based on the generation of aseptic skin blisters, formed by the application of negative pressure, and allows for investigations of the cellular infiltrate as well as of soluble mediators present in the exudate. We believe that this method, combined with modern analysis equipment suitable for small volumes and cell numbers, could be of great use for increasing our understanding of the nature and function of leukocytes that have left circulation and transmigrated to inflamed tissues.
Late divergence of survival curves of treated patients and controls is commonly seen in successful cancer immunotherapy trials. Although late survival curve divergence may be caused by a delayed action of therapy, it may also be related to early effects of the treatment. We suggest that late survival divergence most often reflects a specific benefit of therapy for patients who suffer from a comparatively slow progression of disease. The occurrence of delayed survival curve divergence has important implications for the statistical analysis of immunotherapy trials. Thus, it leads to non-proportional hazard ratios that make commonly used statistical tests, e.g., the logrank test, suboptimal. It is therefore suggested that the statistical analysis of immunotherapy trials primarily should be based on a test that compares the survival curves at or after a prespecified, fixed, late time point.
Microorganisms and microbial products induce the release of reactive oxygen species (ROS) from monocytes and other myeloid cells, which may trigger dysfunction and apoptosis of adjacent lymphocytes. Therefore, T cell-mediated immunity is likely to comprise mechanisms of T cell protection against ROS-inflicted toxicity. The present study aimed to clarify the dynamics of reduced sulfhydryl groups (thiols) in human T cells after presentation of viral and bacterial Ags by dendritic cells (DCs) or B cells. DCs, but not B cells, efficiently triggered intra- and extracellular thiol expression in T cells with corresponding Ag specificity. After interaction with DCs, the Ag-specific T cells acquired the capacity to neutralize exogenous oxygen radicals and resisted ROS-induced apoptosis. Our results imply that DCs provide Ag-specific T cells with antioxidative thiols during Ag presentation, which suggests a novel aspect of DC/T cell cross-talk of relevance to the maintenance of specific immunity in inflamed or infected tissue.
Most patients with acute myeloid leukemia (AML) achieve complete remission (CR) after induction chemotherapy. Despite ensuing courses of consolidation chemotherapy, a large fraction of patients will experience relapses with poor prospects of long-term survival. Histamine dihydrochloride (HDC) in combination with the T-cell-derived cytokine IL-2 was recently approved within the EU as a remission maintenance immunotherapy in AML. HDC reduces myeloid cell-derived suppression of anti-leukemic lymphocytes, and aims to unravel a therapeutic benefit of IL-2 in AML by improving natural killer and T-cell activation. A randomized Phase III trial with 320 AML patients in CR demonstrated a significant reduction of relapse risk after immunotherapy with HDC plus low-dose IL-2 in the post-consolidation phase. HDC is the first approved therapeutic to target the state of immunosuppression in AML; further development in this area may comprise supplementary or alternative counter-suppressive agents with the aim to improve the efficacy of cancer immunotherapy.
The anionic amphiphil sodium dodecyl sulfate (SDS) is commonly used to activate the superoxide-generating NADPH-oxidase complex in cell-free systems, but very little is known about the effects of SDS on intact cells. It was, however, recently shown that SDS causes a translocation and an activation of Rac (a small G-protein) in intact cells, but this signal is not in its own sufficient to activate the oxidase (Nigorikawa et al. (2004) ). We found that SDS acted as an antagonist for FPR1, one of the neutrophil members of the formyl peptide receptor family. Accordingly, SDS reduced superoxide anion production induced by the chemoattractant formylmethionyl-leucyl-phenylalanine (fMLF). The receptor specificity of SDS was fairly high, but the concentration range in which it worked was narrow. The length of the carbohydrate chain as well as the charge of the molecule was of importance for the antagonistic effects. Signaling through FPR2, a closely related receptor also expressed in neutrophils, was not inhibited by SDS. On the contrary, the response induced by the FPR2-specific agonist WKYMVM was primed by SDS. The precise mechanism behind the primed state is not known, but might be related to the effects earlier described for SDS on the small G-protein Rac, that is of importance for a proper transduction of the down-stream signals from the occupied receptor.
Acute myeloid leukemia (AML) is the most common acute leukemia in adults. Although most patients achieve complete remission (CR) after chemotherapy, the majority suffer from subsequent leukemic relapse, which is associated with poor long-term survival. Thus, new therapies to maintain CR are highly warranted. After the completion of chemotherapy, AML patients have a minimal burden of leukemic cells, which are reportedly susceptible to cytotoxic lymphocytes such as NK cells and T cells. A therapy that boosts the function of these effector cells therefore has the potential to eradicate the malignant clone in AML and prevent relapse, Here, we briefly review the literature on the role of the immune system in AML and introduce the rationale for the use of histamine dihydrochloride (HDC) in conjuction with low-dose IL-2 as relapse-preventive immunotherapy for this disease.
NK cells and T cells are commonly dysfunctional in CML, and their status may determine the course of disease. We aimed to define the molecular mechanisms of leukemia-induced immunosuppression with focus on the role of ROS and the PARP-1 pathway of cell death. Malignant granulocytes from patients with BCR-ABL-positive CML expressed the oxygen radical-producing enzyme NOX, produced large amounts of ROS, and triggered extensive cell death in NK cells. Inhibition of PARP-1 maintained NK cell viability in cocultures with suppressive leukemic cells. Under conditions of oxidative stress, PARP-1 inhibition upheld the capacity of NK cells to kill myeloid leukemic cells, in addition to restoring the proliferation and cytokine production of NK cells and cytotoxic T cells. Our findings are suggestive of a novel pathway of relevance to immunosuppression in CML.
Post-consolidation immunotherapy with histamine dihydrochloride and interleukin-2 has been shown to improve leukemia-free survival in acute myeloid leukemia in a phase III trial. For this study, treatment efficacy was determined among 145 trial patients with morphological forms of acute myeloid leukemia as defined by the French-American-British classification. Leukemia-free survival was strongly improved in M4/M5 (myelomonocytic/monocytic) leukemia but not in M2 (myeloblastic) leukemia. We also analyzed histamine H(2) receptor expression by leukemic cells recovered from 26 newly diagnosed patients. H(2) receptors were typically absent from M2 cells but frequently expressed by M4/M5 cells. M4/M5 cells, but not M2 cells, produced reactive oxygen species that triggered apoptosis in adjacent natural killer cells. These events were significantly inhibited by histamine dihydrochloride. Our data demonstrate the presence of functional histamine H(2) receptors on human AML cells and suggest that expression of these receptors by leukemic cells may impact on the effectiveness of histamine-based immunotherapy.
Dysfunction of T cells and natural killer (NK) cells has been proposed to determine the course of disease in acute myeloid leukemia (AML), but only limited information is available on the mechanisms of lymphocyte inhibition. We aimed to evaluate to what extent human malignant AML cells use NADPH oxidase-derived reactive oxygen species (ROS) as an immune evasion strategy. We report that a subset of malignant myelomonocytic and monocytic AML cells (French-American-British [FAB] classes M4 and M5, respectively), recovered from blood or BM of untreated AML patients at diagnosis, expressed the NADPH oxidase component gp91(phox). Highly purified FAB M4/M5 AML cells produced large amounts of ROS on activation and triggered poly-[ADP-ribose] polymerase-1-dependent apoptosis in adjacent NK cells, CD4(+) T cells, and CD8(+) T cells. In contrast, immature (FAB class M1) and myeloblastic (FAB class M2) AML cells rarely expressed gp91(phox), did not produce ROS, and did not trigger NK or T-cell apoptosis. Microarray data from 207 AML patients confirmed a greater expression of gp91(phox) mRNA by FAB-M4/M5 AML cells than FAB-M1 cells (P < 10(-11)) or FAB-M2 cells (P < 10(-9)). Our data are suggestive of a novel mechanism by which monocytic AML cells evade cell-mediated immunity.
Cell death is of utmost importance in immunity, in part as a way to control the development and activity of leukocytes, but also as a strategy employed by leukocytes to rid the body of unwanted cells. Apoptosis is the classic type of programmed cell death involving an ordered sequence of cellular events, resulting in morphological changes that include cleavage/fragmentation of DNA, condensation of nuclei, cell shrinkage, and alterations of the plasma membrane. The apoptotic cell is a nonfunctional, but structurally intact, entity with preserved membrane integrity that is engulfed by surrounding cells (a process known as clearance) in an immunologically silent manner. In contrast, necrotic cells, i.e., nonfunctional cells that have lost membrane integrity, are freely permeable and leak intracellular constituents that may shift immunological homeostasis. Thus, membrane integrity of dead leukocytes is very important from an immunological point of view. For the analysis of leukocyte cell death, a wide variety of assays are available to monitor different events along the cell death pathway; a combination of different methods is advantageous in order to gain a more complete understanding of this dynamic process. In this chapter, we describe several in vitro methods for evaluating leukocyte cell death, mainly focusing on apoptosis in human neutrophils and lymphocytes. Special emphasis is given to assessment of membrane integrity of the cultured cells. Furthermore, a protocol for monitoring clearance of apoptotic neutrophils by monocyte-derived macrophages is provided.
Polymorphonuclear neutrophils (PMN) are potent inflammatory effector cells essential to host defense, but at the same time they may cause significant tissue damage. Thus, timely induction of neutrophil apoptosis is crucial to avoid tissue damage and induce resolution of inflammation. NK cells have been reported to influence innate and adaptive immune responses by multiple mechanisms including cytotoxicity against other immune cells. In this study, we analyzed the effect of the interaction between NK cells and neutrophils. Coculture experiments revealed that human NK cells could trigger caspase-dependent neutrophil apoptosis in vitro. This event was dependent on cell-cell contact, and experiments using blocking Abs indicated that the effect was mediated by the activating NK cell receptor NKp46 and the Fas pathway. CD56-depleted lymphocytes had minimal effects on neutrophil survival, suggesting that the ability to induce neutrophil apoptosis is specific to NK cells. Our findings provide evidence that NK cells may accelerate neutrophil apoptosis, and that this interaction may be involved in the resolution of acute inflammation.
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