Benjamin J Schmiedel

CD137 ligand mediates opposite effects in human and mouse NK cells and impairs NK-cell reactivity against human acute myeloid leukemia cells

Natural killer (NK) cells play an important role in the immunosurveillance of leukemia. Their reactivity is governed by a balance of activating and inhibitory receptors including various members of the tumor necrosis factor receptor (TNFR) family. Here we report that human NK cells acquire expression of the TNFR family member CD137 upon activation, and NK cells of acute myeloid leukemia (AML) patients display an activated phenotype with substantial CD137 expression. CD137 ligand (CD137L) was detectable on leukemic cells in 35% of 65 investigated AML patients, but not on healthy CD34(+) cells, and expression was associated with monocytic differentiation. Bidirectional signaling following CD137-CD137L interaction induced the release of the immunomodulatory cytokines interleukin-10 and TNF by AML cells and directly diminished granule mobilization, cytotoxicity, and interferon-gamma production of human NK cells, which was restored by blocking CD137. Cocultures of NK cells with CD137L transfectants confirmed that human CD137 inhibits NK-cell reactivity, while activating signals were transduced by its counterpart on NK cells in mice. Our data underline the necessity to study the function of seemingly analog immunoregulatory molecules in mice compared with men and demonstrate that CD137-CD137L interaction enables immune evasion of AML cells by impairing NK-cell tumor surveillance in humans.

Glucocorticoid-Induced Tumor Necrosis Factor Receptor–Related Protein Ligand Subverts Immunosurveillance of Acute Myeloid Leukemia in Humans

The reciprocal interaction of tumor cells with the immune system is influenced by various members of the tumor necrosis factor (TNF)/TNF receptor (TNFR) family, and recently, glucocorticoid-induced TNFR-related protein (GITR) was shown to stimulate antitumor immunity in mice. However, GITR may mediate different effects in mice and men and impairs the reactivity of human natural killer (NK) cells. Here, we studied the role of GITR and its ligand (GITRL) in human acute myeloid leukemia (AML). Surface expression of GITRL was observed on AML cells in six of seven investigated cell lines, and 34 of 60 investigated AML patients whereas healthy CD34(+) cells did not express GITRL. Furthermore, soluble GITRL (sGITRL) was detectable in AML patient sera in 18 of 55 investigated cases. While the presence of GITRL was not restricted to a specific AML subtype, surface expression was significantly associated with monocytic differentiation. Signaling via GITRL into patient AML cells induced the release of TNF and interleukin-10 (IL-10), and this was blocked by the inhibition of mitogen-activated protein kinases extracellular signal-regulated kinase 1/2. Furthermore, triggering GITR by surface-expressed and sGITRL impaired NK cell cytotoxicity and IFN-gamma production in cocultures with leukemia cells, and NK cell reactivity could be restored by blocking GITR and neutralization of sGITRL and IL-10. Thus, whereas a stimulatory role of the GITR-GITRL system in mouse antitumor immunity has been reported, our data show that in humans GITRL expression subverts NK cell immunosurveillance of AML. Our results provide useful information for therapeutic approaches in AML, which, like haploidentical stem cell transplantation, rely on a sufficient NK cell response.

Azacytidine impairs NK cell reactivity while decitabine augments NK cell responsiveness toward stimulation

Azacytidine and decitabine are approved for treatment of acute myeloid leukemias and myelodysplastic syndromes. While clinical responses are attributed to epigenetic effects and induction of apoptosis in malignant cells, these azanucleosides also affect antitumor immune responses. NK cells as components of innate immunity may confine development and progression of cancer. Numerous therapeutic strategies presently aim to reinforce NK reactivity against hematopoietic malignancies. We here comparatively analyzed the effect of the two clinically available azanucleosides and report that NK cytotoxicity and IFN‐γ production are significantly impaired by pharmacological concentrations of azacytidine but enhanced by decitabine. This was not due to alterations in the target cells but caused by direct effects on NK cells depending on the chemical modifications by which azanucleosides differ from their physiological analogues. Although azacytidine impaired mRNA synthesis and induced apoptosis in NK cells, decitabine did not per se alter NK cell viability or reactivity but enhanced responsiveness to activating stimuli by inducing transcription of genes involved in NK reactivity. Tantalizingly, these effects were independent of incorporation of the azanucleosides into DNA during cell division. While azacytidine impairs NK antitumor immunity, decitabine augments NK reactivity by yet unidentified mechanisms and may thus serve well in therapeutic strategies combining its effects on malignant cells with its ability to enhance NK functions.

RANKL Expression, Function, and Therapeutic Targeting in Multiple Myeloma and Chronic Lymphocytic Leukemia

Bone destruction is a prominent feature of multiple myeloma, but conflicting data exist on the expression and pathophysiologic involvement of the bone remodeling ligand RANKL in this disease and the potential therapeutic benefits of its targeted inhibition. Here, we show that RANKL is expressed by primary multiple myeloma and chronic lymphocytic leukemia (CLL) cells, whereas release of soluble RANKL was observed exclusively with multiple myeloma cells and was strongly influenced by posttranscriptional/posttranslational regulation. Signaling via RANKL into multiple myeloma and CLL cells induced release of cytokines involved in disease pathophysiology. Both the effects of RANKL on osteoclastogenesis and cytokine production by malignant cells could be blocked by disruption of RANK-RANKL interaction with denosumab. As we aimed to combine neutralization of RANKL with induction of antibody-dependent cellular cytotoxicity of natural killer (NK) cells against RANKL-expressing malignant cells and as denosumab does not stimulate NK reactivity, we generated RANK-Fc fusion proteins with modified Fc moieties. The latter displayed similar capacity compared with denosumab to neutralize the effects of RANKL on osteoclastogenesis in vitro, but also potently stimulated NK cell reactivity against primary RANKL-expressing malignant B cells, which was dependent on their engineered affinity to CD16. Our findings introduce Fc-optimized RANK-Ig fusion proteins as attractive tools to neutralize the detrimental function of RANKL while at the same time potently stimulating NK cell antitumor immunity.

Glucocorticoid-induced TNFR-related protein (GITR) ligand modulates cytokine release and NK cell reactivity in chronic lymphocytic leukemia (CLL)

Natural killer (NK) cells play an important role in the immunosurveillance of hematopoietic malignancies. Their reactivity is influenced by activating and inhibitory signals mediated by tumor-expressed ligands for NK receptors. Many members of the tumor necrosis factor (TNF) family modulate differentiation, proliferation, activation and death of both tumor and immune effector cells. The TNF receptor family member glucocorticoid-induced TNFR-related protein (GITR) stimulates anti-tumor immunity in mice, but available data indicate that GITR may mediate different effects in mice and men and impairs the reactivity of human NK cells. Here, we comprehensively studied the expression and function of GITR ligand (GITRL) in leukemia. Among the different leukemia entities, pronounced expression of GITRL on leukemic cells was observed in chronic lymphocytic leukemia (CLL), and the GITR receptor was expressed at significantly higher levels on NK cells of CLL patients compared with healthy controls. Upon GITR–GITRL interaction, signaling via GITRL into the leukemia cells induced the release of interleukin (IL)-6, IL-8 and TNF, which act as growth and survival factors for CLL cells. In addition, GITRL impaired both direct and Rituximab-induced degranulation, cytotoxicity and interferon-γ production of NK cells, which could be restored by GITR blocking antibodies. Thus, GITRL may contribute to disease pathophysiology and resistance to direct and Rituximab-induced NK reactivity in CLL.

Neutralization of (NK-cell-derived) B-cell activating factor by Belimumab restores sensitivity of chronic lymphoid leukemia cells to direct and Rituximab-induced NK lysis

Natural killer (NK) cells are cytotoxic lymphocytes that substantially contribute to the therapeutic benefit of antitumor antibodies like Rituximab, a crucial component in the treatment of B-cell malignancies. In chronic lymphocytic leukemia (CLL), the ability of NK cells to lyse the malignant cells and to mediate antibody-dependent cellular cytotoxicity upon Fc receptor stimulation is compromised, but the underlying mechanisms are largely unclear. We report here that NK-cells activation-dependently produce the tumor necrosis factor family member ‘B-cell activating factor’ (BAFF) in soluble form with no detectable surface expression, also in response to Fc receptor triggering by therapeutic CD20-antibodies. BAFF in turn enhanced the metabolic activity of primary CLL cells and impaired direct and Rituximab-induced lysis of CLL cells without affecting NK reactivity per se. The neutralizing BAFF antibody Belimumab, which is approved for treatment of systemic lupus erythematosus, prevented the effects of BAFF on the metabolism of CLL cells and restored their susceptibility to direct and Rituximab-induced NK-cell killing in allogeneic and autologous experimental systems. Our findings unravel the involvement of BAFF in the resistance of CLL cells to NK-cell antitumor immunity and Rituximab treatment and point to a benefit of combinatory approaches employing BAFF-neutralizing drugs in B-cell malignancies.

An Fc‐optimized NKG2D‐immunoglobulin G fusion protein for induction of natural killer cell reactivity against leukemia

Recruitment of Fc‐receptor‐bearing effector cells, such as natural killer (NK) cells, is a feature critical for the therapeutic success of antitumor antibodies and can be improved by the modifications of an antibodys Fc part. The various ligands of the activating immunoreceptor NKG2D, NKG2DL) are selectively expressed on malignant cells including leukemia. We here took advantage of the tumor‐associated expression of NKG2DL for targeting leukemic cells by NKG2D–immunoglobulin G (IgG)1 fusion proteins containing modified Fc parts. Compared to NKG2D–Fc containing a wild‐type Fc part (NKG2D–Fc–WT), our mutants (S239D/I332E and E233P/L234V/L235A/ΔG236/A327G/A330S) displayed highly enhanced (NKG2D–Fc–ADCC) and abrogated (NKG2D–Fc–KO) affinity to the NK cell Fc receptor, respectively. Functional analyses with allogenic as well as autologous NK cells and primary malignant cells of leukemia patients revealed that NKG2D–Fc–KO significantly reduced NK reactivity by blocking immunostimulatory NKG2D–NKG2DL interaction. NKG2D–Fc–WT already enhanced antileukemia reactivity by inducing antibody‐dependent cellular cytotoxicity (ADCC) with NKG2D–Fc–ADCC mediating significantly stronger effects. Parallel application of NKG2D–Fc–ADCC with Rituximab caused additive effects in lymphoid leukemia. In line with the tumor‐associated expression of NKG2DL, no NK cell ADCC against resting healthy blood cells was induced. Thus, NKG2D–Fc–ADCC potently enhances NK antileukemia reactivity despite the inevitable reduction of activating signals upon binding to NKG2DL and may constitute an attractive means for immunotherapy of leukemia.

Receptor Activator for NF-κB Ligand in Acute Myeloid Leukemia: Expression, Function, and Modulation of NK Cell Immunosurveillance

The TNF family member receptor activator for NF-κB ligand (RANKL) and its receptors RANK and osteoprotegerin are key regulators of bone remodeling but also influence cellular functions of tumor and immune effector cells. In this work, we studied the involvement of RANK–RANKL interaction in NK cell–mediated immunosurveillance of acute myeloid leukemia (AML). Substantial levels of RANKL were found to be expressed on leukemia cells in 53 of 78 (68%) investigated patients. Signaling via RANKL into the leukemia cells stimulated their metabolic activity and induced the release of cytokines involved in AML pathophysiology. In addition, the immunomodulatory factors released by AML cells upon RANKL signaling impaired the anti-leukemia reactivity of NK cells and induced RANK expression, and NK cells of AML patients displayed significantly upregulated RANK expression compared with healthy controls. Treatment of AML cells with the clinically available RANKL Ab Denosumab resulted in enhanced NK cell anti-leukemia reactivity. This was due to both blockade of the release of NK-inhibitory factors by AML cells and prevention of RANK signaling into NK cells. The latter was found to directly impair NK anti-leukemia reactivity with a more pronounced effect on IFN-γ production compared with cytotoxicity. Together, our data unravel a previously unknown function of the RANK–RANKL molecule system in AML pathophysiology as well as NK cell function and suggest that neutralization of RANKL with therapeutic Abs may serve to reinforce NK cell reactivity in leukemia patients.

Fc-Optimized NKG2D-Fc Constructs Induce NK Cell Antibody-Dependent Cellular Cytotoxicity against Breast Cancer Cells Independently of HER2/neu Expression Status

The ability of NK cells to mediate Ab-dependent cellular cytotoxicity (ADCC) largely contributes to the clinical success of antitumor Abs, including trastuzumab, which is approved for the treatment of breast cancer with HER2/neu overexpression. Notably, only ∼25% of breast cancer patients overexpress HER2/neu. Moreover, HER2/neu is expressed on healthy cells, and trastuzumab application is associated with side effects. In contrast, the ligands of the activating immunoreceptor NKG2D (NKG2DL) are selectively expressed on malignant cells. In this study, we took advantage of the tumor-associated expression of NKG2DL by using them as target Ags for NKG2D-IgG1 fusion proteins optimized by amino acid exchange S239D/I332E in their Fc part. Compared to constructs with wild-type Fc parts, fusion proteins carrying the S239D/I332E modification (NKG2D–Fc–ADCC) mediated highly enhanced degranulation, ADCC, and IFN-γ production of NK cells in response to breast cancer cells. NKG2D–Fc–ADCC substantially enhanced NK reactivity also against HER2/neu-low targets that were unaffected by trastuzumab, as both compounds mediated their immunostimulatory effects in strict dependence of target Ag expression levels. Thus, in line with the hierarchically organized potential of the various activating receptors governing NK reactivity and due to its highly increased affinity to CD16, NKG2D–Fc–ADCC potently enhances NK cell reactivity despite the inevitable reduction of activating signals upon binding to NKG2DL. Due to the tumor-restricted expression of NKG2DL, NKG2D–Fc–ADCC may constitute an attractive means for immunotherapy especially of HER2/neu-low or -negative breast cancer.

Alterations of the RANKL pathway in blood and bone marrow samples of prostate cancer patients without bone metastases

The receptor activator of the NF‐kB ligand (RANKL) pathway is a key mediator of prostate cancer (PC)‐induced bone disease. However, little is known about this pathway in patients with non‐metastatic PC. We aimed to investigate whether changes of RANKL, its inhibitor osteoprotegerin (OPG) and bone marrow‐mesenchymal stromal cells (BM‐MSCs) occur in PC patients without manifest bone metastases.