ALBERT

Description: Abstract 3548 The TNF/TNF receptor (TNFR) family comprises various molecules that substantially influence cellular functions of both tumor and immune effector cells. The TNFR family member OX40 has been shown to influence proliferation and differentiation of T cells in autoimmune diseases. Here we studied the yet unknown role of OX40 in acute myeloid leukemia (AML). Substantial surface expression of OX40 was detected on malignant cells of AML patients in 24 of 60 (40%) investigated cases. Expression of OX40 mRNA and protein by leukemic cells was confirmed by analysis of AML cells lines, which displayed substantial surface levels in 6 of 7 investigated cases. Induction of OX40 signaling into AML cells by recombinant OX40L or agonistic antibodies lead to the release of cytokines like IL-10 and TNF which contribute to AML pathophysiology and stimulated metabolic activity (WST test) of the leukemia cells. Moreover, we found that NK cells, which play an important role in anti-tumor immunity and largely contribute to the clinical success of allogeneic stem cell transplantation (SCT) in AML, express OX40 ligand (OX40L) following activation, and OX40L triggering stimulated NK cell reactivity. Functional analyses with OX40 transfectants and OX40-negative controls confirmed that OX40L signaling promotes NK cell cytotoxicity and IFN-γ production. Furthermore, disruption of OX40-OX40L interaction by blocking OX40 F(ab)2 fragments resulted in reduced cytotoxicity and cytokine production of allogenic NK cells, thereby further confirming the stimulatory effect of OX40L on NK cell anti-leukemia reactivity when interacting with its AML-expressed counterpart. Our data suggest that OX40 is involved in disease pathophysiology of AML and identify OX40-OX40L interaction as previously unknown modulator of NK cell immunosurveillance in AML. Disclosures: No relevant conflicts of interest to declare.

Description: NK cell anti-tumor reactivity is governed by a balance of activating and inhibitory receptors including various TNF receptor (TNFR) family members. Here we report that human tumor cells release a soluble form of the TNF family member Glucocorticoid-Induced TNFR-Related Protein (GITR) ligand (sGITRL), which can be detected in cell culture supernatants. Tumor-derived sGITRL concentration-dependently reduced NK cell cytotoxicity and IFN-γ production, which could be overcome by neutralization of sGITRL using a GITR-Ig fusion protein. Although sGITRL did not induce apoptosis in NK cells, it diminished nuclear localized RelB, indicating that sGITRL negatively modulates NK cell NF-κB activity. Furthermore, we detected substantial levels of sGITRL in sera of patients with various malignancies, but not in healthy controls. Presence of sGITRL-containing patient serum in cocultures with tumor cells significantly reduced NK cell cytotoxicity and IFN-γ production, which could again be restored by neutralization of sGITRL. The strong correlation of tumor incidence and elevated sGITRL levels indicates that sGITRL is released from cancers in vivo, leading to impaired NK cell immunosurveillance of human tumors. Our data suggest that determination of sGITRL levels might be implemented as a tumor marker in patients, and GITRL neutralization may be used to improve immunotherapeutic strategies relying on NK cell reactivity.

Description: Abstract 2164 NK cells play an important role in tumor immunosurveillance, especially of leukemia. Their reactivity is governed by various activating and inhibitory molecules expressed by their targets including multiple members of the TNF family. The TNF family member Receptor Activator of NF-κB ligand (RANKL) and its receptors RANK and osteoprotegerin (OPG) are key regulators of bone remodelling, but recently have also been shown to influence progression of hematopoetic malignancies. Here we studied the yet unkown role of the RANK/RANKL molecule system in NK cells and their reactivity against acute myeloid leukemia (AML). Primary leukemia cells from AML patients were found to substantially express RANKL mRNA and surface protein in 75% of the investigated cases (n=40). Reverse signaling via surface-expressed RANKL into AML blasts induced the release of soluble factors including the immunoregulatory cytokines TNF and IL-10, which impaired NK cell anti-tumor reactivity. Moreover, we observed upregulation of RANK on NK cells among PBMC of healthy donors upon exposure to IL-10. This was not caused by direct effects on NK cells, but was rather due to yet unidentified factors released by monocytes among the PBMC upon IL-10 exposure and could be prevented by the activating cytokine IL-2. Furthermore, functional experiments with NK cells and RANKL transfectants or RANKL-negative controls revealed that forward signaling into RANK-expressing NK cells by tumor-expressed RANKL also directly impaired NK cytotoxicity and IFN-γ production. In line, blocking RANK-RANKL interaction using anti-RANKL antibodies or RANK-Fc fusion protein increased cytotoxicity and cytokine production of allogenic NK cells in cultures with RANKL-positive primary AML cells. Our data indicate that RANKL expression enables immune evasion of leukemia cells both by directly inhibiting reactivity of RANK-expressing NK cells and by orchestrating a reciprocal interplay between AML cells, monocytes and NK cells resulting in an immunosuppressive cytokine milieu. Thus, therapeutic modulation of the RANK/RANKL system, e.g. with Denosumab/AMG162, which is presently being evaluated for treatment of both non-malignant and malignant osteolysis, holds promise to reinforce NK reactivity against hematopoietic malignancies. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3039 Bone resorption is commonly associated with aging, but also with certain cancers. Recent studies identified Receptor Activator of NF-κB (RANK) ligand (RANKL) and its receptors RANK and osteoprotegerin as key regulators of bone remodelling. Multiple myeloma (MM) disrupts the balance within this molecule system towards osteoclastogenesis and bone destruction. Neutralization of RANKL by the monoclonal antibody Denosumab (AMG162) is presently being evaluated for treatment of both non-malignant and malignant osteolysis. We found, in line with previously published data, that primary MM cells (9 of 10) express substantial levels of RANKL at the cell surface and that MM cells directly release RANKL in soluble form (sRANKL). Next we evaluated the possibility to combine neutralization of sRANKL with targeting of MM cells for antibody-dependent cellular cytotoxicity (ADCC) of NK cells utilizing RANK-Ig fusion proteins with modified Fc portions. Compared to wildtype RANK-Fc, our mutants (S239D/I332E and E233P/L234V/L235A/DG236/A327G/A330S) displayed highly enhanced (RANK-Fc-ADCC) and abrogated (RANK-Fc-KO) affinity, respectively, to the NK cell FcγRIIIa, but comparable capacity to neutralize RANKL in binding competition and osteoclast formation assays. Analyses with RANKL transfectants and RANKL-negative controls confirmed the high and lacking potential of the RANK-Fc-ADCC and the RANK-Fc-KO to induce NK ADCC, respectively, and ascertained that the RANK-Fc-ADCC specifically induced NK cell lysis of RANKL-expressing but not RANKL-negative target cells. Most notably, in cultures of NK cells with RANKL-expressing primary MM cells RANK-Fc-ADCC potently enhanced NK cell degranulation, cytokine release and MM cells lysis due to enhanced NK reactivity. Thus, our Fc-engineered RANK-Fc-ADCC fusion protein may both neutralize detrimental effects of sRANKL and enhance NK anti-tumor reactivity by targeting RANKL-expressing malignant cells thereby constituting an attractive immunotherapeutic means for treatment of MM. Disclosures: No relevant conflicts of interest to declare.

Description: NK cells are cytotoxic lymphocytes that play an important role in the immunosurveillance of leukemia and, due to their ability to mediate antibody-dependent cellular cytotoxicity (ADCC), substantially contribute to the therapeutic benefit of antitumor antibodies like Rituximab. Available data indicate that the ability of NK cells to mediate ADCC is compromised in Chronic Lymphoid Leukemia (CLL), but the underlying mechanisms are still unclear. The TNF family member B cell activating factor (BAFF) was described to be aberrantly produced in mature B cell malignancies and contributes to disease pathophysiology e.g. by acting as a growth and survival signal for CLL cells. Here we report that NK cells express and release BAFF, and NK cell activation, notably including FcγRIIIa stimulation by Rituximab, results in increased secretion of BAFF (but not its close relative APRIL). Expression on the cell surface was neither detectable in resting nor in activated state. NK cell-derived BAFF enhanced the metabolic activity of primary CLL cells and protected CLL cells from chemotherapy-induced cell death. Moreover, exposure to BAFF profoundly diminished direct and Rituximab-induced lysis of primary CLL cells by allogeneic and autologous NK cells, while NK activation and degranulation per se remained unaffected. Notably, sensitivity of CLL cells to both chemotherapeutic treatment as well as direct lysis and Rituximab-induced ADCC of NK cells could be restored by the anti-BAFF antibody Belimumab (Benlysta®), which is approved for the treatment of systemic lupus erythematosus. Thus, our data provide evidence for the involvement of BAFF in the resistance of CLL cells to chemotherapy. Moreover, our results offer a functional explanation for the reportedly compromised ability of NK cells to combat lymphoid as compared to myeloid leukemias as well as their impaired ability to mediate ADCC upon Rituximab treatment in CLL patients. Our findings point to a possible benefit of combinatory approaches employing Rituximab and Belimumab for chemo-immunochemotherapy of B cell malignancies. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 1421 In about one third of patients, acute lymphoblastic leukemia (ALL) cells express Her2/neu (p185Her-2). In epithelial malignancies Her2/neu contributes to oncogenic transformation and metastatic potential, and overexpression often is associated with poor prognosis. Previous reports also suggested an inferior prognosis in patients with Her2/neu-positive ALL, but as of now no study has performed survival analyses. In breast cancer patients, targeting of Her2/neu with the monoclonal antibody Trastuzumab results in improved disease outcome, and induction of antibody dependent cellular cytotoxicity (ADCC) and cytokine release of NK cells contributes to the same. Most recently, application of Trastuzumab in patients with relapsed or refractory ALL was shown to yield promising results, but the mechanisms by which Trastuzumab mediates its efficacy have not been studied so far. Here, we studied patient records and flow cytometry data on Her2/neu expression of patients treated for ALL at our hospital between 2000 and 2011 and determined the effect of Trastuzumab and/or Rituximab on NK cell reactivity against ALL blasts in vitro. In 57 patients (37 men, 20 women, median age at diagnosis 42 years, range 17–83 years) data on Her2/neu expression were available. Her2/neu expression was detected in 15/57 patients (26%). Clinical follow up was assessed for all 57 patients. Median follow-up was 44 months (range 0.5–356 months) until death or last follow-up visit. Overall, 60% of the patients had died at the end of follow-up (25 of 42 Her2/neu-negative and 9 of 15 Her2/neu-positive patients, respectively). Median survival times were 43 (Her2/neu-negative, range 0.5–356 months) and 41 months (Her2/neu-positive, range 3.5–138 months; p = 0.81. Kaplan Meyer regression analysis). Also, median time to relapse was comparable in both groups (22.5 months vs. 15.5 months; p = 0.77). In functional analyses with NK cells, incubation of Her2/neu-positive CD20-positive ALL blasts with Trastuzumab or Rituximab comparably induced ADCC and cytokine release of NK cells. Only minor additional effects were observed upon combined application of both antibodies. When blasts from Her2/neu-negative ALL patients were employed (phentotype CD20-positive Her2/neu-negative or CD20-negative Her2/neu-negative), no effect of Trastuzumab was observed, and Rituximab exerted effects solely with blasts from CD20-positive ALL patients. Taken together, in our patient cohort Her2/neu expression on ALL blasts was not associated with time to relapse or overall survival and thus did not appear to be an additional risk factor. Rather, Her2/neu is a promising target for antibody therapy, especially in patients with Her2/neu-positive CD20-negative ALL, as treatment with Trastuzumab is suitable to induce ADCC and cytokine production of NK cells also against Her2/neu-positive CD20-negative ALL blasts. Disclosures: No relevant conflicts of interest to declare.

Description: The cytosine analogues 5-azacytosine (azacytidine) and 2′-deoxy-5-azacytidine (decitabine) display substantial therapeutic potential in patients with AML and MDS. Besides causing DNA demethylation, azanucleosides also mediate cytotoxic effects, and it appears that clinical responses are influenced by both epigenetic alterations and by apoptosis induction. However, the molecular changes induced by these drugs are still poorly understood. NK cells play an important role in tumor-immunosurveillance by confining development and progression of hematopoietic malignancies and are also important after therapeutic intervention like e.g. haploidentical stem cell transplantation. Thus it is important to define how a given therapeutic agent influences NK cell reactivity. Here we studied the effect of pharmacological concentrations of azacytidine and decitabine on NK cell effector functions. After preincubation with the azanucleosides (12h or more), NK cell cytotoxicity was found to be significantly enhanced by decitabine while, in contrast, azacytidine nearly completely abolished NK cell lysis of K562 and Raji target cells (up to 50% increase and 88% reduction, respectively; E:T ratio 10:1). Of note, modulation of NK cell cytotoxicity was also observed when the compounds were, after NK cell pretreatment, absent during the cytotoxicity assays. In contrast, neither the presence of either agent in cytotoxicity assays without preincubation nor pretreatment of target cells with either agent for 4h (corresponding to the time of the cytotoxicity assay) altered NK cell reactivity. These results indicate that azanucleosides are capable to modulate NK cell responsiveness to activating stimuli. In line, after pretreatment with the compounds NK cell IFN-γ production upon stimulation with IL-2 and IL-15 or in cocultures with target cells was found to be enhanced by decitabine but inhibited by azacytidine (up to 46% increase and 85% reduction, respectively). NK cell effector functions were not affected by deoxycytidine and cytidine, the physiological counterparts of the azanucleosides. While azacytidine treatment substantially induced NK cell apoptosis (about 30% after 24h) which may explain its inhibitory effect, no induction of apoptosis by decitabine was observed. Our data demonstrate that azacytidine and decitabine differentially affect NK cell anti-tumor reactivity and suggest that, while azacytidine causes NK cell apoptosis, decitabine enhances NK cell responsiveness via a yet unknown mechanism which is presently under study.

Description: Abstract 4403 Members of the TNF/TNF receptor (TNFR) family of proteins govern differentiation, proliferation, activation, and death of both tumor and immune effector cells and thus play an important role in tumor immunoediting, the reciprocal interaction of tumor cells and anti-tumor immunity. Activation of the TNFR family member GITR has recently been shown to stimulate T cell-mediated anti-tumor immunity in mice. However, available data suggest that GITR mediates different effects in mice and men, and may impair anti-tumor immunity of human NK cells. Here we studied the expression and function of GITR ligand (GITRL) in patients with chronic lymphocytic leukemia (CLL) and the consequences of GITR-GITRL interaction for NK cell reactivity against CLL cells. Substantial GITRL expression was detected on primary B-CLL cells in 38 of 48 (79%) investigated patients. Upon interaction with its cognate receptor, GITRL induced the release of immunoregulatory cytokines like TNF by the leukemia cells, which demonstrated that CLL-expressed GITRL is functional and capable to transduce bidirectional signals. Moreover, disruption of GITR-GITRL interaction in cultures of allogenic NK cells with patient CLL cells by addition of blocking antibody caused a significant increase in NK cell granule mobilization, cytotoxicity and IFN-γ production. The inhibitory effect of tumor-expressed GITRL on the reactivity of human NK cells was also confirmed in cocultures of C1R lymphoma cells transfected to express GITRL with mock transfectants serving as control. In addition, blocking GITR-GITRL interaction also considerably augmented both antibody-dependent cellular cytotoxicity (ADCC) and antibody-induced IFN-γ production of NK cells in cultures with allogenic CLL cells upon Rituximab exposure. Of note, GITR blockade also significantly enhanced anti-leukemia reactivity of autologous NK cells among PBMC of B-CLL patients, and this reinforcement of NK cell effector functions was observed both regarding the direct and, more pronounced, Rituximab-induced anti-leukemia reactivity (both n=10, p

Description: Abstract 1887 NK cells play an important role in anti-tumor immunity. They significantly contribute to the clinical success of allogeneic stem cell transplantation (SCT). Their reactivity as a consequence of an integrative response mediated by various activating and inhibitory surface receptors results in the induction of yet only partially defined signal transduction pathways. One of the major transcriptional regulators in lymphoid cells is NFAT (Nuclear Factor of Activated T Cells). While its role in T cell development and function is meanwhile well defined, surprisingly little is known on its function in NK cells. NFAT seems to be dispensable for NK cell development, but several lines of evidence clearly point to its involvement in NK reactivity and function. Cyclosporin A (CsA) and tacrolimus are immunosuppressive drugs that are widely used in transplant medicine. They mediate their immunosuppressive effects through inhibition of the serine/threonine phosphatase calci-neurin, which dephosphorylates and thereby activates NFAT. Here we studied the role of NFAT in NK cells and found that all five NFAT family members are expressed in NK cells with their levels being dependent on NK cell activation state. CsA and tacrolimus, but not mycophenolic acid which mediates its immunosuppressive effects by inhibiting inosine monophosphate dehydrogenase, reduced activation and degranulation of NK cells, resulting in impaired cytotoxicity and IFN-γ production in response to leukemia targets. NK reactivity was also suppressed by the specific NFAT inhibitors VIVIT and INCA-6, indicating that the calcineurin inhibitors CsA and tacrolimus in fact modulate NK reactivity by inhibition of NFAT proteins and not by potential “off target”-effects. These results provide evidence for the critical involvement of the transcription factor NFAT in NK cell reactivity and also indicate that potential effects on NK cell immunosurveillance should be considered upon choice and dosing of immunosuppressive treatment regimens after SCT. Disclosures: No relevant conflicts of interest to declare.