NK Cell Anti-tumor Reactivity Research Articles

Crosstalk between NFAT and NFκB Signaling Regulates NK Cell Immunosurveillance

One of the major transcriptional regulators in lymphoid cells is NFAT (Nuclear Factor of Activated T Cells), controlling lymphocyte development and activity. The role of NFAT signaling is well defined in T cells, the cytotoxic lymphocytes of the adaptive immunity. However, surprisingly little is known regarding the relevance of this transcription factor family in NK cells as effector cells of the innate immunity. Available data indicate that NFAT activity is dispensable for development of NK cells, whereas effects of the immunosuppressive drugs cyclosporin A and tacrolimus, that inhibit calcineurin and consecutively NFAT, implicate an involvement of the NFAT family in NK cell function. We here employed different genetic mouse models and functional analyses to unravel the role of NFAT1 (NFATc2) and NFAT2 (NFATc1) in NK cell reactivity. In vitro knockout (KO) of NFAT1 (NFATc2) or NFAT2 (NFATc1) enhanced NK cell degranulation and resulted in increased production of granzyme B and perforin upon stimulation of activating receptors like NK1.1 or Nkp46 or upon co-culture with different leukemia and solid tumor cells. In line, cytotoxicity assays revealed increased lysis of YAC-1 and B16F10 tumor cells by both NFAT1- and NFAT2-deficient NK cells as compared to wildtype (WT) controls. The inhibitory effect of NFAT transcription factors on NK cell effector function could also be confirmed in vivo by employing WT and NFAT KO animals in syngeneic B16F10 melanoma, RMA-S lymphoma and LL/2-luc lung carcinoma models, which revealed a significantly reduced tumor burden in NFAT1 and NFAT2 KO mice. Furthermore, we detected higher numbers of tumor infiltrating NK cells in NFAT KO mice, which suggested improved NK cell migration and homing to the tumor side. Comparative analyses with single NFAT as well as NFAT1+NFAT2 double KO and WT animals further confirmed the inhibitory effect of NFAT1 and NFAT2 and pointed to additive effects of NFAT1 and NFAT2 in NK cell tumor immunosurveillance. Proteomics analysis of NK cells from the different NFAT KO strains revealed altered expression of molecules which are regulating chemokine receptor expression as well as a decreased expression of NFκB inhibitory molecules, like NFKBIB (NFKB inhibitor beta) Commd6 (COMM domain containing 6). NFκB signaling was shown to regulate granzyme B and perforin synthesis and expression. Thus, we suggest that NFAT1 and NFAT2 inhibit NFκB signaling in NK cells, and NFAT1 and NFAT2 KO reinforce NFκB induced granzyme B and perforin expression and thereby increase NK cell cytotoxicity. Taken together, our results identify NFAT as a negative regulator of NK cell function. In addition, we provide the first evidence for a direct functional involvement of NFAT1 and NFAT2 in NK cell antitumor reactivity by regulation tumor infiltration and cytotoxicity of NK cell.

Abstract 2104: NK cell immunosurveillance of tumors is regulated by NFAT1 and NFAT2

Abstract NK cells are lymphoid components of innate immunity and play an important role in tumor immunosurveillance. One of the major transcriptional regulators in lymphoid cells is NFAT (Nuclear Factor of Activated T cells), controlling lymphocyte development and activity. However, while the role of NFAT signaling is well defined in T cells, the cytotoxic lymphocytes of adaptive immunity, surprisingly little is known regarding the relevance of this transcription factor family in NK cells as effector cells of innate immunity. Available data indicate that NFAT activity is dispensable for development of NK cells. However, several lines of evidence including reports on the effects of the immunosuppressive drugs cyclosporin A and tacrolimus, which mediate their effects through inhibition of calcineurin and consecutively NFAT, implicate an involvement of the NFAT family in NK cell function. We here employed different genetic mouse models to unravel the role of NFAT1 (NFATc2) and NFAT2 (NFATc1) in NK cell reactivity. When NK cells with knockout (KO) of NFAT1 or NFAT2 compared to NK cells of control mice (WT) were employed in in vitro analyses, lack of either NFAT was found to surprisingly result in enhanced NK cell degranulation as well as increased production of granzyme B and perforin upon stimulation of activating receptors like NK1.1 or Nkp46 or upon co-culture with different leukemia and solid tumor tumor cells. In line, cytotoxicity assays revealed increased lysis of YAC-1 and B16F10 tumor cells by both NFAT1- and NFAT2-deficient NK cells as compared to WT controls. The inhibitory effect of NFAT transcription factors on NK cell effector function could also be confirmed in vivo by employing WT and NFAT KO animals in syngeneic B16F10 melanoma and RMA-S flank tumor models, which revealed a significantly reduced tumor burden in NFAT1 and NFAT2 KO mice. Comparative analyses with single NFAT as well as NFAT1+NFAT2 double KO and WT animals further confirmed the inhibitory effect of NFAT1 and NFAT2 and pointed to additive effects of NFAT1 and NFAT2 in NK cell tumor immunosurveillance. Taken together, our results provide the first evidence for the direct functional involvement of NFAT1 and NFAT2 in NK cell antitumor reactivity and, in contrast to T and B cells, identify NFAT as a negative regulator of NK cell function. Citation Format: Melanie Märklin, Samuel Holzmayer, Kübra Kaban, Martin R. Müller, Helmut R. Salih. NK cell immunosurveillance of tumors is regulated by NFAT1 and NFAT2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2104.

Inhibition of NK Reactivity Against Solid Tumors by Platelet-Derived RANKL.

NK cells play an important role in tumor immunosurveillance. Their reactivity is governed by various activating and inhibitory surface receptors, which include several members of the TNF/TNF receptor family. For more than 50 years, it has been recognized that tumor immunosurveillance and in particular NK cell antitumor reactivity is largely influenced by platelets, but the underlying mechanisms remain to be fully elucidated. Here we report that upon activation, which reportedly occurs following interaction with cancer cells, platelets upregulate the TNF family member RANKL. Comparative analysis of the expression of RANK among different NK cell subsets and RANKL on platelets in cancer patients and healthy volunteers revealed a distinct malignant phenotype, and platelet-derived RANKL was found to inhibit the activity of normal NK cells against cancer cells. Notably, NK cell antitumor reactivity could be partially restored by application of denosumab, a RANKL-neutralizing antibody approved for treatment of benign and malignant osteolysis. Together, our data not only unravel a novel mechanism of tumor immune evasion mediated by platelets, but they also provide a functional explanation for the clinical observation that denosumab, beyond protecting from bone loss, may prolong disease-free survival in patients with solid tumors.

Involvement of NFAT Transcription Factors in NK Cell Reactivity

NK cells are lymphoid components of innate immunity and play an important role in tumor immunosurveillance. One of the major transcriptional regulators in lymphoid cells is NFAT (Nuclear Factor of Activated T Cells), as highlighted by its important role in T and B cell development and function. With regard to NK cells, available data indicate that NFAT is dispensable for development. However, several lines of evidence including the observation that the immunosuppressive drugs cyclosporin A and tacrolimus, which mediate their effects through inhibition of calcineurin and consecutively NFAT, influence NK reactivity implicate a role of this family of transcription factors in NK cell reactivity and function. Here we employed different genetic mouse models on the C57BL/6 background to directly study the functional role of NFAT in NK cells.We found that except for NFAT3 mRNA and protein of all family members (NFAT 1, 2, 4 and 5) was expressed in resting NK cells of wild type (WT) mice with NFAT1, 2 and 4 being most abundantly detectable. When we employed NK cells with knockout (KO) of NFAT 1, 2, and 4 in comparative in vitro analyses, we surprisingly found that lack of NFAT resulted in enhanced NK cell activation, degranulation and release of immunomodulatory cytokines like IFN-γ after co-culture with YAC-1 target cells as well as increased production of granzyme B and perforin after cytokine activation. The inhibitory effect of NFAT on NK cell effector function was further confirmed in vivo by employing WT and germ line NFAT KO animals in the syngeneic B16 melanoma model, which revealed a significantly reduced metastatic burden in NFAT KO mice. Depletion of NK cells in this model system in turn resulted in increased metastasis, however, with WT animals displaying significantly higher metastatic burden compared to NFAT KO mice. As this pointed to the fact that NFAT influences metastasis via both NK-dependent and independent mechanisms, we further generated mice with a NK cell-specific (conditional Ncr1-Cre dependent) NFAT2 KO. When these animals were employed again in analyses of B16 lung metastasis, comparative analyses with WT animals confirmed the inhibitory effect of NFAT on NK tumor immunosurveillance.Taken together, these results provide the first direct evidence for the functional involvement of NFAT in NK cell antitumor reactivity and, in contrast to T and B cells, identify NFAT as a negative regulator of NK cell function. DisclosuresNo relevant conflicts of interest to declare.

Cannabinoid receptor CB1 regulates STAT3 activity and its expression dictates the responsiveness to SR141716 treatment in human glioma patients' cells.

Herein we show that a majority of human brain tumor samples and cell lines over-expressed cannabinoid receptor CB1 as compared to normal human astrocytes (NHA), while uniformly expressed low levels of CB2. This finding prompted us to investigate the therapeutic exploitation of CB1 inactivation by SR141716 treatment, with regard to its direct and indirect cell-mediated effects against gliomas. Functional studies, using U251MG glioma cells and primary tumor cell lines derived from glioma patients expressing different levels of CB1, highlighted SR141716 efficacy in inducing apoptosis via G1 phase stasis and block of TGF-β1 secretion through a mechanism that involves STAT3 inhibition. According to the multivariate role of STAT3 in the immune escape too, interestingly SR141716 lead also to the functional and selective expression of MICA/B on the surface of responsive malignant glioma cells, but not on NHA. This makes SR141716 treated-glioma cells potent targets for allogeneic NK cell-mediated recognition through a NKG2D restricted mechanism, thus priming them for NK cell antitumor reactivity. These results indicate that CB1 and STAT3 participate in a new oncogenic network in the complex biology of glioma and their expression levels in patients dictate the efficacy of the CB1 antagonist SR141716 in multimodal glioma destruction.

An Fc-optimized NKG2D-Ig fusion protein for induction of NK cell reactivity against breast cancer.

3054 Background: The anti-tumor activity and clinical success of the monoclonal antibody trastuzumab, approved for treatment of HER2/neu-overexpressing breast cancer, is at least partially mediated by induction of antibody dependent cellular cytotoxicity (ADCC). However, only about 20% of patients show HER2/neu overexpression, and trastuzumab treatment is associated with side effects. The ligands of the activating immunoreceptor NKG2D (NKG2DL) are widely expressed on malignant cells, but generally absent on healthy tissue. We aimed to take advantage of this tumor-restricted expression by using NKG2DL as target-antigens on breast cancer cells. To this end we generated NKG2D-Ig fusion proteins with modified Fc moieties and studied their ability to induce NK cell anti-tumor reactivity. Methods: The Fc parts within the constructs were modified by amino acid exchange as previously described (Lazar 2006; Armour 1999). Direct effects on tumor cell viability as well as induction of NK cell activation, degranulation, cytotoxicity and IFN-γ release in cultures with breast cancer cell lines expressing different HER2/neu levels were determined. Results: Compared to NKG2D-Fc containing a wildtype Fc part (NKG2D-Fc-WT) or trastuzumab, 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. In contrast to trastuzumab, no direct effect of the constructs on tumor cell viability was observed. In cultures of NK cells and breast cancer cells, NKG2D-Fc-KO significantly reduced NK reactivity due to blocking immunostimulatory NKG2D-NKG2DL interaction. NKG2D-Fc-WT substantially enhanced NK reactivity by induction of ADCC, while the effects of NKG2D-Fc-ADCC by far exceeded that of NKG2D-Fc-WT and, in case of HER2/neu-low targets also that of Herceptin. Conclusions: Fc-engineered NKG2D-Ig fusion protein effectively target breast cancer cells for NK anti-tumor reactivity. 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.

RANKL Expressed by Acute Myeloid Leukemia Cells Impairs NK Cell-Mediated Immune Surveillance

AbstractAbstract 2164NK 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.

Pseudoexpression of Glucocorticoid-Induced TNF-Related (GITR) Ligand Upon Coating of Cancer Cells by Platelets Impairs NK Cell Anti-Tumor Reactivity

AbstractAbstract 3193NK cells as part of the innate immune system substantially contribute to cancer immune surveillance. They prevent tumor progression and metastasis due to their ability to mediate cellular cytotoxicity and to produce cytokines like IFN-γ, which, among others, stimulates subsequent adaptive immune responses. NK reactivity results from an integrative response emerging upon recognition of multiple ligands for activating and inhibitory NK cell receptors including various members of the TNFR family. Apart from the direct interaction with their target cells, NK cell activity is further influenced by the reciprocal interplay with various other hematopoietic cells like e.g. dendritic cells. Metastatic tumor spread in experimental animals is dramatically reduced in thrombopenic mice. Additional depletion of NK cells reverses this effect, indicating that platelets may impair NK anti-tumor reactivity. However, the underlying mechanisms have not been fully elucidated, especially in humans. Recently, we demonstrated that NK anti-tumor immunity is impaired by platelet-derived TGF-β, which is released upon interaction of platelets with tumor cells (Kopp et al., Cancer Res. 2009). Here we report that the ligand for the TNFR family member GITR (GITRL) is upregulated on megakaryocytes during maturation resulting in substantial GITRL expression by platelets. Since we recently identified GITR as inhibitory NK receptor involved in tumor immune escape (e.g., Baltz et al., Blood 2008, Baessler et al., Cancer Res. 2009) we investigated how platelet-derived GITRL influences platelet function and NK immune surveillance. Signaling via GITRL into platelets upon interaction with NK-expressed GITR or recombinant GITR-Ig fusion protein did not alter platelet activation as revealed by analysis of the activation marker CD62P and release of TGF-β. Interestingly, we found that GITRL-negative tumor cells rapidly get coated by platelets, which confers a seemingly GITRL-positive phenotype. “GITRL pseudoexpression” on tumor cells caused a substantial reduction of NK cell cytotoxicity and cytokine production. This reduced NK reactivity was not due to induction of apoptosis via GITR and could be restored by addition of a blocking GITR antibody. Thus, coating of tumor cells by platelets inhibits NK reactivity, which is in part mediated by platelet-derived GITRL. Our data provide a functional basis for the previously observed finding that platelets increase metastasis i.e. by enabling evasion of tumor cells from NK-mediated immune surveillance. Disclosures:No relevant conflicts of interest to declare.

A Fc-Engineered RANK-Fc Fusion Protein Neutralizes Soluble RANK Ligand and Targets Malignant Hematopoietic Cells for NK Cell Reactivity.

Abstract 411Bone resorption is commonly associated with aging, but also with certain cancers. Recent studies identified Receptor Activator of NFκB (RANK) ligand (RANKL) and its receptors RANK and osteoprotegerin (OPG) as key regulators of bone resorption. Multiple myeloma (MM) disrupts the balance within this molecule system, and severe bone destruction due to inappropriate osteoclastogenesis is a prominent feature of this disease. Besides MM cells, other malignant hematopoietic cells have also been found to express RANKL at the cell surface and to release this molecule in soluble form (sRANKL). Neutralization of RANKL using RANK-Fc fusion protein or monoclonal antibody (Denosumab/AMG162), which mimics the RANKL-neutralizing endogenous effects of osteoprotegerin, decreases osteolysis in multiple in vivo models and is presently being evaluated as a means to treat both non-malignant and malignant osteolysis. We here confirmed and extended previously published data and report that all investigated MM cell lines (n = 5) as well as primary leukemic cells of CLL patients (n = 12) displayed substantial levels of RANKL mRNA and surface expression. Moreover, we report that substantial levels of sRANKL can be detected in culture supernatants of MM and primary CLL cells, but not in supernatants of healthy PBMC. Next we engineered RANK-Fc fusion proteins with modified affinity to FcγR by mutating amino acids in the Fc portion as previously described (Lazar et al., PNAS 2006; Armour et al., Eur J Immunol 1999). Compared to wildtype RANK-Fc (RANK-Fc-wt), our mutants (S239D/I332E and E233P/L234V/L235A/DeltaG236/A327G/A330S) displayed highly enhanced and abrogated (RANK-Fc-ADCC+ and RANK-Fc-KO, respectively) affinity to FcγRIIIa expressed on NK cells, which play an important role in anti-tumor immunity due to their ability to lyse target cells directly and to mediate antibody-dependent cellular cytotoxicity (ADCC) upon application of therapeutic antibodies. The RANK-Fc-ADCC+ displayed similar capacitiy to neutralize sRANKL compared to the RANK-Fc-KO and the RANK-Fc-wt as revealed by binding competition assays. Next we cultured NK cells with L cells or P815 cells transfected to express RANKL and the parental, RANKL-negative controls in the presence or absence of the different RANK-Fc constructs. Addition of RANK-Fc-KO or RANK-Fc-wt did not substantially alter NK cell reactivity against the target cells. However, presence of the RANK-Fc-ADCC+ dramatically enhanced NK cell cytotoxicity and cytokine production in cultures with the RANKL-expressing target cells (increase from 20% to 89%, E:T ratio 30:1 and 12 pg/ml to 290 pg/ml respectively; both p<0.01, Student's t-test). Neither of the three RANK-Fc proteins altered NK cell cytotoxicity and cytokine production in cultures with the RANKL negative controls demonstrating that the RANK-Fc-ADCC+ specifically induced NK cell reactivity against RANKL-expressing malignant cells. Moreover, treatment with RANK-Fc-ADCC+ also significantly augmented NK cell anti-tumor reactivity in cultures with RANKL-expressing primary CLL cells of patients, and this was observed both in settings using allogenic NK cells and analyzing autologous NK cells among PBMC of the leukemia patients (both p<0.01, Student's t-test). Taken together, our Fc-engineered RANK-Fc-ADCC+ fusion protein may neutralize detrimental effects of sRANKL, can target RANKL-expressing malignant cells for NK cell anti-tumor reactivity and may thus constitute an attractive immunotherapeutic means for treatment of hematopoietic malignancies. Disclosures:No relevant conflicts of interest to declare.

The Role of the PI3K-AKT-mTOR Pathway in NK Cell Anti-Tumor Reactivity.

Abstract 3690Poster Board III-626The phosphatidylinositol 3-kinase – protein kinase B – mammalian target of rapamycin (PI3K – AKT – mTOR) pathway was found to be abnormally activated in many malignancies. Thus, protein kinase (PK) inhibitors (PKI) targeting different signaling molecules of this pathway are presently under clinical evaluation e.g. in sarcoma, multiple myeloma, or renal cell cancer. However, PK are also responsible for most of the signal transduction in immune effector cells and control various effector mechanisms including proliferation, cellular cytotoxicity, and cytokine release. Among those immunoregulatory signaling pathways, the PI3K – AKT – mTOR pathway was found to play a central role in TLR-mediated release of cytokines in macrophages and DC as well as in the regulation of T cell functions. Little is known about the role of this pathway in NK cell-mediated anti-tumor reactivity. Here we analyzed the tumor cell-induced activation of PI3K, AKT, and mTOR in NK cells and the consequences of an inhibition of these molecules by therapeutic PKI for NK cell anti-tumor reactivity. We found that, in response to tumor target cells, PI3K, AKT, and mTOR are consecutively activated in NK cells as revealed by western blot analyses using phospho-specific antibodies. Presence of the specific PI3K-inhbitor BKM-120 concentration-dependently inhibited cytotoxicity and IFN-g production of NK cells, which is in line with available data defining PI3K as a central regulator of NK cell target recognition. The mTOR inhibitors Sirolimus, Temsirolimus, and Everolimus did not alter cytotoxicity but significantly impaired NK cell IFN-γ production. In contrast, Triciribine, a compound which inhibits the phosphorylation and thus activation of AKT, did not influence cytotoxicity and, tantalizingly, even enhanced NK cell IFN-γ production. Thus, after target cell recognition and the activation of proximal PK like PI3K, different and at least partially independent signaling events govern NK cell cytokine production and cellular cytotoxicity. While the activity of PI3K followed by the activation of mitogen-activated PK is known to be crucial for NK cell cytotoxicity, we here identified the AKT – mTOR pathway as a yet unknown central component in the regulation of NK cell IFN-γ production. Moreover, in light of the important role of NK cells in tumor immune surveillance our data indicate that the choise and dosing of the most suitable PKI for a given cancer patient requires careful consideration. In the future it will be critical to define potential differences in immunosuppressive and immunostimulatory side effects of different compounds among the rapidly growing assortment of multi-targeted PKI to enable therapeutic approaches combining targeting of crucial signaling pathways in tumor cells with immunotherapy. Disclosures:No relevant conflicts of interest to declare.

Coating of Tumor Cells by Platelets Confers Expression of Immunoregulatory Molecules Which Impair NK Cell Anti-Tumor Reactivity.

Abstract 2993Poster Board II-970NK cells play an important role in cancer immunosurveillance and may prevent tumor progression and metastasis due to their ability to mediate direct cellular cytotoxicity and by releasing immunoregulatory cytokines which shape adaptive immune responses. Their reactivity is governed by various activating and inhibitory molecules expressed on target cells and reciprocal interactions with other hematopoietic cells like dendritic cells. Platelets contribute to tumor immune escape, metastasis, and angiogenesis (e.g. Jin et al Nature Med. 2006). In mice, thrombocytopenia inhibits metastasis, and this is reversed by NK cell depletion suggesting that platelets are an important additional player in NK cell-tumor interaction. However, the knowledge regarding the molecular mechanisms by which platelets influence NK cells is fragmentary at best. We found recently that platelet release soluble factors, most notably TGF-β, upon interaction with tumor cells which mediates NK cell silencing through downregulation of the activating immunoreceptor NKG2D (Kopp et al., Cancer Res 2009, in press). However, immunoregulatory molecules residing in the platelet membrane may also modulate NK cell anti-tumor responses. We report here that presence of platelets causes coating of tumor cells, and this markedly reduces NK cell lysis of tumor cells. This is mediated by conferment of “pseudoexpression” of platelet-expressed immunoregulatory molecules to tumor cells which are absent on the tumor cells alone. Among those immunregulatory molecules we identified various ligands for NK cell receptors like MHC class I, GITR ligand or CD62P. To establish the functional significance of tumor cell pseudoexpression of platelet molecules we employed functional analyses of tumor cells and NK cells with or without coating by autologous platelets. The impaired anti-tumor reactivity of NK cells against coated tumor cells was restored by blocking MHC class I on the coating platelets, while isotype control had no effect. Moreover, coating of tumor cells by platelets was validated by ex vivo analyses of primary leukemic cells from patients which also revealed substantial coating by platelets and confered expression of NK cell-modulating molecules. Our data indicate that platelets enable a molecular mimicry of tumor cells, which enables tumor cells to escape NK cell-mediated tumor immunosurveillance. Disclosures:No relevant conflicts of interest to declare.

Platelet-Derived Transforming Growth Factor-β Down-Regulates NKG2D Thereby Inhibiting Natural Killer Cell Antitumor Reactivity

Natural killer (NK) cells play an important role in cancer immunosurveillance and may prevent tumor progression and metastasis due to their ability to mediate direct cellular cytotoxicity and by releasing immunoregulatory cytokines, which shape adaptive immune responses. Their reactivity is governed by various activating and inhibitory molecules expressed on target cells and reciprocal interactions with other hematopoietic cells such as dendritic cells. In mice, thrombocytopenia inhibits metastasis, and this is reversed by NK cell depletion, suggesting that platelets are an important additional player in NK cell-tumor interaction. Moreover, it has been shown that metastasizing tumor cells do not travel through the blood alone but are rapidly coated by platelets. However, the knowledge about the molecular mechanisms by which platelets influence NK cells is fragmentary at best. Here we show that platelet-derived soluble factors, secreted on coating of tumor cells or after stimulation with classic platelet agonists, impair NK cell antitumor reactivity resulting in diminished granule mobilization, cytotoxicity, and IFN-gamma production. The impaired NK cell reactivity was not due to induction of apoptosis but mediated by down-regulation of the activating immunoreceptor natural killer group 2, member D (NKG2D) on NK cells by platelet-derived transforming growth factor beta (TGF-beta). Neutralization of TGF-beta in platelet releasate not only prevented NKG2D down-regulation but also restored NK cell antitumor reactivity. Thus, our data elucidate the molecular basis of the previously described influence of platelets on NK cell antitumor reactivity and suggest that therapeutic intervention in tumor cell-platelet interaction and the resulting TGF-beta release by platelets may serve to enhance antitumor immunity.

5-Azacytosine (Azacytidine) and 2′-Deoxy-5-Azacytidine (Decitabine) Mediate Opposite Effects on NK Cell Anti-Tumor Reactivity

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.

Differential Effects of the BCR-ABL-Inhibitors Imatinib, Nilotinib and Dasatinib on NK Cell Reactivity against Chronic Myeloid Leukemia (CML)

CML is characterized by the BCR-ABL fusion protein, which mediates the oncogenic signaling. This led to the development of BCR-ABL inhibitors revolutionizing therapy of CML. However, as recently reported for Dasatinib (Schade et al., Blood 111:1366 (2008); Blake et al., Blood 111:4415 (2008)), these agents may impair the activity of immune effector cells like NK cells and T cells. After initiating oncogenic events, development and progression of clinically apparent malignancy is dependent on the evasion of the tumor cells from immunosurveillance. In light of the important role of NK cell reactivity against leukemia we compared the influence of Imatinib, Nilotinib and Dasatinib on the reactivity of both resting and IL-2 activated NK cells against CML cells to identify the compound with the least immuno-compromising side effects. First, the effects of the compounds on NK cell reactivity in concentrations corresponding to plasma peak levels were studied. Dasatinib (200nM) completely abolished NK cell granule mobilization, cytotoxicity and IFN-γ production, while no substantial inhibition was observed with Imatinib (5μM) and Nilotinib (3.6μM) mediated a minor but significant inhibition (p<0.05, Student's T-test). Presence of the compounds in concentrations corresponding to IC50 levels (Imatinib 600nM, Nilotinib 30nM, Dasatinib 10nM) revealed no influence of Imatinib and Nilotinib, while Dasatinib still significantly reduced NK cell cytotoxicity and IFN-γ production up to 60%. Since Dasatinib, in addition to BCR-ABL, potently inhibits SRC kinases, which are involved in the activation of MAPK pathways and thus crucial for NK cell cytotoxicity, we determined the influence of the compounds on ERK phosphorylation. While no inhibitory effect was observed using Imatinib and Nilotinib, Dasatinib markedly reduced ERK phosphorylation in NK cells. Our data demonstrate that NK cell anti-tumor reactivity is not inhibited by clinically relevant concentrations of Imatinib. While Nilotinib may mediate a minor effect, Dasatinib substantially impairs NK cell reactivity by inhibition of signaling pathways crucial for NK cell effector functions. For a given patient, the choice and dosing of the most suitable BCR-ABL inhibitor may thus require careful consideration of its influence on the immune system, especially in view of the important role of NK cells in the immunesurveillance of residual leukemia.

NK Cell Anti-Tumor Reactivity Is Impaired by Platelet-Derived TGF-B through NKG2D Downregulation

NK cells can recognize and eliminate malignant cells thereby preventing both local tumor progression and metastatic spread. Besides by the direct interaction with their target cells, NK cell activity is influenced by the interplay with other hematopoietic cells influencing the activity of both involved cell types. Recently, we and others demonstrated that thrombocytopenia inhibits metastasis in mice, which is dependent on the presence of NK cells as shown by NK cell depletion experiments (Jin et al., Nat Med 2006, 12: 557; Nieswandt et al., Cancer Res 1999, 59: 1295). These data suggest that thrombocytopenia indirectly inhibits tumor dissemination by allowing NK cells to exert their anti-tumor effector functions. However, yet nothing is known regarding the molecular mechanisms underlying platelet-NK cell interaction, especially in humans. Here we report that human platelets express high levels of MHC class I, and incubation of MHC class I-negative tumor cells with platelets induced formation of tumor cell-platelet aggregates resulting in tumor MHC class I “pseudoexpression” which can inhibit NK cell reactivity via KIR triggering. Furthermore, tumor cell-induced platelet aggregation, alike stimulation with classical platelet agonists such as thrombin or collagen, resulted in secretion of platelet-derived soluble factors (releasate) containing, among others, high levels of the immunoregulatory cytokine TGF-b. Presence of releasate in cocultures of NK cells and tumor target cells was found to mediate a substantial inhibition of NK cell cytotoxicity and IFN-g production. Of note, platelet releasate caused a marked downregulation of the activating immunoreceptor NKG2D expressed on NK cells, which was partially restored by a neutralizing TGF-b antibody. Confirming the functional relevance of this finding, neutralization of TGF-b in platelet releasate by the same antibody also partially restored both the impaired NK cell cytotoxicity and IFN-g production in cocultures with tumor cells. Taken together, our data demonstrate that platelets may substantially influence NK cell anti-tumor reactivity by various mechanisms including inhibition of NKG2D-mediated NK cell immunosurveillance. Further elucidation of platelet ability to confer expression of immunregulatory surface molecules to tumor cells and the various soluble factors carrying NK cell-modulatory activity may reveal novel targets to prevent tumor dissemination.

Neutralization of tumor-derived soluble Glucocorticoid-Induced TNFR-Related Protein ligand increases NK cell anti-tumor reactivity

AbstractNK 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.

The kinase inhibitors Sunitinib and Sorafenib differentially affect reactivity of NK cells against Renal Cell Cancer

Sunitinib and Sorafenib are multi‐kinase inhibitors licensed for the treatment of patients with advanced renal cell cancer (RCC) and target, among others, VEGF receptors, PDGF receptors, c‐kit and Flt3. Beyond that, Sorafenib inhibits c‐RAF and B‐RAF. Here we report that Sorafenib but not Sunitinib significantly (p&lt;0.05, Mann‐Whitney U‐test) reduce cell surface CD107a expression (about 50%) on NK cells as a surrogate marker for degranulation in response to tumor targets. In line, pharmacological levels of Sorafenib but not Sunitinib significantly (p&lt;0.05) diminish cytotoxicity of primary NK cells against the RCC cell lines A498, ACHN and CAKI‐2 (46% to 14%, 52% to 22% and 63% to 42%, respectively) in chromium release assays. While Sunitinib had no effect, Sorafenib, alike the specific c‐RAF inhibitor ZM336372 or the specific MEK inhibitor PD98059, markedly reduces ERK phosphorylation which regulates the movement of lytic granula towards target cells. Our results indicate that NK cell anti‐tumor reactivity is impaired by Sorafenib but not by Sunitinib due to the additional inhibitory effect of Sorafenib on the ERK pathway, which has been demonstrated to dictate NK cell cytotoxicity. We therefore conclude that Sunitinib is exquisitely suitable for combination with immunotherapeutic approaches in RCC patients.

The Kinase Inhibitors Sunitinib (Sutent®) and Sorafenib (Nexavar®) Differentially Affect Reactivity of NK Cells Against Renal Cell Cancer.

Sunitinib (Sutent®) and Sorafenib (Nexavar®) are multikinase inhibitors licensed for the treatment of patients with advanced renal cell cancer (RCC) because of their direct and indirect anti-tumor and anti-angiogenic effects. Sunitinib and Sorafenib target, among others, vascular endothelial growth factor receptors (VEGFR), platelet-derived growth factor receptors (PDGFR), the stem cell factor receptor (cKIT) and Fms-like tyrosine kinase-3 (Flt3). Beyond that, Sorafenib additionally inhibits c-RAF and B-RAF. NK cells play an important role in the immune surveillance of tumors. Their reactivity is governed by different surface receptors leading to activation of various kinases, phosphatases and transcription factors. Up to now, nothing is known regarding the influence of Sunitinib and Sorafenib on NK cells. Here we report that Sunitinib and Sorafenib differentially affect NK cell cytotoxicity. Analysis of peripheral blood mononuclear cells from 10 independent donors revealed that Sorafenib but not Sunitinib significantly (p<0.05, Mann-Whitney U-test) reduced cell surface CD107a expression (about 50%) on NK cells as a surrogate marker for degranulation in response to tumor targets. In line, pharmacological levels of Sorafenib significantly (p<0.05, Mann-Whitney U-test) diminished cytotoxicity of primary NK cells against the RCC cell lines A498, ACHN and CAKI-2 (from 46% to 14%, from 52% to 22% and from 63% to 42%, respectively) in chromium release assays. Again, no inhibition of cytotoxicity was observed in the presence of pharmacological concentrations of Sunitinib. Since activation of MAP kinases leading to ERK phosphorylation regulates the movement of lytic granula towards target cells, which is responsible for NK cell cytotoxicity we investigated the influence of Sunitinib and Sorafenib on ERK phosphorylation. While Sunitinib had no effect, Sorafenib, alike the specific c-RAF inhibitor ZM336372 or the specific MEK inhibitor PD98059, markedly reduced ERK phosphorylation as determined by Western Blot. Our results indicate that NK cell anti-tumor reactivity is impaired by Sorafenib but not by Sunitinib due to the additional inhibitory effect of Sorafenib on the ERK pathway, which has been demonstrated to dictate NK cell cytotoxicity. We therefore conclude that Sunitinib is exquisitely suitable for combination with immunotherapeutic approaches in RCC patients.

Serotherapy with thymoglobulin and alemtuzumab differentially influences frequency and function of natural killer cells after allogeneic stem cell transplantation

Although thymoglobulin and alemtuzumab are frequently used in hematopoietic stem cell transplantation (HSCT), little is known of their effects on NK cells, which mediate important functions in post-transplantation immunology. In the present study, we determined NK cell death in vitro using propidium iodide and Annexin V. The NK cell activity in 34 patients at day +30 after allogeneic HSCT was assessed using the CD107a assay. Alemtuzumab and thymoglobulin were similarly very potent in inducing NK cell death in vitro. Even in low concentrations (<1 microg/ml) the antibodies induced apoptosis and necrosis in a relevant percentage of NK cells (>30%). However, the number of tumor reactive (CD107a+) NK cells was 13.16 per mul and 1.15 per microl (mean) in patients receiving T-cell depletion with 6 mg/kg thymoglobulin and in patients receiving 100 mg alemtuzumab, respectively (P=0.02). Although thymoglobulin and alemtuzumab are equally NK cell toxic in vitro, the recovery of NK cell frequency and anti-tumor reactivity is reduced in recipients of alemtuzumab. Our findings can be explained by a longer half-life of alemtuzumab as compared to active thymoglobulin under therapeutic conditions. Prolonged immunosuppression with increased risk of infections and tumor relapse are a potential threat to patients undergoing HCST and receiving alemtuzumab as T-cell depletion.