NKL Cells Research Articles

Resveratrol Induces Cell Cycle Arrest and Apoptosis in Malignant NK Cells Via JAK2/STAT3 Pathway Inhibition

Abstract 1343Natural killer (NK)-cell malignancies, particularly aggressive NK-cell leukemias/lymphomas, have poor prognoses. Although recent regimens that include L-asparaginase substantially improve outcomes, novel therapeutic approaches are needed to improve clinical responses. Recent reports have shown that the signal transducer and activator of transcription 3 (STAT3) pathway is critical for proliferation and survival of malignant NK cells. Resveratrol is a naturally-occurring polyphenol that has been extensively studied for its anti-inflammatory, cardioprotective, and anti-cancer activities. In this study, we investigated the potential anti-tumor activities of resveratrol against the NK cell lines KHYG-1, NKL, NK-92, and NK-YS. Resveratrol significantly suppressed cell proliferation in a dose- and time-dependent manner in these four cell lines. Flow cytometry analysis with annexin V/propidium iodide staining showed a variable but consistent induction of apoptosis in the four cell lines treated with resveratrol for 48 hours, ranging from 57.1±6.9% of apoptosis in the L-asparaginase resistant cell line KHYG-1 to 53.4±9.7%, 28.8±3.3%, and 51.7±6.7% in NKL, NK-92, and NK-YS cells respectively (Fig. 1a). Notably, the anti-tumor activity of resveratrol against NK cell lines was p53 independent as demonstrated by equal efficacy of resveratrol against NK cell lines pretreated with the p53 inhibitor Pifithrin-α. Immunoblot analysis to study intracellular signaling in resveratrol-treated cells showed suppression of constitutively active STAT3 in all four cell lines 24 hours after treatment. Remarkably, resveratrol inhibited JAK2 phosphorylation, but had no effect on other known upstream mediators of STAT3 activation such as PTEN and Tyk2 (Fig. 1b). Resveratrol induced robust G1 cell cycle arrest and down-regulation of two anti-apoptotic proteins, Mcl-1 and survivin, both of which are downstream effectors of the STAT-3 pathway. Furthermore, resveratrol enhanced the pro-apoptotic and anti-proliferative activities of L-asparaginase against NKL and NK-92 cells by 32% and 126% respectively.These data indicate that resveratrol possesses a potent anti-tumor effect via inactivation of the STAT3 pathway in malignant NK cells. These mechanistic findings suggest that resveratrol may have therapeutic potential against NK cell malignancies. Our finding that resveratrol is a bonafide JAK2 inhibitor extends its therapeutic potential to other diseases with deregulated JAK2 signaling. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

NK Cell Proliferation and Cytokine Production Depends on the mTOR- and Calcineurin-Pathway but not p38 MAP-Kinase Pathway

The role of NK cells during solid organ rejection is still nearly unexplored. Therefore we investigated the effect of routinely given immunosuppressants on the proliferation and cytokine production of fresh NK cells and NKL, a NK cell leukemia line. Methods: eFluor 670 stained NKL cells were treated with medium supplemented with DMSO, mTOR-inhibitor Sirolimus (Sir), calcineurin-inhibitors Tacrolimus (Tac) and Cyclosporin A (CsA) or MEK1/2- inhibitor U0126 (10 or 20 μM) in the presence or absence of IL-2. Supernatants were harvested at day 3 for the analysis of cytokines/chemokines by the Bioplex-technique and proliferative capacity was assessed by FACS-analyses at day 0 to day 3. Results: Sir, Tac and CsA inhibit the proliferation of NK and NKL cells in a dose-dependent manner. The anti-proliferative effect of the inhibitors can be partly recovered by IL-2. Sir causes the strongest anti-proliferative effect on NKL cells. Although CsA and Tac are both calcineurin-inhibitors, Tac shows a less inhibitory capacity regarding NKL proliferation. In contrast MEK1/2 inhibitor U0126 does not affect NKL proliferation in the presence of IL-2. Some of the cytokines/chemokines produced by NKL cells like GM-CSF or CCL5 are secreted in an IL-2- independent manner. Others like VEGF, CXCL8, IFN-g or IL-10 could only be detected in higher amounts in the presence of IL-2. Interestingly, the influence of these inhibitors on the cytokine/chemokine secretion does not show the same pattern as for NKL proliferation. The most potent inhibitor of NKL proliferation, Sir, only decreases VEGF-production, whereas CsA inhibits the secretion of VEGF, CXCL8 and IL-10. Surprisingly, MEK1/2 inhibitor U0126 induces a strong production of CXCL8, CCL5 and CCL2. Conclusions: Our results show that CNI as well as mTOR inhibitors effect proliferation of NKL cells which depends clearly on IL-2. Interestingly the responsiveness to IL-2 via mTOR inhibition plays a more critical role than the production of IL-2 itself since CsA and Tac do not inhibit NKL proliferation cells as strong as Sir. The MAP kinase pathway is not involved in this proliferation process in the presence of IL-2 as U0126 does not reduce the proliferation of NKL cells. Taken together, proliferation and cytokine/chemokine-production of NK cells are regulated differently by calcineurin and other pathways. Thus, immunosuppressants with high anti-proliferative capacity must not be automatically a good suppressor of cytokine production. Therefore, the efficacy of immunosuppressants should also be addressed with respect to cytokine and chemokine secretion.

Cyclosporine Induces Up-regulation of Immunoglobulin-like Transcripts 3 and 4 Expression on and Activity of NKL Cells

BackgroundImmunoglobulin-like transcripts (ILTs), which belong to a kind of receptor family discovered recently, are differentially expressed on myeloid and lymphoid cells. Most of them play important roles to regulate human immune responses by interacting with ligands. Cyclosporine (CsA) is frequently used to prevent graft-versus-host disease and treat autoimmune diseases. There are some studies about the effects of CsA on various human immunologic reactions, but its impact on ILT3 and ILT4 expression on natural killer (NK) cells is less well understood. MethodsAn NKL cell line was exposed to CsA (5, 10, 15, or 20 mg/L) for 12, 24, or 36 hours before real-time quantitative polymerase chain reaction and flow cytometry were used to detect alterations in ILT3 and ILT4 mRNA and protein expressions. NKL cells treated for 36 hours with or without CsA (15 mg/L) and then coincubated with BGC-823 or JEG-3 cells, in cytolytic and proliferative systems measured by Thiazoyl blue tetrazolium bromide assays. ResultsAfter CsA treatment both RNA and protein levels of ILT3 and ILT4 on NKL cells were increased for 12, 24, or 36 hours. CsA at various concentrations inhibited the proliferation of NKL cells to varying degrees; at 36 hours CsA (15 mg/L) showed greater effects on ILT3 and ILT4 expression and less influence on NKL growth. The ability of NKL cells primed with CsA (15 mg/L) for 36 hours to kill tumor cells was decreased markedly. ConclusionsCsA up-regulated the expression of ILT3 and ILT4 on NKL cells, which influenced their cytotoxicity against tumor cells with different expression of HLA-G and proliferation of NKL cells.

MiR‐30c‐1* promotes natural killer cell cytotoxicity against human hepatoma cells by targeting the transcription factor HMBOX1

Natural killer (NK) cells play a critical role in antitumor immunity, and the activation of NK cells is regulated by a series of NK cell receptors. Here, we show that crosslinking CD226, an important NK cell receptor, with the anti-CD226 mAb LeoA1 on NKL cells, regulated the expression of several microRNA and transmembrane tumor necrosis factor-α. Among them, miR-30c-1(*) was noticed because overexpression of miR-30c-1(*) triggered upregulation of transmembrane tumor necrosis factor-α expression and enhanced NK cell cytotoxicity against hepatoma cell lines SMMC-7721 and HepG2. Furthermore, we proved that the inhibitory transcription factor HMBOX1, which depressed the activation of NK cells, was the direct target gene of miR-30c-1(*). In conclusion, our results revealed a novel regulatory mechanism: miR-30c-1(*) promoted NK cell cytotoxicity against hepatoma cells by targeting HMBOX1.

Cervical cancer cell lines expressing NKG2D-ligands are able to down-modulate the NKG2D receptor on NKL cells with functional implications

BackgroundCervical cancer represents the third most commonly diagnosed cancer and the fourth leading cause of cancer-related deaths in women worldwide. Natural killer (NK) cells play an important role in the defense against viruses, intracellular bacteria and tumors. NKG2D, an activating receptor on NK cells, recognizes MHC class I chain-related molecules, such as MICA/B and members of the ULBP/RAET1 family. Tumor-derived soluble NKG2D-ligands have been shown to down-modulate the expression of NKG2D on NK cells. In addition to the down-modulation induced by soluble NKG2D-ligands, it has recently been described that persistent cell-cell contact can also down-modulate NKG2D expression. The goal of this study was to determine whether the NKG2D receptor is down-modulated by cell-cell contact with cervical cancer cells and whether this down-modulation might be associated with changes in NK cell activity.ResultsWe demonstrate that NKG2D expressed on NKL cells is down-modulated by direct cell contact with cervical cancer cell lines HeLa, SiHa, and C33A, but not with non-tumorigenic keratinocytes (HaCaT). Moreover, this down-modulation had functional implications. We found expression of NKG2D-ligands in all cervical cancer cell lines, but the patterns of ligand distribution were different in each cell line. Cervical cancer cell lines co-cultured with NKL cells or fresh NK cells induced a marked diminution of NKG2D expression on NKL cells. Additionally, the cytotoxic activity of NKL cells against K562 targets was compromised after co-culture with HeLa and SiHa cells, while co-culture with C33A increased the cytotoxic activity of the NKL cells.ConclusionsOur results suggest that differential expression of NKG2D-ligands in cervical cancer cell lines might be associated with the down-modulation of NKG2D, as well as with changes in the cytotoxic activity of NKL cells after cell-cell contact with the tumor cells.

Expression of IL-15 in NK cells results in rapid enrichment and selective cytotoxicity of gene-modified effectors that carry a tumor-specific antigen receptor

Natural killer (NK) cells hold promise for adoptive cancer immunotherapy but are dependent on cytokines such as interleukin (IL)-2 for growth and cytotoxicity. Here, we investigated the consequences of ectopic expression of IL-15 in human NK cells. IL-2 and IL-15 belong to the common γ chain family of cytokines and have overlapping activities. Transduction of clinically applicable NK-92 cells with lentiviral vectors encoding human IL-15 resulted in predominantly intracellular expression of the cytokine, and STAT5 activation, proliferation and cytotoxicity of the producer cells in the absence of IL-2. Growth of non-transduced bystander cells was not supported, allowing rapid enrichment of gene-modified cells solely by IL-2 withdrawal. This was also the case upon transduction of NK-92 and NKL cells with a bicistronic lentiviral vector encoding IL-15 and a chimeric antigen receptor (CAR) targeting the pancarcinoma antigen EpCAM. Effector cells co-expressing CAR and IL-15 continued to proliferate in the absence of exogenous cytokines and displayed high and selective cell-killing activity against EpCAM-expressing breast carcinoma cells that were resistant to the natural cytotoxicity of unmodified NK cells. This strategy facilitates rapid isolation and continuous expansion of retargeted NK cells and may extend their potential clinical utility.

Syntaxin 11 marks a distinct intracellular compartment recruited to the immunological synapse of NK cells to colocalize with cytotoxic granules

The syntaxin 11 (STX11) gene is mutated in a proportion of patients with familial haemophagocytic lymphohistiocytosis (FHL) and exocytosis of cytotoxic granules is impaired in STX11-deficient NK cells. However, the subcellular localization, regulation of expression and molecular function of STX11 in NK cells and other cytotoxic lymphocytes remain unknown. Here we demonstrate that STX11 expression is strictly controlled by several mechanisms in a cell-type-specific manner and that the enzymatic activity of the proteasome is required for STX11 expression in NK cells. In resting NKL cells, STX11 was localized in the cation-dependent mannose-6-phosphate receptor (CD-M6PR)-containing compartment, which was clearly distinct from cytotoxic granules or Rab27a-expressing vesicles. These subcellular structures appeared to fuse at the contact area with NK-sensitive target cells as demonstrated by partial colocalization of STX11 with perforin and Rab27a. Although STX11-deficent allo-specific cytotoxic T-lymphocytes efficiently lysed target cells and released cytotoxic granules, they exhibited a significantly lower extent of spontaneous association of perforin with Rab27a as compared with STX11-expressing T cells. Thus, our results suggest that STX11 promotes the fusion of Rab27a-expressing vesicles with cytotoxic granules and reveal an additional level of complexity in the spatial/temporal segregation of subcellular structures participating in the process of granule-mediated cytotoxicity.

Human Leukocyte Antigen E Contributes to Protect Tumor Cells from Lysis by Natural Killer Cells

The nonclassic class I human leukocyte antigen E (HLA-E) molecule engages the inhibitory NKG2A receptor on several cytotoxic effectors, including natural killer (NK) cells. Its tissue distribution was claimed to be wider in normal than in neoplastic tissues, and surface HLA-E was undetectable in most tumor cell lines. Herein, these issues were reinvestigated taking advantage of HLA-E-specific antibodies, immunohistochemistry, and biochemical methods detecting intracellular and surface HLA-E regardless of conformation. Contrary to published evidence, HLA-E was detected in a few normal epithelia and in a large fraction (approximately 1/3) of solid tumors, including those derived from HLA-E-negative/low-normal counterparts. Remarkably, HLA-E was detected in 30 of 30 tumor cell lines representative of major lymphoid and nonlymphoid lineages, and in 11 of 11, it was surface-expressed, although in a conformation poorly reactive with commonly used antibodies. Coexpression of HLA-E and HLA class I ligand donors was not required for surface expression but was associated with NKG2A-mediated protection from lysis by the cytotoxic cell line NKL and polyclonal NK cells from healthy donors, as demonstrated by antibody-mediated relief of protection in 10% to 20% of the tested target-effector combinations. NKG2A-mediated protection of additional targets became evident on NK effector blocking with antibodies to activating receptors (DNAM-1, natural cytotoxicity receptors, and NKG2D). Thus, initial evidence that the long-elusive HLA-E molecule is enhanced by malignant transformation and is functional in tumor cells is presented here, although its importance and precise functional role remain to be addressed in the context of a general understanding of the NK ligand-receptor network.

Ab-IL2 fusion proteins mediate NK cell immune synapse formation by polarizing CD25 to the target cell-effector cell interface.

The huKS-IL2 immunocytokine (IC) consists of IL2 fused to a mAb against EpCAM, while the hu14.18-IL2 IC recognizes the GD2 disialoganglioside. They are under evaluation for treatment of EpCAM(+) (ovarian) and GD2(+) (neuroblastoma and melanoma) malignancies because of their proven ability to enhance tumor cell killing by antibody-dependent cell-mediated cytotoxicity (ADCC) and by antitumor cytotoxic T cells. Here, we demonstrate that huKS-IL2 and hu14.18-IL2 bind to tumor cells via their antibody components and increase adhesion and activating immune synapse (AIS) formation with NK cells by engaging the immune cells' IL-2 receptors (IL2R). The NK leukemia cell line, NKL (which expresses high affinity IL2Rs), shows fivefold increase in binding to tumor targets when treated with IC compared to matching controls. This increase in binding is effectively inhibited by blocking antibodies against CD25, the α-chain of the IL2R. NK cells isolated from the peritoneal environment of ovarian cancer patients, known to be impaired in mediating ADCC, bind to huKS-IL2 via CD25. The increased binding between tumor and effector cells via ICs is due to the formation of AIS that are characterized by the simultaneous polarization of LFA-1, CD2 and F-actin at the cellular interface. AIS formation of peritoneal NK and NKL cells is inhibited by anti-CD25 blocking antibody and is 50-200% higher with IC versus the parent antibody. These findings demonstrate that the IL-2 component of the IC allows IL2Rs to function not only as receptors for this cytokine but also as facilitators of peritoneal NK cell binding to IC-coated tumor cells.

Differential internalization of hu14.18-IL2 immunocytokine by NK and tumor cell: impact on conjugation, cytotoxicity, and targeting

The hu14.18-IL2 (EMD 273063) IC, consisting of a GD(2)-specific mAb genetically engineered to two molecules of IL-2, is in clinical trials for treatment of GD(2)-expressing tumors. Anti-tumor activity of IC in vivo and in vitro involves NK cells. We studied the kinetics of retention of IC on the surface of human CD25(+)CD16(-) NK cell lines (NKL and RL12) and GD(2)(+) M21 melanoma after IC binding to the cells via IL-2R and GD(2), respectively. For NK cells, ∼ 50% of IC was internalized by 3 h and ∼ 90% by 24 h of cell culture. The decrease of surface IC levels on NK cells correlated with the loss of their ability to bind to tumor cells and mediate antibody-dependent cellular cytotoxicity in vitro. Unlike NK cells, M21 cells retained ∼ 70% of IC on the surface following 24 h of culture and maintained the ability to become conjugated and lysed by NK cells. When NKL cells were injected into M21-bearing SCID mice, IT delivery of IC augmented NK cell migration into the tumor. These studies demonstrate that once IC binds to the tumor, it is present on the tumor surface for a prolonged time, inducing the recruitment of NK cells to the tumor site, followed by tumor cell killing.

Interactions of NK Cell Receptor KIR3DL1*004 with Chaperones and Conformation-Specific Antibody Reveal a Functional Folded State As Well As Predominant Intracellular Retention

Variable interaction between the Bw4 epitope of HLA-B and the polymorphic KIR3DL1/S1 system of inhibitory and activating NK cell receptors diversifies the development, repertoire formation, and response of human NK cells. KIR3DL1*004, a common KIR3DL1 allotype, in combination with Bw4(+) HLA-B, slows progression of HIV infection to AIDS. Analysis in this study of KIR3DL1*004 membrane traffic in NK cells shows this allotype is largely misfolded but stably retained in the endoplasmic reticulum, where it binds to the chaperone calreticulin and does not induce the unfolded protein response. A small fraction of KIR3DL1*004 folds correctly and leaves the endoplasmic reticulum to be expressed on the surface of primary NK and transfected NKL cells, in a form that can be triggered to inhibit NK cell activation and secretion of IFN-γ. Consistent with this small proportion of correctly folded molecules, trace amounts of MHC class I coimmunoprecipitated with KIR3DL1*004. There was no indication of any extensive intracellular interaction between unfolded KIR3DL1*004 and cognate Bw4(+) HLA-B. A similarly limited interaction of Bw4 with KIR3DL1*002, when both were expressed by the same cell, was observed despite the efficient folding of KIR3DL1*002 and its abundance on the NK cell surface. Several positions of polymorphism modulate KIR3DL1 abundance at the cell surface, differences that do not necessarily correlate with the potency of allotype function. In this context, our results suggest the possibility that the effect of Bw4(+) HLA-B and KIR3DL1*004 in slowing progression to AIDS is mediated by interaction of Bw4(+) HLA-B with the small fraction of cell surface KIR3DL1*004.

SiRNA Inactivation of the Inhibitory Receptor NKG2A Augments the Anti-Tumor Effects of Adoptively Transferred NK Cells In Tumor-Bearing Hosts

AbstractAbstract 1015Natural killer (NK) cells are a component of the innate immune system that target both tumors and virally infected cells. NK cell killing of tumors is regulated by a delicate balance of activating and inhibitory receptors. These inhibitory receptors bind HLA ligands which prevent NK cell targeting of normal “self” cells. Up regulation of surface expression of HLA molecules has been utilized by tumors as a method to evade NK cell cytotoxicity. Disrupting the function or expression of inhibitory receptors on NK cells could potentially be used as a method to overcome this effect. While most inhibitory receptors are present in only a subset of NK cells, NK cells universally express the HLA-E binding inhibitory receptor NKG2A. We hypothesized that siRNA inactivation of NK cell NKG2A would could be used as a therapeutic approach to enhance NK cell tumor cytotoxicity in vivo.The human natural killer cell line NKL was transduced with lentiviral vectors encoding shRNA targeting various regions of the NKG2A transcript. Following lentiviral transduction, knockdown of receptor expression was confirmed by flow cytometry and RT-qPCR. Compared to wild type (WT) and GFP-transduced NKL controls, NKG2A silenced NKL cells had increased secretion of IFN-gamma and Fas-L by ELISA and increased granzymes A and B and Nkp30 expression by flow cytometry. In contrast, expression of NKG2D, Nkp44, Nkp46, LFA-1, DNAM, and TRAIL was not altered by NKG2A silencing. Chromium-based cytotoxicity assays showed shRNA knockdown of NKG2A significantly enhanced NK cell cytotoxicity of tumor cells: at a 20:1 effector to target ratio, NKG2A knockdown NKLs, WT NKLs and GFP-transduced NKLs induced 68.9%, 8.2% and 8.3% lysis respectively of 721.221 EBV-LCL tumor targets (p=0.001). Remarkably, NKG2A silencing enhanced NKL killing of both HLA-E positive (721.221 EBV-LCL and 526 melanoma cells) and HLA-E negative (K562) tumor cell lines, suggesting NKG2A inactivation increased NK cell cytotoxicity through both HLA-E dependent and independent mechanisms. Using a xenogeneic model, we next explored the in vivo effects of transferring NKG2A silenced NK cells in tumor bearing mice. Immunodeficient NSG mice were injected with 1 million human luciferase transduced 721.221 HLA-E expressing EBV-LCL tumor cells. Twenty-four hours later, tumor-bearing mice were injected with 2–5 million WT NKL cells, GFP-control-transduced NKL, or NKG2A silenced NKL cells, then received IL-2 sq for 10 days to induce in vivo NK cell proliferation. NKL numbers in blood were subsequently analyzed by flow cytometry and tumor burden was assessed by luciferase-based bioluminescence imaging (BLI). At 16 and 21 days following adoptive NK cell transfer, BLI showed that recipients of NKG2A silenced NKL cells had slower tumor growth and significantly smaller tumor burden compared to NKL wt and NKL-GFP transduced controls (figure). Importantly, no toxicity related to infusing NKG2A inactivated NK cells was observed. These in vitro and in vivo data suggest shRNA knockdown of the NKG2A inhibitory receptor could be used as a method to augment NK cell tumor cytotoxicity in patients with hematological malignancies. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

NOVEL Pathways Modulate TUMOR CELL Susceptibility to NK CELLS.

Abstract 277NK cells play an important role in innate immune responses directed against autologous cells that have undergone viral or malignant transformation. The ability of NK cells to lyse targets is primarily dependent on the expression of various inhibitory or activating receptors. However, transformed cells have also developed mechanisms to evade immune surveillance and the molecular basis for target resistance to immune-mediated lysis is not well understood. To address this issue we undertook a genetic screening approach to identify novel pathways that modulate tumor cell susceptibility to NK cell lysis. Our genetic screen utilized a subset of the TRC1 lentiviral shRNA library developed at the Broad Institute of Harvard and MIT. The library subset targeted 476 protein kinases and 180 phosphatases that represent 88% and 80%, respectively, of known NCBI sequences with these functions. The library also targeted 372 genes representing tumor suppressors, DNA binding proteins as well as irrelevant shRNAs as controls. Each gene was targeted by 5 or more independent shRNAs tested individually in a 384 well format using robotic manipulations. A total of 6,144 individual shRNAs were incubated with IM-9 myeloma cells and subsequently tested for their ability to modulate response by NKL effector cells (an IL-2 dependent human NK cell line). The top 10 percentile of shRNAs inducing increased secretion of interferon-gamma (INF-γ) from NKL cells was identified. To reduce the likelihood of off-target effects, only genes that were positive for 2 or more independent shRNAs were selected for further analysis. Among the genes that increased target cell susceptibility to NK activity we found 2 members of the Jak family (Jak1 and Jak2) with Jak1 being one of the strongest hits in our screen. IM-9 myeloma cells with stable expression of at least 2 independent shRNAs targeting Jak1 and Jak2 were established and tested for their sensitivity to NKL, NK92 or primary NK cells using INF-γrelease and 51Cr release cytotoxicity assays. Stable suppression of both Jak1 and Jak2 in IM-9 cells induced a significant increase of INF-γsecretion from NK cells and increased sensitivity in cytotoxicity assay when compared to parental IM-9 or cells expressing irrelevant shRNAs. Western blot analysis showed a selective decrease of Jak1 and Jak2 protein in IM-9 cells expressing specific shRNAs but not irrelevant shRNAs. While target cells with reduced expression of Jak1 and Jak2 were more susceptible to NK cell activity, no effect was observed when Jak3 and TYK2 were silenced. We then tested the NK susceptibility of different tumor cell lines with reduced expression of Jak1 and Jak2. Seven additional tumor cell lines representing other hematologic malignancies expressing Jak1 and Jak2 shRNAs were established: myeloma (KM12BM), chronic myeloid leukemia (K562), Burkitt's lymphoma (Daudi), acute myeloid leukemia (U937, ML2 and KG1) and acute T cell leukemia (Jurkat). These experiments confirmed that Jak1 silencing can induce increased susceptibility to NK cell activity. However, this effect was more pronounced in some cases (IM-9, KM12BM, U937, KG1) compared to other cell lines where this effect was limited (K562, ML-2, Jurkat) or absent (Daudi). To investigate the mechanism for modulation of target cell susceptibility to NK cells by Jak1, we compared gene expression profiles of IM-9-Jak1-KO with IM-9 parental cells and IM-9 cells infected with an irrelevant shRNA. No difference in expression was found for ligands of activating NKG2D receptors (MICA, MICB, ULPB1, 2, 3) or ligands for NK inhibitory receptors (HLA Class I genes A, B, C, E). One of the most up-regulated genes in IM-9-Jak1-KO cells was TNFSFR10A (TRAIL-R1), a gene that is known to induce apoptotic signals upon TRAIL engagement. In contrast, FAIM3, an inhibitor of FAS (CD95) signaling, was significantly down-regulated. IM-9-Jak1-KO cells also over-expressed several GALNT genes, recently shown to be markers of TRAIL sensitivity. These results suggest that Jak1 and possibly Jak2 can modulate susceptibility of some tumor cells to NK cell lysis. The mechanism for this effect appears to be at least partly through increased sensitivity to engagement of the TRAIL/FAS extrinsic apoptotic pathway. Disclosures:No relevant conflicts of interest to declare.

KIR2DS4 is a product of gene conversion with KIR3DL2 that introduced specificity for HLA-A*11 while diminishing avidity for HLA-C

Human killer cell immunoglobulin-like receptors (KIRs) are distinguished by expansion of activating KIR2DS, whose ligands and functions remain poorly understood. The oldest, most prevalent KIR2DS is KIR2DS4, which is represented by a variable balance between “full-length” and “deleted” forms. We find that full-length 2DS4 is a human histocompatibility leukocyte antigen (HLA) class I receptor that binds specifically to subsets of C1+ and C2+ HLA-C and to HLA-A*11, whereas deleted 2DS4 is nonfunctional. Activation of 2DS4+ NKL cells was achieved with A*1102 as ligand, which differs from A*1101 by unique substitution of lysine 19 for glutamate, but not with A*1101 or HLA-C. Distinguishing KIR2DS4 from other KIR2DS is the proline–valine motif at positions 71–72, which is shared with KIR3DL2 and was introduced by gene conversion before separation of the human and chimpanzee lineages. Site-directed swap mutagenesis shows that these two residues are largely responsible for the unique HLA class I specificity of KIR2DS4. Determination of the crystallographic structure of KIR2DS4 shows two major differences from KIR2DL: displacement of contact loop L2 and altered bonding potential because of the substitutions at positions 71 and 72. Correlation between the worldwide distributions of functional KIR2DS4 and HLA-A*11 points to the physiological importance of their mutual interaction.

Tyrosine nitration in the human leucocyte antigen‐G‐binding domain of the Ig‐like transcript 2 protein

Ig-like transcript 2 (ILT2) is a suppressive receptor that participates in the control of the autoimmune reactivity. This action is usually carried out in a proinflammatory microenvironment where there is a high production of free radicals and NO. However, little is known regarding whether these conditions modify the protein or affect its suppressive functions. The present study aimed to investigate the suppressive response of the ILT2 receptor under oxidative stress. To address this topic, we treated the ILT2-expressing natural killer cell line, NKL, with the NO donor N-(4-[1-(3-aminopropyl)-2-hydroxy-2-nitrosohydrazino]butyl)propane-1,3-diamine (DETA-NO). We observed that DETA-NO caused ILT2 protein nitration. MS analysis of the chimeric recombinant human ILT2-Fc protein after treatment with the peroxynitrite donor 3-(morpholinosydnonimine hydrochloride) (SIN-1) showed the nitration of Tyr35, Tyr76 and Tyr99, which are involved in human leucocyte antigen-G binding. This modification is selective because other Tyr residues were not modified by SIN-1. Recombinant human ILT2-Fc treated with SIN-1 bound a significantly higher quantity of human leucocyte antigen-G than untreated recombinant human ILT2-Fc. DETA-NO did not modify ILT2 mRNA expression or protein expression at the cell surface. Preincubation of NKL cells with DETA-NO decreased the cytotoxic lysis of K562-human leucocyte antigen-G1 cells compared to untreated NKL cells (P < 0.05) but increased cytotoxicity against K562-pcDNA cells (P < 0.05). Intracellular tyrosine phosphorylation produced after human leucocyte antigen-G binding was not affected by DETA-NO cell pretreatment. These results support the hypothesis that the ILT2human leucocyte antigen-G interaction should have a central role in tolerance under oxidative stress conditions when other tolerogenic mechanisms are inhibited.

The Traffic of the NKG2D/Dap10 Receptor Complex during Natural Killer (NK) Cell Activation

NKG2D is an important activating receptor for triggering the NK cell cytotoxic activity, although chronic engagement of specific ligands by NKG2D is also known to provoke decreased cell surface expression of the receptor and compromised NK cell function. We have studied the dynamics of surface NKG2D expression and how exposure to the specific ligand major histocompatibility complex class I chain-related molecule B (MICB) affects receptor traffic and fate. While in the NKL cell line and "resting" NK cells NKG2D was found principally at the cell surface, in activated primary NK cells an intracellular pool of receptor could also be found recycling to the plasma membrane. Exposure of NK cells to targets expressing MICB resulted in degradation of approximately 50% of total NKG2D protein and lysosomal degradation of the DAP10 adaptor molecule. Consistent with these observations, confocal microscopy experiments demonstrated that DAP10 trafficked to secretory lysosomes in both transfected NKL cells and in activated primary NK cells upon interaction with MICB-expressing target cells. Interestingly, polarization to the synapse of secretory lysosomes containing DAP10 was also observed. The implications of the intracellular traffic of the NKG2D/DAP10 receptor complex for NK cell activation are discussed. We propose that the rapid degradation of NKG2D/DAP10 observed coincident with recruitment of the receptor to the cytotoxic immune synapse may explain the loss of NKG2D receptor expression after chronic exposure to NKG2D ligands.

MicroRNA-30c is down-regulated during NKL cell activation and cytotoxicity induced by CD226 crosslinking (136.20)

Abstract MicroRNAs have been implicated as negative regulators controlling diverse biological processes at the level of posttranscriptional repression. There have been little report on NK cell associated miRs until now. CD226 is a kind of activation receptors located on NK cells and involved in the activation and cytotoxixity of NK cells. Previous study showed that CD226 cross-linking increased the CD107a expression on NKL cells. In the present study, we identified miRs induced in NKL cells after exposure to CD226 crosslinking using microarray and quantitive RT-PCR technology. It has been found that miR-30c level was down-regulated in NKL cells after CD226 crosslinking. Furthermore transfection of miR-30c into NKL cells decreased the CD107a expression on NKL cells induced by CD226 crosslinking, suggesting that miR-30c plays a role in NKL activation and cytotoxicity induced by CD226 crosslinking. Meanwhile miR-30c was predicted to base-pair with sequences in the 3' UTRs of the HMBOX gene. Altogether, our studies propose that miR-30c may play a role in control of CD226 signalling in NKL cells through a negative feedback regulation loop involving in down-regulation of HMBOX expression.

Intratumoral (IT) injection of hu14.18-IL2 immunocytokine (IC) enhances antitumor activity and augments NK infiltration into tumors: analyses of cellular internalization of hu14.18-IL2 by human NK cells (41.44)

Abstract Hu14.18-IL2 (EMD 273063) IC, consisting of GD2-specific mAb and IL2, is in trials for treatment of melanoma and neuroblastoma. Preclinical studies showed augmented antitumor activity when IC is injected IT vs. IV. Human IL2R+CD16+NK cells mediate ex vivo ADCC in the presence of IC that binds them to GD2+ tumor cells. We studied the kinetics of IC presence on the surface of human M21 melanoma and CD25+CD16-NK cell lines, NKL and RL12, after IC binding to the cells via GD2 and IL2R, respectively. For NK cells, ~50% of IC was internalized by 6 h, and ~90% by 24 h of cell culture. Excess free IL2 in the culture did not slow internalization kinetics for these NK cells. IL2-deprivation for 24 h prior to IC arming of NK cells decreased IL2Rα expression and overall IC binding but did not affect internalization kinetics of the bound IC. For NK cells that were prelabeled with IC, the decrease of surface IC levels over 24 h correlated with reduction of ADCC in vitro. Unlike NK cells, M21 cells retained ~70% of IC on the surface following 24 h of culture. When CFSE-labeled NKL cells were injected IV into M21-bearing SCID mice, they migrated better into the tumor when IC was delivered IT than when they were pre-armed with IC before implantation. These preliminary studies suggest that once IC gets to tumor sites, it may stay on the tumor surface for a prolonged time, and that IT injections of IC enhance the recruitment of NK cells to the tumor site.

Human IgG1 antibodies antagonizing activating receptor NKG2D on natural killer cells

NKG2D is a surface receptor expressed on NK cells but also on CD8+ T cells, γδ T cells, and auto-reactive CD4+/CD28- T cells of patients with rheumatoid arthritis. Various studies suggested that NKG2D plays a critical role in autoimmune diseases, e.g., in diabetes, celiac disease and rheumatoid arthritis (RA), rendering the activating receptor a potential target for antibody-based therapies. Here, we describe the generation and characteristics of a panel of human, high-affinity anti-NKG2D IgG1 monoclonal antibodies (mAbs) derived by phage display. The lead molecule mAb E4 bound with an affinity (KD) of 2.7 ± 1.4 x 10-11 M to soluble and membrane-bound human NKG2D, and cross-reacted with NKG2D from cynomolgus macaque, indicating potential suitability for studies in a relevant primate model. MAb E4 potently antagonized the cytolytic activity of NKL cells against BaF/3-MICA cells expressing NKG2D ligand, and blocked the NKG2D ligand-induced secretion of TNFα, IFNγ and GM-CSF, as well as surface expression of CRTAM by NK cells cultured on immobilized MICA or ULBP-1 ligands. The antibody did not show a detectable loss of binding to NKG2D after 7 days in human serum at 37°C, and resisted thermal inactivation up to 70°C. Based on these results, anti-human NKG2D mAb E4 provides an ideal candidate for development of a novel therapeutic agent antagonizing a key receptor of NK and cytotoxic T cells with implications in autoimmune diseases.

NKG2D Ligands Expression and NKG2D-Mediated NK Activity in Sezary Patients

Sezary syndrome (SS) is a rare lymphoma characterized by the clonal expansion in the skin and in blood of CD4(+)CD158k(+) T cells. Natural killer (NK) activation against tumors in leukemia models is partly based on the recognition of the target through the NKG2D/NKG2D ligands interactions. We analyzed ex vivo SS malignant lymphocytes for the expression of the NKG2D ligands such as the major histocompatibility complex class I-related molecules (MIC) A and B and the UL16 binding proteins (ULBP). The expressions of NKG2D, the natural cytotoxicity receptors (NKp30, NKp44, and NKp46) and the activating receptor DNAM-1 were simultaneously investigated on circulating patients NK and CD8(+) nonmalignant lymphocytes. Interestingly, although at least one of the NKG2D ligands was expressed on the circulating malignant lymphocytes of 9 out of 10 patients, NKG2D was expressed on effector lymphocytes. We found that soluble MICA in patient's sera was increased, which may constitute a mechanism to escape the immune response. In vitro, SS tumor lymphocytes induced the degranulation of perforin by the NKL cell line. More importantly, NKG2D expressed on SS patients NK cells is functional and capable to induce degranulation. Altogether, these data could suggest that stimulating NK function in SS patients may be a promising strategy to reduce tumor invasion.