NK-92MI Cell Research Articles

Magnetic Nanoparticles Attached to the NK Cell Surface for Tumor Targeting in Adoptive Transfer Therapies Does Not Affect Cellular Effector Functions.

Adoptive cell transfer therapy is currently one of the most promising approaches for cancer treatment. This therapy has some limitations, however, such as the dispersion of in vivo-administered cells, causing only a small proportion to reach the tumor. Nanotechnological approaches could offer a solution for this drawback, as they can increase cell retention and accumulation in a region of interest. In particular, strategies employing magnetic nanoparticles (MNPs) to improve targeting of adoptively transferred T or NK cells have been explored in mice. In vivo magnetic retention is reported using the human NK cell line NK-92MI transfected with MNPs. Primary NK cells are nonetheless highly resistant to transfection, and thus we explore in here the possibility of attaching the MNPs to the NK cell surface to overcome this issue, and examine whether this association would affect NK effector functions. We assessed the attachment of MNPs coated with different polymers to the NK cell surface, and found that APS-MNP attached more efficiently to the NK-92MI cell surface. In association with MNPs, these cells preserved their main functions, exhibiting a continued capacity to degranulate, conjugate with and lyse target cells, produce IFN-γ, and respond to chemotactic signals. MNP-loaded NK-92MI cells were also retained in an in vitro capillary flow system by applying an EMF. A similar analysis was carried out in primary NK cells, isolated from mice, and expanded in vitro. These primary murine NK cells also maintained their functionality intact after MNP treatment and were successfully retained in vitro. This work therefore provides further support for using MNPs in combination with EMFs to favor specific retention of functional NK cells in a region of interest, which may prove beneficial to adoptive cell-therapy protocols.

Focused Ultrasound Improves NK-92MI Cells Infiltration Into Tumors.

The efficiency of natural killer (NK) cells, adoptively transferred, for treatment against solid tumors is hindered by their difficulty to enter tumors from the blood circulation as well as their inability to prolong viability in the absence of IL-2. Among different sources of NK cells, we used genetically modified NK-92MI cells, a suitable candidate which can release IL-2 to maintain their viability and overcome undesirable side effects caused by systemic administration of exogenous IL-2. In this study, we evaluated whether the combination of focused ultrasound (FUS) and microbubbles can improve adoptively NK-92MI cell infiltration into ovarian tumors through biodistribution, immunofluorescence, and flow cytometry. The treatment effects of using this strategy twice a week were explored. The potential molecular mechanism of FUS assisting NK cell therapy was also initially explored through evaluating the expression of ICAM1 and CX3CL1 by qRT-PCR. Our results indicated that FUS and microbubbles can improve NK-92MI cells’ infiltration into tumors, and the combination of FUS and NK-92MI cells had a better treatment effect compared to the PBS group, but not compared to the NK-92MI group. The qRT-PCR results also showed that CX3CL1 may be involved in the process of FUS-assisted NK cell infiltration. These results indicate that further optimization of the FUS-assisted strategy is still needed to achieve therapeutic benefit.

Enhancement of natural killer cell cytotoxicity by using static magnetic field to increase their viability

ABSTRACTNatural killer (NK) cells are innately immune to the body’s immune system and can actively recognize and kill cancer cells. This study explores the potential for enhancing the killing ability of NK cells by co-culturing the NK cells with the target cells under a static magnetic field (SMF). In this study, NK92-MI cell lines were cultured in the presence of a 0.4-T SMF. The effect of the SMF on NK cell viability was evaluated by means of an MTT assay. Culturing tests were performed with inhibitors of the DAG/IP3, STAT3, ERK, JNK and p38 pathways in order to examine the possible signaling cascade responsible for the SMF effect on the NK92-MI cell viability. Finally, the effect of the SMF on the cytotoxicity of the NK92-MI cells was evaluated by co-culturing the NK cells with K562 leukemia cell lines. The results showed that the application of a 0.4-T SMF significantly increased (p < 0.05) the viability of the NK92-MI cells. Furthermore, the inhibitor tests indicated that the SMF affected cell viability by activating multiple MAPK signaling pathways (ERKs, JNKs, and p38-MAPK). Finally, SMF pre-exposure for 48 hr significantly improved the killing activity of the NK92-MI cells (p < 0.05). That is, pre-exposure to SMF increased the viability of the NK92-MI cells and improved their killing ability against K562 tumor cells. In general, the present results suggest that NK cells pre-exposed to 0.4-T SMF show potential as a tool for immune-therapy treatment of cancer.

Natural killer cell–mediated anticancer effects of an arabinogalactan derived from rice hull in CT26 colon cancer–bearing mice

Rice hull polysaccharides (RHPS) have been reported to activate innate immunity in mice. This study investigated the effects of RHPS on natural killer (NK) cell–mediated cytotoxicity in vitro and the possible underlying anticancer mechanisms in vivo. The results showed that sustained exposure to RHPS increased NK-92MI cell–mediated cytotoxicity in a time- and concentration-dependent manner. In addition, RHPS upregulated the expression of Fas ligand, TNF-related apoptosis-inducing ligand, perforin, and granzyme B of NK-92MI cells and induced the secretion of IFN-γ and TNF-α. In the in vivo experiment, colon cancer CT26–bearing mice were used to investigate the effects of RHPS in cytotoxicity and anticancer. The results revealed that RHPS inhibited cancer weight and volume in CT26-bearing mice and significantly upregulated splenic cytotoxicity and NK-cell population. Moreover, RHPS treatment increased NK-cell infiltration in tumors. Thus, RHPS can enhance NK-cell activation in vivo and in vitro, thereby exhibiting anticancer activity.

Application of Chimeric Antigen Receptor-Modified NK Cells in Multiple Myeloma

To explore the killing effect of CAR (CD138-CD28-CD3ζ)-NK cells on myeloma cells through construction of CAR(CD138-CD28-CD3)-NK cells. The antiCD138scFv-CD28-CD3 zeta plasmid pcDNA3.1 was constructed, which then together with 3 plasmid lentiviral packaging system were transfected into 293T cells, the virus was collected. Furthermore, in order to get the stably transfected cell line, the NK92MI cell line was infected by the virus, then the positive cells were screened by puromycin. The expression of the CARNK cells were verified by RT-PCR and Western blot. At last the ability of secreting cytokine CD107a was detected by flow cytometry, and the statistical analysis was carried out to verify the anti-myeloma effect of CAR-NK cells. Gene fragment of the CAR(antiCD138scFv-CD28-CD3ζ) was constructed successfully by gene engineering technique in vitro, and the gene sequence was verified to be correct by sequencing. By virus packaging technology, the virus expressing the protein of the CAR was obtained. PCR and Western blot verified the expression of CAR fusion protein on the sufurce of NK cells. The cell killing experiment confirmed that the CAR-NK cells possessed the ability to secrete cytokine CD107a superior to control cells and showed the obvious killing effect on multiple myeloma cells. The CAR can be constructed in vitro, and express on NK92 cells. The CAR-NK cells can kill the multiple myeloma cells expressing CD138 antigen, thereby plays an antimyeloma effect.

A Developed NK-92MI Cell Line with Siglec-7neg Phenotype Exhibits High and Sustainable Cytotoxicity against Leukemia Cells.

Altered sialic acid processing that leads to upregulation of cell surface sialylation is recognized as a key change in malignant tissue glycosylation. This cancer-associated hypersialylation directly impacts the signaling interactions between tumor cells and their surrounding microenvironment, especially the interactions mediated by immune cell surface sialic acid-binding immunoglobulin-like lectins (Siglecs) to relay inhibitory signals for cytotoxicity. First, we obtained a Siglec-7neg NK-92MI cell line, NK-92MI-S7N, by separating a group of Siglec-7neg cell population from an eight-month-long-term NK-92MI in vitro culture by fluorescence-activated cell sorting (FACS). The effect of Siglec-7 loss on NK-92MI-S7N cells was characterized by the cell morphology, proliferation, and cytotoxic activity via FACS, MTS assay, cytotoxic assay, and natural killer (NK) degranulation assay. We found the expression levels of Siglec-7 in NK-92MI were negatively correlated with NK cytotoxicity against leukemia cells. This NK-92MI-S7N cell not only shared very similar phenotypes with its parental cells but also possessed a high and sustainable killing activity. Furthermore, this Siglec-7neg NK line was unexpectedly capable of eliminating a NK-92MI-resistant leukemia cell, THP-1, through enhancing the effector-target interaction. In this study, a NK cell line with high and sustainable cytotoxicity was established and this cell may provide a potential application in NK-based treatment for leukemia patients.

Effects of total flavonoids of sea buckthorn ( Hippophae rhamnoides L.) on cytotoxicity of NK92-MI cells.

Sea buckthorn (Hippophae rhamnoides L.) has multifarious medicinal properties including immunoregulatory effect. The total flavonoids of Hippophae rhamnoides L. (TFH) are the main active components isolated from berries of sea buckthorn. The aim of this study was to evaluate the effects of TFH on the cytotoxicity of NK92-MI cells and its possible mechanisms. NK92-MI cells were treated with TFH (2.5 or 5.0 mg/L) or phosphate-buffered saline (PBS) for 24 h, the cytotoxicity against K562 was detected by measuring the release of lactate dehydrogenase (LDH), expression levels of NCRs (NKp30, NKp44, NKp46) and NKG2D were detected by flow cytometry, and expression levels of perforin and granzyme B were detected by western blot. Cytokine Antibody Arrays with 80 cytokine proteins were used to profile the effect of TFH on cytokines. Western blot was adopted to detect the effects of TFH on STAT1, STAT4, and STAT5 signal pathway. Compared with the normal control group, TFH could significantly enhance NK92-MI cell cytotoxicity against K562 cells, upregulate expressions of NKp44, NKp46, perforin, and granzyme B. TFH could upregulate expressions of IL-1α, IL-2, IL-7, IL-15, CSF-2, CSF-3, MCP-1, MIG, IFN-γ, TNF-α, and TNF-β and downregulate expressions of IL-16, MIP-1β, CX3CL-1, and MIF. TFH could increase expressions of phospho-STAT1 and phospho-STAT5. The results suggest that TFH stimulated NK92-MI cells to activate and enhance cytotoxicity of NK92-MI cells.

Chromatin state dynamics during NK cell activation.

Studies of Natural Killer (NK) cell cytotoxicity have mainly focused on the balance of activating and inhibitory receptors, signaling transduction, calcium influx, formation of immune synapse, and cytolytic degranulation. However, little is known about the chromatin state of NK cells and the impact of its changes during target recognition. In this study, we investigate the contribution of chromatin state dynamics during NK cell activation by comprehensively analyzing a set of microarray data and two sets of Chromatin Immunoprecipitation-Sequencing (ChIP-seq) data. We find that the expression of several histone demethylases and methyltransferases was influenced upon stimulation. Furthermore, we notice that a series of genes, including PI3KCA, NFATC1and TNFSF9, which play important roles during NK cell activation, were at ‘poised’ state prior to activation, and that modifications of H3K4me3 and H3K27me3 on these promotors were sensitive to stimulation with Phorbol Myristate Acetate (PMA) and Ionomycin (Iono) in the NK92MI cell line. Finally, we demonstrate that a series of small molecule inhibitors, which are specific to H3K4 and H3K27 modification, enhance degranulation or the expression levels of IFN-γ and TNF-α. Our results suggest that the histone modification state has a profound impact on NK cell activation, and provide novel insights into the regulation of NK cellular cytotoxicity and immunoregulatory function by chromatin state dynamics.

Natural Killer Cell Cytotoxicity Against SKOV3 after HLA-G Downregulation by shRNA.

HLA-G is a nonclassical HLA class I molecule which, when elevated in tumor cells, is one of the main factors involved in tumor evasion of immune responses including NK and T cells. To evaluate the effect of HLA-G downregulation on NK cell cytotoxicity in tumor cell lines. The expression level of HLA-G was measured by real-time PCR and flowcytometry after transfection of SKOV3 by shRNA.1, which targets specific sequences in exon 4, or shRNA.2, which targets both exons 4 and 6. NK-92MI cell cytotoxicity against transfected or untransfected target cell lines was measured with the lactate dehydrogenase (LDH) release assay. The Jeg-3 cell line was used as a positive control. Membrane-bound HLA-G expression levels decreased significantly in both cell lines after transfection with both shRNAs compared to their corresponding untransfected cells (p<0.05). Jeg-3 cells were better lysed than SKOV3 cells by NK cells during the first 48 h after transfection with both shRNAs. Compared to untransfected cells, shRNA.1-transfected SKOV3 cells were significantly more lysed by NK cells 24 h post-transfection (p=0.043). As a clinical approach, HLA-G downregulation with shRNA may be effective in cancer therapy by improving immune cell activation.

Immunologic Function and Molecular Insight of Recombinant Interleukin-18.

In recent years, cytokine-mediated therapy has emerged as further advance alternative in cancer therapy. Interleukin-18 (IL-18) has exhibited interesting anti-cancer properties especially when combined with IL-12. We engineered IL-18 in order to improve its activity using single point mutagenesis. IL-18 mutants were constructed according to binding residues and polarity which we tried to increase polarity in M33Q and M60Q, enhanced cationicity in E6K, and flexibility in T63A. All IL-18 proteins were expressed in Pichia pastoris, purified, and then measured the activity by treating with the NK-92MI cell line to evaluate interferon-γ (IFN-γ) stimulation. The E6K and T63A mutant forms showed higher activity with respect to native proteins at the concentration of 200 ng mL-1 by inducing the expression of IFN-γ, about factors of 9 and 4, respectively. Meanwhile, M33Q and M60Q had no significant activity to induce IFN-γ. Interestingly, the combination of E6K and T63A mutations could synergize the induction activity of IL-18 to be 16 times at 200 ng mL-1. Furthermore, molecular dynamics studies have elucidated the effect due to mutation on conformation of the binding site of IL-18. The results turn out that E6K provides structural perseverance against mutation, while M33Q and M60Q promote vivid overall change in protein conformation, especially at the binding site. For T63A, mutation yields small difference in structure but clearly increases structural flexibility. However, a small structural change was observed when T63A was combined with E6K. Our research resulted in a novel version of IL-18 which could be a new key candidate for cytokine-mediated therapy.

Aryl hydrocarbon receptor activation and IL-2 have synergistic effects that enhance human natural killer cell cytotoxicity.

Abstract The aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor. Initial studies of AHR concentrated on its role in the detoxification of xenobiotics. However, recent research has focused on its role in the immune system. AHR has been shown to be involved in the differentiation and maintenance of different immune cell subsets. Little is known about how AHR regulates immune cell effector function. In this study, we were able to show that AHR activation significantly increased IL-2-producing human NK-92MI cell degranulation capacity and cytotoxicity. However, for the FACS-sorted primary human NK cells from blood donor PBMC, AHR activation alone had no influence on NK cell degranulation and cytotoxic functions. We observed similar effector function enhancement of primary NK cells only with the addition of AHR agonist 6-Formylindolo (3,2-b)carbazole (FICZ) in conjunction with IL-2. Moreover, AHR activation significantly increased signaling factor MAPK1 expression in the NK-92MI cells, but not MAPK3 and NF-κB. Surprisingly, FICZ stimulation had no direct impact on human NK cell TNFα production, pre-formed proteins Perforin and Granzyme B production, as well as IL-2 receptor expression, but inhibited IFN-γ production slightly. In summary, AHR agonist FICZ acted synergistically with IL-2 to enhance human NK cell degranulation capacity and cytotoxicity function, possibly through activation of the MAPK1 signaling pathway which is involved in the polarization process of microtubule organizing center(MTOC) and lytic granules, a prerequisite for killing the target. Overall, the modulation of human NK cell response from AHR activation has revealed its potential therapeutic value.

Clinically applicable magnetic-labeling of natural killer cells for MRI of transcatheter delivery to liver tumors: preclinical validation for clinical translation.

To test the hypothesis that MRI can monitor intraportal vein (IPV) transcatheter delivery of clinically applicable heparin-protamine-ferumoxytol (HPF) nanocomplex-labeled natural killer (NK) cells to liver tumor. Liver tumor rat models underwent catheterization for IPV infusion of HPF-labeled NK cells (NK-92MI cell line). MRI measurements within tumor and adjacent liver tissues were compared pre- and post-NK cell infusion. Histology studies were used to identify NK cells in the target tumors. For first time, we demonstrated that MRI tracks HPF-labeled NK cells migration within liver following IPV delivery. IPV transcatheter infusion permitted selective delivery of NK cells to liver tissues and MRI allowed tracking NK cell biodistributions within the tumors.

Effect of carbamate pesticides on perforin, granzymes A-B-3/K, and granulysin in human natural killer cells.

We previously found that ziram, a carbamate pesticide, significantly reduced perforin, granzyme A (GrA), granzyme B (GrB), granzyme 3/K (Gr3/K), and granulysin (GRN) levels in NK-92MI cells, a human natural killer (NK) cell line. To investigate whether other carbamate pesticides also show similar toxicity on human NK cells, we conducted further experiments with NK-92CI cells, a human NK cell line, using a more sensitive assay. We previously confirmed that NK-92CI cells express CD56, perforin, GrA, GrB, Gr3/K, and GRN and are highly cytotoxic to K562 cells in a chromium release assay, which are more sensitive to organophosphorus pesticides and ziram than the NK-92MI cell line. NK-92CI cells were treated with ziram, thiram, maneb, or carbaryl at various concentrations for 4-24 h at 37°C in vitro. Thereafter, intracellular levels of perforin, GrA, GrB, Gr3/K, and GRN were determined by flow cytometry. It was found that all carbamate pesticides significantly reduced the intracellular levels of perforin, GrA, GrB, Gr3/K, and GRN in NK-92CI cells in a dose-dependent manner. However, the strength of the effect differed among the pesticides, and the order was thiram > ziram > maneb > carbaryl. In addition, it was also found that the degree of the reductions differed among the five proteins, with perforin more sensitive to pesticides than GRN, GrA, GrB, and Gr3/K, and the order was perforin > GRN > Gr3/K ≒ GrA ≒ GrB.

Vanadium pentoxide prevents NK-92MI cell proliferation and IFNγ secretion through sustained JAK3 phosphorylation

Vanadium is a major air pollutant with toxic and carcinogenic effects; it also exercises immunosuppressive effects on the adaptive immune response. Its effect on the innate immune response is poorly explored. The aim of this study was to identify if vanadium pentoxide (V2O5) impairs the function of immunoregulatory NK cells and to determine possible mechanisms associated with this effect. Interleukin-2-independent NK-92MI cells were exposed to different V2O5 concentrations for 6, 12, or 24 h periods. Cell proliferation was then evaluated using CFSE staining, apoptosis by Annexin V binding, and necrosis by 7-AAD staining. The release of IL-2, -4, -6, -10, -17A, IFNγ, and TNFα by the cells were assessed using a human CBA kit. Expression of CD45, SOCS1, JAK3, pJAK3, STAT5, pSTAT5, IL-2R, IL-15R, Fas, and FasL in/on the cells was determined by flow cytometry; JAK3 and pJAK3 expression were also evaluated via confocal microscopy. The results indicated that V2O5 could inhibit NK-92MI cell proliferation and induce cell apoptosis in a dose- and time-related manner. V2O5 also inhibited IL-2, IL-10, and IFNγ secretion but mostly only after 24 h of exposure and with primarily the higher doses tested. V2O5 had no effect on expression of JAK3 and STAT5, but did cause an increase in pJAK3 and appeared to lead (trend) to reductions in levels of phosphorylated STAT5. V2O5 increased the expression of IL-2R, IL-15R, Fas, and FasL at concentrations above the 50–100 µM range. V2O5 had no effect on expression of the CD45 membrane phosphatase, but it did cause an increase in the expression of SOCS1. These results indicate that a key toxic effect of V2O5 on NK cells is a dysregulation of signaling pathways mediated by IL-2. These effects could help to explain the previously-reported deleterious effects on innate immune responses of hosts exposed to inhaled V2O5.

Co-culture with NK-92MI cells enhanced the anti-cancer effect of bee venom on NSCLC cells by inactivation of NF-κB

In the present study we experimented on a multimodal therapeutic approach, such as combining chemotherapy agent (Bee venom) with cellular (NK-92MI) immunotherapy. Previously bee venom has been found to show anti-cancer effect in various cancer cell lines. In lung cancer cells bee venom showed an IC(50) value of 3 μg/ml in both cell lines. The co-culture of NK-92MI cell lines with lung cancer cells also show a decrease in viability upto 50 % at 48 h time point. Hence we used bee venom treated NK-92MI cells to co-culture with NSCLC cells and found that there is a further decrease in cell viability upto 70 and 75 % in A549 and NCI-H460 cell lines respectively. We further investigated the expression of various apoptotic and anti-apoptotic proteins and found that Bax, cleaved caspase-3 and -8 were increasing where as Bcl-2 and cIAP-2 was decreasing. The expression of various death receptor proteins like DR3, DR6 and Fas was also increasing. Concomitantly the expression of various death receptor ligands (TNFalpha, Apo3L and FasL) was also increasing of NK-92MI cells after co-culture. Further the DNA binding activity and luciferase activity of NF-κB was also inhibited after co-culture with bee venom treated NK-92MI cell lines. The knock down of death receptors with si-RNA has reversed the decrease in cell viability and NF-κB activity after co-culture with bee venom treated NK-92MI cells. Thus this new approach can enhance the anti-cancer effect of bee venom at a much lower concentration.

NK Cell Mediated Cytotoxicity Against Malignant Promyelocytes Enhanced By Arsenic Trioxide: Potential Clinical Relevance

Abstract NK cells are primary effectors of the innate immune response against cells that have undergone malignant transformation. Several lines of evidence indicate that the expression level of NK ligands on leukemic cells affects the sensitivity of the leukemic cells to cytolytic activity by NK cells. Various agents have been evaluated for their ability to induce these ligands on leukemic cells to augment the NK cell mediated anti-leukemia effect. There is substantial evidence that has established the importance of the adaptive immune system in the treatment of acute promyelocytic leukemia (APL) (Rose Ann Padua et al. Nat Med 2003). While there is significant data which address the mechanisms of arsenic trioxide (ATO) on malignant promyelocytes, limited data is available of its effect on the innate and adaptive immune system. We undertook a series of experiments to address the impact of ATO on NK cell receptor and malignant promyelocyte ligand expression and its effect on NK cell mediated cytotoxicity. We also evaluated NK cell reconstitution in patients treated with ATO and the impact of KIR genotypes on relapse. We first evaluated the cytotoxic activity of NK92MI (NK cell line) against 5 different myeloid (K562, U937, HL60, UF1, NB4) and 2 lymphoid cell lines (Jurkat E6.1, SUP-B15) by CFSE/ 7AAD cytotoxicity assay. Target (T) cells (1x 105/100 µL/well) pre-treated with CFSE were co-cultured with effector NK cells (E) at a E:T ratio of 1:1, 2:1 and 5:1 for 5 hours at 37°C in 96 well plates. The percentage cytolytic activity of the NK cells was then calculated after adding 7AAD and acquired in FACS Calibur (Becton Dickinson, San Jose, CA, USA). Significant cytolytic activity was noted against K562 and NB4 cell lines. At the highest E:T ratio there was a median 22% cytolytic activity against NB4 (N=5). We observed that NB4 when treated overnight with 1µM ATO (&gt;99% viability retained after this exposure) significantly increased the cytotoxic effect of NK92MI cell line at all the E:T ratios as shown in figure 1A (n=5; P=0.0023). No other cell line showed a similar increase in cytotoxic effect following exposure to ATO at these concentrations (data not shown). We next evaluated the effect of exposure of NB4 cells to ATO at 1µM for 6 hours on NK ligand expression by flowcytometry. As shown in figure 1B there was a significant increase in activating ligand MICA/B in NB4 cell lines (n=3; P=0.016) which was not seen in any of the other cell lines. Similar significant increased expression of Nectin-2 (DNAM-1 ligand) and HLA Class I was seen. Exposure of NK92MI to ATO for 6 hours at 1uM (non cytotoxic dose:IC50-3.8uM) resulted in increased expression of activating receptors NKG2D, NKP30 and KIR2DS4 (figure 1C) and inhibitory receptor NKG2A and decrease in inhibitory receptors KIR3DL1/DL2. There were no changes in the expression of NKP46, KIR2DL1, KIR2DL2 and DNAM1 receptors. We undertook a prospective study to evaluate the pattern of NK (CD56+CD3-) reconstitution in patients with newly diagnosed APL treated at our center with a single agent ATO regimen (Mathews et al. JCO 2011). The mean NK cell counts in patients were below the 2SD deviation level of the normal range even after completion of therapy (approximately a year)(figure 1D). All other subsets evaluated (CD4, CD8, CD3, CD19, CD56+CD3+, CD4CD45RO) had returned to levels within the normal range by the end of consolidation therapy (approximately 3 months from diagnosis). KIR genotyping was done on 55 patients with APL who received treatment with single agent ATO based regimen. The median follow up of this cohort was 20 months and 14 cases relapsed following initial therapy. The presence or absence of 17 KIR genes was done by PCR-SSP method (KIR Typing kit, Miltenyi Biotech Inc, CA). There was no association with any specific genotype or haplotype with risk of relapse. In summary we have noted that there is up regulation of receptors on NK cells and ligands on malignant promyelocytes following exposure to ATO that favors NK cell mediated cytotoxicity. In-vitro we have demonstrated a significant increase in NK cell mediated cytolytic activity against malignant promyelocytes exposed to ATO even at relatively low E:T ratios. This could be an important mechanism by which ATO induces durable remissions in patients with APL. The delayed NK cell recovery following treatment with ATO raises the possibility of using NK cell therapy to augment the effect of ATO in the treatment of patients.Figure 1Figure 1. Disclosures: No relevant conflicts of interest to declare.

NK cells extract HLA-G1 antigen from K562-G1 cells via trogocytosis

Objective To investigate the mechanism of acquisition of HLA-G1 antigen by NK cells.Methods K562 cells stably expressing HLA-G1 antigen （K562-G1） were constructed.K562-G1 cells, K562 cells and shed HLA-G1 were respectively co-cultured with NK-92MI cells to observe the acquisi-tion of HLA-G by NK cells.To further investigate the mechanism , NK-92MI cells with blockage HLA-G re-ceptors were further co-cultured with K562-G1 cells and HLA-G1 proteins expressing on K 562-G1 cells were blocked and then co-cultured with NK-92MI cells. Acquisition of HLA-G 1 by NK-92MI cells was analyzed by flow cytometry and fluorescence microscopy .The effects of HLA-G1 expression on the cytotoxicity of NK-92MI cell were evaluated by flow cytometry analysis based on CD 107a labeling.R esults NK-92MI cells could quickly acquire HLA-G1 from K562-G1 cells in co-culture experiments .Blockade of HLA-G1 or its re-ceptors KIR2DL4 and ILT2 with specific mAbs did not affect the acquisition of HLA-G1 by NK-92MI cells. Moreover, HLA-G1 could significantly inhibit the cytotoxicity of NK cell （ P〈0.01）.Conclu sion NK-92MI cells acquire HLA-G1 from K562-G1 cells via trogocytosis , which is not associated with affinity be-tween receptor and ligand , extracellular domain of HLA-G1 or passive adhesion . Key words: HLA-G1; NK cell; Trogocytosis

Retargeting NK‐92 for anti‐melanoma activity by a TCR‐like single‐domain antibody

The efficacy of immunotherapy based on natural killer (NK) cells is hampered by intrinsic non-specific cytotoxicity and insufficient activation of NK cells. Here, we confer the T-cell receptor-like (TCR-like) specificity on NK cells, taking advantage of both the innate and adaptive immune arms of the immune response to generate enhanced anti-melanoma activity. The TCR-like antibody (Ab) GPA7 was selected against melanoma-associated gp100/human leukocyte antigen (HLA)-A2 complex and then fused to intracellular domain of CD3-ζ chain. This fusion construct was incorporated into NK-92MI cell line and expressed as a chimeric antigen receptor on the surface of the cell. The anti-tumour activity of the transgenic NK-92MI-GPA7-ζ cell line was assessed against melanoma in vitro and in vivo. The engineered NK-92MI-GPA7-ζ cells could recognize melanoma cells in the context of HLA-A2 and showed enhanced killing of both melanoma cell lines and primary melanoma. Furthermore, adoptively transferred NK-92MI-GPA7-ζ cells significantly suppressed the growth of human melanoma in a xenograft model in mice. Collectively, these results demonstrate that the TCR-like Ab, GPA7, could redirect NK cells to target the intracellular antigen gp100 and enhance anti-melanoma activity, providing a promising immunotherapeutic strategy to prevent and treat melanoma.

Regulation of NK92‐MI Cell Cytotoxicity by Substance P

The neuropeptide substance P (SP) can regulate a number of immunological functions in vitro and in vivo and may regulate natural killer (NK) cell activity. Here, we investigated whether SP has a role in regulating NK92-MI cell function in vitro, and how it influences NK cell activity. We found that SP dose dependently increased the cytotoxicity of NK92-MI cells and had a maximal effect at a concentration of 10(-12) and 10(-10) m. Furthermore, the expression of cytotoxic-associated molecules (perforin, granzyme) and activating receptor NKp46 [a member of natural cytotoxicity receptors (NCRs)] was observed to be upregulated by SP at optimal concentration, at which SP enhanced the cytotoxicity of NK92-MI cells. Neurokinin-1 receptor (NK-1R), a functional receptor of SP, was found on NK92-MI cells, and the observed effects of SP on NK92-MI cells could be more partially blocked by an NK-1R antagonist. Our data suggest that SP induces NK92-MI cell cytotoxicity by directly increasing the expression of cytotoxic granules and upregulates NK92-MI cell receptor-mediated functions indirectly. Thus, SP may regulate NK cell function mainly through NK-1R.

Abstract 3656: Therapeutic effects of anti-KIR antibodies against metastatic cancer cells with aberrant expression of Natural Killer-Cell Immunoglobulin-like Receptors (KIRs)

Abstract Background: The aim of this study is to investigate the mechanism of immune-escape and cancer metastasis due to an aberrant expression of the natural killer (NK) cell immunoglobulin-like receptors (KIRs) on human lung cancer. We previously reported that metastatic cancer cells acquired immune-resistance by aberrantly expressing KIRs on their surface (AACR 2009 and 2010). Here we report the effects of anti-KIR antibodies on the cancer cells with aberrant expression of KIRs enhanced cytolytic killing of cancer cells by human Natural Killer cells. Method: KIR2DL1 (LL454) and KIR3DL1 (LL456) plasmids were used to transfect target cells (H2122-GFP) for individual KIR subtype or both KIR subtype expression. Stable transformants (termed as 2DL1 or 3DL1) were enriched by cell sorting. H2122 parental and KIR expressing cells were pre-treated with two different anti-KIR antibodies respectively (DX9 or DX27) and then exposed to various amounts of human NK-92MI cell line. Anti-proliferative effects by NK cells were accessed by a fluorescent microplate reader. IC50s were determined based on the Effector (NK) / Target Ratio (ETR). Result: When exposed to various amount of NK-92MI cell line, the parental line was most sensitive to NK-92MI killing with an IC50 ETR=1.12, followed by 2DL1(1.8), KIR3+ (1.8) and 3DL1 (3.5). After these cancer cells were pretreated with anti-KIR2DL2/3 antibodies (DX27) or anti-KIR3DL1 antibodies (DX9) respectively, sensitivities (based on IC50 ETR) to NK killing were all downshifted when compared to the untreated samples respectively. (a) After DX27 pretreatment: parent decreased by 3.3 folds (ETR 1.10 → 0.33), KIR3+ decreased by 3.3 folds (ETR 1.9 → 0.57), 2DL1 decreased by 5 folds (ETR 1.8 → 0.36), and 3DL1 decreased by 7.9 folds (ETR 3.4 → 0.43). (b) After DX9 pretreatment: parent decreased by 1.9 folds (ETR 1.13 → 0.58), KIR3+ decreased by 2.71 folds (ETR 1.9 → 0.7), 2DL1 decreased by 3.27 folds (ETR 1.8 →0.55) and 3DL1 decreased by 6.3 folds (ETR 3.5 → 0.56) respectively. Our data clearly demonstrated that aberrant expression of KIR on lung cancer cells either by in vivo immune selection in orthotopic nude rat model or by forced expression of KIRs confers resistance to NK cytolytic killing. Pretreatment with anti-KIR antibodies not only reversed resistance but increased their sensitivity to NK cytolytic killing. Conclusion: We have discovered a novel mechanism of immune resistance to NK cells due to aberrant expressions of KIRs on cancer cells. This immune resistance can be reversed by the treatment with anti-KIR antibodies, suggesting that anti-KIR antibodies have clinical potential for the treatment of aggressive and immune resistant cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3656. doi:10.1158/1538-7445.AM2011-3656