Enriched NK Cells Research Articles

Enhanced IL-15-mediated NK cell activation and proliferation by an ADAM17 function-blocking antibody involves CD16A, CD137, and accessory cells

BackgroundNatural killer (NK) cells are being extensively studied as a cell therapy for cancer. These cells are activated by recognition of ligands and antigens on tumor cells. Cytokine therapies, such...

Modakafusp Alfa (TAK-573), a Novel CD38-Targeting Attenuated Interferon-Alpha Immunocytokine, Kills MM Cells Via NK Cell Activation

Background Despite recent success of monoclonal antibodies (mAb) and T cell redirecting therapies, relapsed/refractory (RR) multiple myeloma (MM) patients remain in urgent need of new therapies with distinct mechanisms of action (MoA). Modakafusp alfa, previously known as TAK-573, is a novel immune-targeted attenuated cytokine specifically designed to deliver interferon-alpha to CD38-expressing cells via a CD38-targeting IgG4 mAb. Preliminary results of a first in human phase 1 trial with modakafusp alfa monotherapy (1.5mg/kg, every four weeks) in RRMM patients (90% anti-CD38 mAb refractory; 37% anti-BCMA agent exposed) indicate a promising 40% overall response with manageable toxicity (Kaufman et al. EHA 2022 abstract S181). Prior studies suggest a unique and multimodal MoA of modakafusp alfa, including anti-proliferative effects on tumor cells as well as stimulation of CD38-positive immune effector cells. Towards a thorough understanding of these immunomodulatory effects, specifically regarding the activation of innate immunity, we here studied the effects of modakafusp alfa on the anti-MM activity of NK cells, which highly express CD38. Aims We assessed the ex vivo activity of modakafusp alfa on NK cell activation, degranulation, and antibody-independent cytotoxicity. In addition, we investigated the impact of modakafusp alfa on NK cell-mediated, antibody-dependent cellular cytotoxicity (ADCC) against MM cells using daratumumab (anti-CD38) or elotuzumab (anti-SLAMF7). Methods NK cell activation was explored by flow cytometry after 24-hour incubation of peripheral blood mononuclear cells (PBMC) from healthy donors with modakafusp alfa or negative/specificity controls. These pre-incubated PBMCs were also used to examine NK cell degranulation after subsequent 4 hour stimulation with K562 or PMA/Ionomycin, or in a 16-hour cytotoxicity assay against K562. In addition, modakafusp alfa pre-incubated PBMCs or enriched NK cells were assessed for antibody-independent cytotoxicity or daratumumab/elotuzumab-mediated ADCC in 24-48 hour assays against MM cell lines RPMI-8226, MM.1S or UM9. Alternatively, modakafusp alfa was directly added to the assays without pre-incubation steps. Results We found that incubation of PBMCs with modakafusp alfa for 24 hours resulted in upregulation of activation markers CD38 and CD69 on the surface of NK cells. In addition, modakafusp alfa pre-incubation significantly improved the degranulation of NK cells after 4-hour stimulation with K562 or PMA/Ionomycin. Importantly, overnight pre-incubation of PBMCs or enriched NK cells with modakafusp alfa showed enhanced antibody-independent cytotoxicity against K562 tumor cells and MM cell lines RPMI-8226, MM.1S or UM9. Consequently, this also resulted in augmented killing of MM cells in the presence of daratumumab or elotuzumab. We observed similar effects when modakafusp alfa was directly added to the cytotoxicity assays without pre-incubation of immune effector cells. Conclusions Modakafusp alfa rapidly activates NK cells, and improves their degranulation and anti-MM effectivity. This important MoA also results in improved efficacy of daratumumab or elotuzumab in short-term cytotoxicity assays. Our findings warrant clinical investigation of combining modakafusp alfa with NK cell-exploiting immunotherapies.

Cell Density of NK Cells during Ex Vivo Expansion Impacts NK Cell Surface TRAIL Expression

Introduction Natural Killer (NK) cells are an emerging form of cancer immunotherapy currently being tested in clinical trials world-wide. NK cells are innate immune cells that can kill tumor cells via release of cytotoxic granules and via surface expression of the death receptor ligands tumor-related apoptosis-inducing ligand (TRAIL) and Fas ligand. We and others have recently shown that the proteasome inhibitor bortezomib sensitizes tumor cells to NK cell TRAIL-mediated killing by upregulation of death receptor 5. In a recent phase I NK cell dose-escalation study conducted at the NIH (NCT00720785) we have attempted to exploit TRAIL sensitization by administering ex vivo-expanded autologous NK cells to patients with solid tumors or hematologic malignancies that have been pretreated with bortezomib. Ex vivo cultures used to expand clinical grade NK cells for this trial utilize irradiated EBV-LCL feeder cells and IL-2 containing media which upregulates surface expression of TRAIL, substantially augmenting NK cell killing of bortezomib-treated tumors in vitro. Here we characterize the impact of specific expansion conditions used to generate high numbers of NK cells for clinical use on NK cell TRAIL expression. Methods To generate clinical grade ex vivo-expanded NK cells, we first isolated NK cells from patient apheresis products by CD3+ depletion followed by CD56+ selection, and stimulated these enriched NK cells with irradiated EBV-LCL feeder cells at a ratio of 1:10 in X-VIVO 20 supplemented with 10% inactivated human AB serum and recombinant human IL-2 (500 IU/ml). The clinical trial evaluated 8 escalating NK cell dose levels (Figure 1). Cohorts 1-4 received a single infusion of ex vivo-expanded NK cells on day 0 in a dose-escalating fashion (3-6 pts per cohort) and cohorts 5-7 received 1 x 108 NK cells/kg on day 0 and a second escalating dose of NK cells infused on day +5. A "closed bag" Baxter PL732 culture system was used for cohorts 1-7 which was later changed to a GREX500-CS (Wilson Wolf) system in cohorts 7-8. Using flow cytometry, we monitored surface expression of TRAIL on the day NK cells were harvested and infused fresh into patients. We also assessed TRAIL expression on NK cells from a single patient cultured at 6 different cell densities (range: 2.03-16.95 x 106/cm2) using culture conditions mimicking the phase I trial. Results A total of 137 NK cell cultures were harvested and administered fresh to 32 patients. NK cells on the day of harvest expanded a median of 198-fold, 895-fold, and 3637-fold on culture days 14-16, 19-22, and 24-27, respectively. NK cells at harvest contained a median of 99.7% CD3−/CD56+ NK cells, were 68.65% CD16+ and had a median of 88% viability. TRAIL was assessed by mean fluorescence intensity (MFI) with a median surface expression of of 1245 (range 132-4913) at the time of infusion (Figure 1). Expansions for cohort 8 generated 10-14 x109 (1 vessel) and 50-70 x109 NK cells (4-5 vessels) for fresh infusion, enough to support the target dose level of 1x108 (1st harvest) and 5x108 (2nd harvest) NK cells/kg. Remarkably, NK cells grown at higher cell density to reach the target cell numbers for cohort 8 exhibited substantially reduced TRAIL expression (median: 255, range 132-691). Subsequent experiments conducted on NK cells expanded in vitro for 14 days at different cell densities/concentrations showed TRAIL expression (MFI range: 319-1627) inversely correlated with both cell density and concentration (Figure 2). NK cells grown at the highest cell density (16.95 x 106/cm2) and concentration (4.23 x 106/mL) expressed the least amount of TRAIL (MFI 319), in contrast to those cultured at the lowest cell density (2.03 x 106/cm2) and concentration (0.51 x 106/mL), which demonstrated a TRAIL MFI of 1627. Conclusions Although ex vivo cultures using feeder cells make it possible to expand large numbers of NK cells for clinical use in humans, the higher concentrations and density of cells in these cultures reduce NK cell surface expression of TRAIL. In vitro, TRAIL expression appears to inversely correlate with cell density. These data highlight the need to avoid overly concentrating ex vivo expanded NK cells to maximize TRAIL surface expression as a method to potentiate the anticancer effects of adoptively infused NK cells. Disclosures No relevant conflicts of interest to declare.

Abstract B108: Hypoxia induces phenotypic and functional changes in NK cells which negatively impacts antitumor immunity in the solid tumor microenvironment

Abstract Natural killer (NK) cells are key effectors in cancer immunosurveillance, and they can eliminate tumor cells through the release of cytotoxic granules triggered by interactions with natural ligands or through antibody-dependent cellular cytotoxicity (ADCC). NK cell-based treatment has had therapeutic success for hematologic malignancies, but strategies to treat solid tumors have been more limited. One of the biggest barriers to NK cell immunotherapy is immunosuppression within the tumor microenvironment. An important aspect of this immunosuppression is the highly hypoxic environment created by proliferating tumor cells. To further study the effect of hypoxia on NK cell function, our lab has been collaborating with XCell Biosciences to use their novel incubators that precisely control oxygen and pressure levels. We incubated NK cells isolated from peripheral blood mononuclear cells (PBMCs) at three different oxygen (O2) levels: 12% O2 to mimic the peripheral blood, 5% O2 to mimic the bone marrow, and 1% O2 to mimic the solid tumor microenvironment. All comparisons were made against a standard incubator condition. NK cells were incubated for three different time points—24 hours, 72 hours, and 7 days—and activated with low dose IL-15. We conducted a mass cytometry analysis to assess phenotype of PBMCs and enriched NK cells incubated at the different oxygen conditions. NK cells from the 1% O2 condition express fewer activating receptors (NKG2D, Nkp30, Nkp46, DNAM-1) and less perforin and granzyme than NK cells from the higher oxygen conditions. However, the NK cells from 1% O2 conditions express high levels of the activating receptors CD69 and CD16 (which triggers ADCC). This altered activation state is reflected in cell-killing assays using an IncuCyte machine. Here, fluorescently labeled target cells and NK cells are incubated together and images are taken for 48 hours to measure target cell death. We observed that natural cytotoxicity against K562 targets is severely impaired in the NK cells at the lowest oxygen condition but ADCC responses using the antibody rituximab against Raji targets remain intact. We observe a sharp decrease in proliferation, as measured by CellTrace dilution and Ki-67 expression in the NK cells in the 1% O2 condition. We also detect a decrease in glycolytic and mitochondrial metabolism, as measured by a Seahorse Analyzer, as the oxygen levels decrease. Supernatants collected from NK cells in the lower oxygen conditions (12% O2 and 5% O2) contain more inflammatory cytokines (IFNγ, TNFα, GM-CSF) and chemokines (CCL3, CCL4, CCL5) than NK cells in high oxygen conditions, indicating a shift from cytotoxic to a more immunomodulatory state. These results indicate that hypoxia changes the phenotype and function of NK cells. Overall, the insights gained from these experiments can help overcome hypoxia-induced immune suppression in the tumor microenvironment and enhance NK cell-based immunotherapy for solid tumors. Citation Format: Upasana Sunil Arvindam, Pippa Kennedy, Bri Ettestad, Peter Hinderlie, Jeffrey S. Miller, Martin Felices. Hypoxia induces phenotypic and functional changes in NK cells which negatively impacts antitumor immunity in the solid tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B108.

Distinct HLA-E Peptide Complexes Modify Antibody-Driven Effector Functions of Adaptive NK Cells

Adaptive NK cells are characterized by profound alterations in multiple signaling molecules, transcription factors, and epigenetic modifications compared with canonical NK cells. Although their existence is associated with prior exposure to human cytomegalovirus (HCMV), key questions regarding their regulation and function remain. A large proportion of adaptive NK cells express the activating receptor CD94/NKG2C, binding to human leukocyte antigen E (HLA-E), that presents a limited set of peptides. We show that adaptive NK cells discriminate differences between HLA-E-peptide complexes with exquisite specificity. Prolonged exposure to an environment displaying the HLA-E peptide ligand VMAPRTLFL, derived from the leader sequence of HLA-G, enriched adaptive NK cells with low FcεRγ expression, upregulated CD25 expression, increased proliferative activity, and resulted in elevated antibody-dependent cellular cytotoxicity and IFN-γ responses compared with other HLA-E peptide complexes. Our study demonstrates that recognition of alterations in the HLA-E ligandome via an activating receptor can influence heterologous effector mechanisms and proliferation in adaptive NK cells.

Identification and functional analysis of NKp46+ natural killer cells from peripheral blood of squirrel (Saimiri sps.) and owl monkey (Aotus sps.)

Abstract Natural Killer (NK) cells as minor subsets of peripheral blood lymphocytes (PBL) are innate in nature to clear invading pathogens and tumor cells. Small rodent species to nonhuman primates and humans, immune systems consist of a spectrum of NK cell subsets with phenotype and functional diversity. This study examined NK cell subsets and expression of NKp46 in squirrel and owl monkeys, which are both New World primate species. Squirrel and owl monkeys are recommended by the World Health Organization as excellent experimental models for studies of malaria infection due to their susceptibility to some of the same strains that cause disease in humans and endemic viruses that are analogues of opportunistic human viruses. NK cells were identified from CD3 negative lymphocytes and sub-phenotyped by CD16 and CD8 expression, to classify CD8+ and CD8− NK cells and absolute numbers in peripheral blood were compared. Analysis of expression of NCR (Natural Cytotoxicity Receptors) proteins, NKp30 and NKp46 showed that NK cells from NHP had preferential expression of NKp46 over NKp30 and expression is mostly restricted to subset of CD8+ NK cells. Peripheral blood lymphocytes, enriched based on NKp46 expression, were further phenotyped as classic CD3−CD16+CD8+NK cells. Functional analysis of enriched cells was focused to verify whether subsets differ in cytolitic and/or “innate” in nature and to correlate with NCR protein expression. Enriched NK cells were co-cultured for stimulation with standard target cells including tumor cells and analyzed for cytokine production. The potential plasticity of peripheral blood NK cells in NHP based on steady state and/or inflammatory/pathogenic status of species will be analyzed and discussed.

Super-charged NK cells inhibit growth and progression of stem-like/poorly differentiated oral tumors in vivo in humanized BLT mice; effect on tumor differentiation and response to chemotherapeutic drugs

ABSTRACTTherapeutic role of NK cells in solid tumors was challenged previously even though their role in hematological malignancies has clearly been established. Furthermore, functions and numbers of NK cells are greatly suppressed in oral cancer patients necessitating effective future NK immunotherapeutic strategies to aid in the control of disease. The humanized-BLT (hu-BLT) mice were used to implant stem-like/undifferentiated oral tumors to study the role of super-charged NK cells with and without feeding with AJ2 probiotic bacteria. Implanted CSC/undifferentiated tumors resected from NK-injected mice exhibited differentiated phenotype, grew slowly, and did not cause weight loss, whereas those from tumor-bearing mice without NK-injection remained relatively more stem-like/poorly-differentiated, grew faster, and caused significant weight loss. Moreover, in vitro NK-differentiated tumors were sensitive to chemotherapeutic drugs, and when implanted in the oral-cavity grew no or very small tumors in mice. When NK-mediated differentiation of tumors was blocked by IFN-γ and TNF-α antibodies before implantation, tumors grew rapidly, remained stem-like/poorly-differentiated and became resistant to chemotherapeutic drugs. Loss of NK cytotoxicity and decreased IFN-γ secretion in tumor-bearing mice in PBMCs, splenocytes, bone marrow derived immune cells and enriched NK cells was restored by the injection of super-charged NK cells with or without feeding with AJ2. Much greater infiltration of CD45+ and T cells were observed in tumors resected from the mice, along with the restored secretion of IFN-γ from purified T cells from splenocytes in NK-injected tumor-bearing mice fed with AJ2 probiotic bacteria. Thus, super-charged NK cells prevent tumor growth by restoring effector function resulting in differentiation of CSCs/undifferentiated-tumors in hu-BLT mice.

Evaluation of antibody-dependent cell-mediated cytotoxicity activity and cetuximab response in KRAS wild-type metastatic colorectal cancer patients.

To investigate the prognostic role of invariant natural killer T (iNKT) cells and antibody-dependent cell-mediated cytotoxicity (ADCC) in wild type KRAS metastatic colorectal cancer (mCRC) patients treated with cetuximab. Forty-one KRAS wt mCRC patients, treated with cetuximab and irinotecan-based chemotherapy in II and III lines were analyzed. Genotyping of single nucleotide polymorphism (SNP)s in the FCGR2A, FCGR3A and in the 3' untranslated regions of KRAS and mutational analysis for KRAS, BRAF and NRAS genes was determined either by sequencing or allelic discrimination assays. Enriched NK cells were obtained from lymphoprep-peripheral blood mononuclear cell and iNKT cells were defined by co-expression of CD3, TCRVα24, TCRVβ11. ADCC was evaluated as ex vivo NK-dependent activity, measuring lactate dehydrogenase release. At basal, mCRC patients performing ADCC activity above the median level (71%) showed an improved overall survival (OS) compared to patients with ADCC below (median 16 vs 8 mo; P = 0.026). We did not find any significant correlation of iNKT cells with OS (P = 0.19), albeit we observed a trend to a longer survival after 10 mo in patients with iNKT above median basal level (0.382 cells/microliter). Correlation of OS and progression-free survival (PFS) with interesting SNPs involved in ADCC ability revealed not to be significant. Patients carrying alleles both with A in FCGR2A and TT in FCGR3A presented a trend of longer PFS (median 9 vs 5 mo; P = 0.064). Chemotherapy impacted both iNKT cells and ADCC activity. Their prognostic values get lost when we analysed them after 2 and 4 mo of treatment. Our results suggest a link between iNKT cells, basal ADCC activity, genotypes in FCGR2A and FCGR3A, and efficacy of cetuximab in KRAS wt mCRC patients.

CD30/CD16A Tandab AFM13-Induced Target Cell Lysis By NK-Cells Is Enhanced By CD137 Co-Stimulation and Blocking PD-1

Introduction: AFM13 is a CD30/CD16A bispecific tetravalent TandAb antibody that recruits and activates NK-cells by specific binding to CD16A for targeted lysis of CD30+ tumor cells. Given promising clinical activity and safety profile of AFM13 and proof-of-mechanism demonstrating dependence on the immune response, potential synergy of AFM13 and checkpoint modulators was evaluated.Methods: Efficacy of AFM13 alone or in combination with anti-CTLA-4, anti-PD-1, or anti-CD137 antibodies was assessed by in vitro cytotoxicity assays with human PBMCs or enriched NK-cells and CD30+ target cells as well as patient-derived xenograft in vivo models with autologous PBMC.To evaluate NK-cell-mediated lysis of CD30+ lymphoma cell lines, 4 hour cytotoxicity assays were performed with PBMCs or enriched NK-cells as effector cells in the presence of suboptimal concentrations of AFM13 alone, and in combination with anti-CTLA-4, anti-PD-1, or anti-CD137 antibodies. For the in vivo model tumor fragments derived from surgical specimens of newly diagnosed patients with CD30+ Hodgkin Lymphoma were xenografted (PDX) in immuno-deficient mice. After 28 days mice were reconstituted with autologous patient-derived PBMC and treated with AFM13 alone and in combination with anti-CTLA-4, anti-PD-1, or anti-CD137 antibodies weekly for a total of three weeks. Tumor size, tumor-infiltrating human lymphocytes and intra-tumoral cytokines were evaluated on day 58.Results: AFM13 as a single agent at suboptimal concentrations induced effector-to-target cell-dependent lysis of CD30+ lymphoma cells up to 40% using enriched NK-cells as effector cells in a 4 hour in vitro assay. Immune-modulating antibodies alone mediated substantially lower lysis (<25%). However, the addition of anti-PD-1 or anti-CD137 to AFM13 strongly enhanced specific lysis up to 70%, whereas the addition of anti-CTLA-4 to AFM13 showed no beneficial effect. The most impressive increase of efficacy was observed when AFM13 was applied together with a combination of anti-PD-1 and anti-CD137. In vivo, reduction of tumor growth was observed when AFM13 and anti-PD-1 were used as single agents or when AFM13 was combined with anti-CD137. Synergy was most impressive in these PDX models for the combination of AFM13 and anti-PD-1 which led to a very strong reduction of tumor size. Of note, reduction of tumor growth was strongly correlated with infiltrating NK- and T-cells and intra-tumoral cytokines.Conclusions:The combination trials performed with companion intra-tumoral assessment of lymphocytes and cytokines may enhance the efficacy of AFM13 in patients. This may be explained by a potential cross-talk between NK-cells and T-cell which was enhanced when AFM13 was used in combination with checkpoint modulators. DisclosuresNo relevant conflicts of interest to declare.

Abstract LB-151: A novel TGFβ 1/2 specific antibody suppresses 4T1 tumor growth by enhancing NK cell recruitment and function

Abstract TGFβ can play an important role in tumor progression. Whether expressed by the tumor, or the tumor microenvironment, it has a profound impact on not only the tumor itself but also the host immune system. There are three isoforms of TGFβ: TGFβ1, TGFβ2 and TGFβ3. The functions of TGFβ1 and 2 have been widely studied. However, little is known about how TGFβ3 affects both tumor growth and the host. Here we used a novel human antibody that neutralizes only TGFβ1 and 2, while leaving TGFβ3 activity untouched. By specifically blocking TGFβ1 and 2 functions, we were able to investigate the role of TGFβ3 in modulating the immune compartment, and its ability to combat tumor growth. We used the 4T1 murine breast carcinoma model, which secretes all three isoforms of TGFβ. Tumor-bearing mice were treated with 10mg/kg the novel anti-TGFβ1/2 antibody, which inhibited 25% of 4T1 tumor growth after 20 days. We evaluated the immune cells from tumors and found there was a significant increase (14%) in the NK cells that infiltrated the tumor site in the antibody-treated group. To explore the mechanism underlying this phenomenon, we evaluated the effect of tumor-secreted TGFβ isoforms on NK cells in vitro in a co-culture system. When 4T1 tumor cells were co-cultured with enriched NK cells, the TGFβ1/2 antibody elevated the levels of IFNγ, GzmB and perforin. However, the expression levels of NKG2D, CD69, Ly49A and C were unchanged. We also detected a significant upregulation of FasL on NK cells treated with the TGFβ1/2 antibody. Moreover, NK cells treated with the TGFβ1/2 antibody in the co-culture system showed improved killing activity towards Yac-1 cells, which was confirmed to be mediated by GzmB. In summary, a novel therapeutic antibody candidate that specifically neutralizes TGFβ1 and 2 inhibits 4T1 tumor growth by (i) changing the tumor microenvironment to attract NK cells to tumor sites; and (ii) by increasing NK cell function via upregulation of FasL and GzmB. Citation Format: Ou Li, Shireen Khan, Nerissa Mendoza, Sandra Vanegas, Cat Tran, Amer Mirza. A novel TGFβ 1/2 specific antibody suppresses 4T1 tumor growth by enhancing NK cell recruitment and function. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-151. doi:10.1158/1538-7445.AM2014-LB-151

Effects of tributyltin exposures on secretion of interleukin 1 beta from human immune cells (1012.1)

Tributyltin (TBT) is an organotin compound used as a biocide in a variety of industrial applications such as wood preservation and as an antifungal. It has been found in human blood samples. Interleukin 1 beta (IL‐1β) is produced by monocytes and macrophages and promotes cell growth, tissue repair, and regulates immune response. IL‐1β appears to increase the invasiveness of certain tumors. The aim of the current study is to determine if exposures to TBT modify secretion of IL‐1β from increasingly reconstituted preparations of human immune cells. TBT concentrations of 25 to 200 nM at 24h, 48h, or 6 days were examined in highly enriched human NK cells, monocyte‐depleted human peripheral blood mononuclear cells (PBMCs), PBMCs, granulocytes, and a combination of PBMCs and granulocytes. IL‐1β was measured using an enzyme‐linked immunosorbent assay (ELISA). Results show TBT alters IL‐1β secretion from all cells preparations. 200 nM concentrations of TBT blocked the secretion of IL‐1β, while lower concentrations of TBT elevated secretion of IL‐1β. Concentrations and lengths of exposure to TBT that caused statistically significant increases in IL‐1β secretion from human immune cells varied from one donor to the next. The data indicates that TBT‐induced alterations of IL‐1β secretion from immune cells may be a significant consequence of TBT exposures that may potentially affect immune competence and cancer invasiveness.Grant Funding Source: Supported by NIH grant 5U54CA163066‐02

Low gene expression levels of activating receptors of natural killer cells (NKG2E and CD94) in patients with fulminant type 1 diabetes

Fulminant type 1 diabetes is an independent subtype of type 1 diabetes characterized by extremely rapid onset and absence of islet-related autoantibodies. However, detailed pathophysiology of this subtype is poorly understood. In this study, a comprehensive approach was applied to understand the pathogenesis of fulminant type 1 diabetes. We determined the genes that were differentially expressed in fulminant type 1 diabetes compared with type 1A diabetes and healthy control, using gene expression microarray in peripheral blood cells. Using volcano plot analysis, we found reduced expression of killer cell lectin-like receptor subfamily C, member 3 (KLRC3) which encodes NKG2E, a natural killer (NK) cell activating receptor, in fulminant type 1 diabetes, compared with healthy controls. This difference was confirmed by real-time RT-PCR among NK-enriched cells. The expression of KLRD1 (CD94), which forms heterodimer with NKG2E (KLRC3), was also reduced in NK-enriched cells in fulminant type 1 diabetes. Furthermore, flow cytometry showed significantly lower proportion of NK cells among peripheral blood mononuclear cells (PBMCs) in fulminant type 1 diabetes than in healthy controls. In patients with fulminant type 1 diabetes, the relative proportion of NK cells correlated significantly with the time period between onset of fever to the appearance of hyperglycemic-related symptoms. We conclude the presence of reduced NK activating receptor gene expression and low proportion of NK cells in fulminant type 1 diabetes.

98-P: KIR mRNA EXPRESSION IN PBMC AND ENRICHED NK CELLS ASSAYED BY QUANTITATIVE REAL-TIME PCR

To develop assays to quantify the mRNA transcribed from KIR genes utilizing a quantitative real-time PCR (qPCR) method. To study the KIR mRNA expression patterns in PBMC and enriched NK cells among healthy volunteers. PBMCs were obtained from freshly drawn whole blood (maximum 48 hours) from 14 healthy volunteers using a Ficoll method. NK cell enrichment from PBMC was performed by negative selection using a magnetic bead kit. mRNA extraction and cDNA synthesis was followed by EvaGreen based qPCR. Relative KIR mRNA expression was quantified by 2-ΔΔCt algorithm with GAPDH as reference. qPCR with a Cq ⩽ 38.0 was considered positive. 10 mRNA expression assays for the 9 KIR genes 2DL1, 2DL5A, 2DL5B, 2DS1, 2DS2, 2DS3, 2DS4 (2DS4-full length and 2DS4-truncated), 2DS5 and 3DS1 have been developed and validated. Among the 14 individuals, KIR mRNAs were detected in all samples carrying respective KIR genes and were negative for KIR gene negative samples. The mRNA expression varied among individuals. In PBMC, the relative quantification (RQ) of cDNA showed the largest variation for 2DL1 (14-fold, n = 14) and the smallest for 3DS1 (1.8-fold, n = 3) among volunteers who were positive for the respective genes. 2DL5B cDNA was not detected, as expected for this gene. More KIR cDNA was detected in the enriched NK cell fraction when compared to PBMC, with the largest difference observed for 2DL1 (NK : PBMC ratio, mean ± SD: 37.8 ± 25.0, n = 14). Development of qPCR assays with high specificity and easy applicability will be useful, because all known 15 KIR genes share > 90% identity at the genomic level. We established 10 assays for the mRNA expression of 9 KIR genes. KIR mRNA expression varied among healthy individuals carrying the same KIR genotype. Because NK cells are the predominant, but not the only source of KIR mRNA, analyzing PBMC and NK cells will allow establishing an overall and NK cell specific KIR mRNA expression signature from an individual.

Selective expansion of NK cells from PBMCs for cancer immunotherapy (156.28)

Abstract NK cells have the ability to kill transformed or tumor cells without any prior stimulation. Despite this ability, the function of NK cells in tumor patients is greatly suppressed due to multiple factors. Therefore, efforts have been focused on isolating and expanding NK cells from tumor patients and re-infusing them after in vitro activation. These processes require multiple procedures to isolate and purify NK cells from the patients' PBMCs and high cost cytokines. To devise a simple and low-cost method to expand and activate NK cells with high purity, we have screened numerous feeder cell lines. Among these cell lines, we discovered KL-1 cell line, selectively enriched NK cells from whole PBMCs. After 15 days of culture, the number of CD56+ CD3- NK cells increased approximately 130-fold with the NK cell purity of over 90%. Expanded NK cells mounted superior cytotoxicity against NK-sensitive K562 cells as well as KL-1 cells. These results indicated that KL-1 feeder cell line could be exploited to generate NK cells for adoptive transfer with this cost-effective yet highly functional NK expansion method.

Introduction of Differential KIR Activation Mediated Patters of NK Cell Cytotoxicity Against Myeloma

AbstractAbstract 5178 Background:Natural Killer cells (NK cells) are part of the innate immune system. These cells have the ability to recognise and kill malignant cells, like myeloma cells. NK cell activation is tightly regulated by different activating or inhibiting receptors. Killer immunoglobulin like receptors (KIR, CD158) are a family of receptors which have activating as well as inhibitory function. KIR molecules are thought to recognize the HLA-C molecules, which then lead to NK cell signal transduction. Our knowledge about KIR expression and impact on tumor cell control has developed over the last years, but still our understanding of how the receptors are activated in multiple myeloma is limited. We therefore, investigated three different model systems for NK cell alloreactivity: 1) HLA-C/ HLA-C interaction model (KIR-Ligand model), 2) HLA-C/ KIR receptor interaction model, and 3) impact of donor KIR haplotype. Material and Methods:Three different myeloma cell lines (KMS12BM [C1/C1], MOLP8 [C1/C2] and RPMI8266 [C2/C1]) and a NK cell line (NKL) were cultured under standard conditions. NKL cells were transfected with human KIR2DL1 and KIR2DL3 alleles, respectively. For RNAi experiments two siRNA and two control siRNA were used. NK cells from healthy donors were isolated by magnetic end labeling. Enriched NK cells were HLA-typed for expression of HLA-C molecules, KIR receptor expression or KIR haplotype. Thereafter, NK cells were transiently transfected with the siRNA or control siRNA against the KIR2DL1 or KIR2DL3 receptors for up to 48 h. Functional analysis of the NK cell cytotoxicity was measured using a LDH release assay, based on their killing ability against the three fore mentioned myeloma cell lines. Results:Using NK cells that have been HLA-C genotyped as HLA-C1/C1, we observed a rescue of C1/C1 positive meyeloma cell lines in contrast to the C2/C1 myeloma cell line (12% cytotoxicity vs. 38% and 45% cytotoxicity, respectively, p <0.05). KIR receptors, by recognition of HLA-C molecules, are believed to reduce NK cell cytotoxicity. We used NK cells from healthy donors, which express KIR2DL1 (recognizing C2) to evaluate whether receptors' RNAi knockdown could increase the ability of NK cells to lyse target cells. Knockdown of KIR2DL1 in NK cells results in a highly efficient killing of a C2/C2 target cell line (RNAi treated NK cells showed a cytotoxicity up to 70%, p<0.05). To evaluate whether KIR receptors are major inhibitory molecules, we used the NK cell line (wt NKL, a non expressor of KIR receptors) that exhibited high cytotoxic potential against all tested target cell lines (>65% cytotoxicity). This NK cell line was transfected with KIR2DL1 or KIR2DL3 (recognizing C2, or C1). C2 positive myeloma cell lines were significantly rescued when exposed to KIR2DL1 transfected NK cells (wt NKL 72% vs. KIR2DL1 NKL 29%, p <0.05). Similar results were obtained when C1 positive target cell line was co- cultured with KIR2DL3 transfected NK cells. RNAi mediated silencing of KIR overexpression restores protection observed. As last model for the regulation of NK cell cytotoxicity we used KIR haplotyped NK cells and co-cultured them with a C1/C2 myeloma cell line. NK cells with an AA KIR haplotype showed the lowest killing capacity against the myeloma cell line, while BB KIR haplotype had higher killing potential. (46% vs 59%, p=0,005). Conclusions:We have shown that NK cell cytotoxicity is regulated by the HLA-C expression on the target cell as well as the expression of a specific KIR receptor and various KIR haplotypes. All tested models showed significant killing effect on myeloma cells. These data underline the importance of KIR receptor modulation on NK cell alloreactivity against myeloma cells. Disclosures:No relevant conflicts of interest to declare.

Role of sympathetic nervous system in the entrainment of circadian natural-killer cell function

Previous research in our laboratory has demonstrated robust circadian variations of cytokines and cytolytic factors in enriched NK cells from rat spleen, strongly suggesting these functions may be subject to circadian regulation. The SCN mediates timing information to peripheral tissues by both humoral and neural inputs. In particular, noradrenergic (NE) sympathetic nervous system (SNS) terminals innervate the spleen tissue communicating information between central and peripheral systems. However, whether these immune factors are subject to timing information conveyed through neural NE innervation to the spleen remained unknown. Indeed, we were able to characterize a circadian rhythm of NE content in the spleen, supporting the role of the SNS as a conveyor of timing information to splenocytes. By chemically producing a local splenic sympathectomy through guanethidine treatment, the splenic NE rhythm was abolished or shifted as indicated by a blunting of the expected peak at ZT7. Consequently, the daily variations of cytokine, TNF-α, and cytolytic factors, granzyme-B and perforin, in NK cells and splenocytes were altered. Only time-dependent mRNA expression of IFN-γ was altered in splenocytes, but not protein levels in NK cells, suggesting non-neural entrainment cues may be necessary to regulate specific immune factors. In addition, the rhythms of clock genes and proteins, Bmal1 and Per2, in these tissues also displayed significantly altered daily variations. Collectively, these results demonstrate rhythmic NE input to the spleen acts as an entrainment cue to modulate the molecular clock in NK cells and other spleen cells possibly playing a role in regulating the cytokine and cytolytic function of these cells.

Abstract 2170: Genomic profiling and pathway significances of natural killer cell lymphoma (NKL): Implication for NK lymphomagenesis and genomic targeting therapeutics

Abstract Natural killer cell lymphoma (NKL) is a highly aggressive form of lymphoid malignancy (Suzuki et al. BLOOD 2009). We hypothesized that the comparison of differentially expressed genes of NKL vs normal NK cells would be a rational approach for enhancing our understanding of NK lymphomagenesis and lead to the discovery of genomic markers and potential therapeutic targets. Isolated RNA from highly enriched peripheral blood NK cells (CD3-, CD56+; PBNK) and human IL2-independent NKL cell line (NK-92MI) were subjected to microarray studies using Affymetrix U133A_2. Gene data were subjected to Agilent GeneSpring analyses. Welch test were used to perform statistical analysis and fold change of &amp;lt; 2.0 and values of p&amp;lt;0.05 were considered to be significant. NK-92MI NKL vs PBNK expressed high CD3ε (3.4F UP), presumably due to the constitutive expression of IL-2 in NK-92MI. The gene array studies identified 1284 genes that were upregulated (&amp;gt;2F) and 1558 genes that were downregulated (&amp;gt;2F) in NKL vs PB NKs. The pathway mapping studies identified 22 pathways (p&amp;lt;0.05), including cytokine-cytokine receptor interaction (CCRI) (12 genes), pathways in cancer (PIC) (26 genes) and p53 signaling (13 genes), involved in up-regulation, whereas 26 pathways (p&amp;lt;0.05), including antigen processing and presentation (APP) (12 genes), CCRI pathway (29 genes), and MAPK signaling (36 genes), involved in down-regulation. Altered genes in CCRI pathway included CCL18 (1257F UP), CXCL2 (583F DOWN), IL7R (379F DOWN), TNFRSF25 (276F DOWN), IL1B (198F DOWN), CX3CR1 (181F DOWN). Altered genes in MAPK pathway included DUSP4 (330F DOWN), IL1B (198F DOWN), CD14 (120F DOWN), CACNA2D2 (51F DOWN). Altered genes in p53 pathway included IGF1 (465F UP), RRM2 (144F UP), CDKN2A (56F UP). Altered genes in PIC pathway included BIRC5 (117F UP), FN1 (73F UP), RALBP1 (158F DOWN), TCF7L2 (137F DOWN), CTBP2 (122F DOWN), PTK2 (107F DOWN). NK cell mediated cytotoxicity pathway was not significantly changed (p &amp;lt;0.2). However, 16 and 9 genes were demonstrated to be up and down regulated, including NCR2 (211F UP), NFATC1 (100F DOWN), DAP-12/TYROBP (64F DOWN), and IFNGR2 (40F DOWN). CCL18 chemokine is a known non-invasive biomarker in many diseases. Granzyme protein, which activates caspase-3 (CASP3), was down-regulated in NKL. BIRC5 (survivin) gene, which inhibits CASP3, was increased in NKL. CASP3 and BIRC5 counteract a default induction of apoptosis in G2/M phase. NCR2 and DAP-12 specifically regulate NK mediated cytotoxicity. These data suggest that CCRI, p53, APP, and MAPK signaling pathways may be involved in NK lymphomagenesis. The 108 altered genes in these pathways may serve in part to characterize the genetic signature in NKL. Furthermore, CCL18, NCR2, BIRC5, and DAP-12 together may serve as genomic markers to identify NKL and may be candidate targets for novel therapeutics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2170.

Interleukin-21 can efficiently restore impaired antibody-dependent cell-mediated cytotoxicity in patients with oesophageal squamous cell carcinoma

Background:We previously reported that Trastuzumab- and Cetuximab-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) in cancer patients was impaired in comparison with that in healthy donors because of NK-cell dysfunction. In this study, we evaluated whether IL-21 could improve the impairment of ADCC in patients with oesophageal squamous cell carcinoma (ESCC), as IL-21 was reported to have the ability to activate NK cells.Methods:We examined Trastuzumab- and Cetuximab-mediated ADCC of peripheral blood mononuclear cells (PBMCs) or of enriched NK cells derived from ESCC patients (n=20) and healthy donors (n=16) in the presence of IL-21. We further analysed ADCC-related molecules (perforin, granzyme-B, and CD247) on NK cells in response to IL-21.Results:Trastuzumab- and Cetuximab-mediated ADCC of PBMCs or of enriched NK cells was enhanced by the addition of IL-21 in a dose-dependent manner and the levels of ADCC enhanced by IL-21 in patients were high enough in comparison with those in healthy donors, paralleling the upregulation of CD247 on NK cells.Conclusion:IL-21 could efficiently restore impaired ADCC in ESCC patients with the upregulation of CD247 molecules.

Proteomic Analysis for the Identification of Disease Biomarkers and Therapeutic Targets in Natural Killer Cell Lymphoma.

Natural killer cell lymphoma (NKL) is a highly aggressive form of hematolymphoid malignancy that arises from natural killer cells, a critical component of the innate immune system. Early diagnosis of the tumor is critical however there are no biomarkers which are useful currently for the diagnosis and monitoring of patients with NKLs. We hypothesized that comprehensive analysis of differentially expressed proteins by NKL and normal NK cells would be a rational strategy for the discovery of NKL biomarkers and lead to better understanding of pathogenetic mechanisms of NKL. We used total cell lysates obtained from highly enriched peripheral blood NK cells (CD3-, CD56dim) and an Epstein-Barr virus negative, IL-2 dependent cell line (NK-92MI) for quantitative proteomic analysis. Isotopecoded affinity tags (ICATTM)-labeling followed by liquid chromatography-tandem mass spectrometry analyses (LC-MS/MS) identified 114 differentially expressed proteins in NKL compared to the normal NK cells. Out of these, 80 were upregulated 1.5-fold or greater and 34 were downregulated 1.5-fold or greater in NKL. Proteins within diverse cellular functional categories including cell signaling, transcriptional regulation, DNA metabolism, and immune response were differentially expression. Furthermore, known proteins relevant to the function of NK cells such as perforin, granzyme and defensins were downregulated in the NKL cell line. We selected eight proteins to validate the changes identified by ICAT-LC-MS/MS using Western blot analysis. HSP90, PCNA, alpha-enolase, GSTP1, galectin-1, and PARK7 were upregulated, and lactoferrin and annexin A1 were downregulated by western blot analysis demonstrating high concordance with the results of ICAT-LC-MS/MS analysis. We validated the functional significance of HSP90 as a potential therapeutic target in NKL by investigating the effect of inhibitors of HSP90 (geldanamycin) on the survival of an NKL cell line. Inhibition of HSP90 led to significant decrease in cell viability and apoptotic cell death as confirmed by western blotting demonstration of PARP-1 induction and detection of its cleavage products. This study represents the first global quantitative proteomic cataloguing of proteins expressed by NKL and normal NK cells, and reveals potential candidate proteins that may be useful for the diagnosis and function as therapeutic targets in patients with NKL.

Decreased NK cell frequency in chronic hepatitis C does not affect ex vivo cytolytic killing

Prior studies have suggested that natural killer (NK) cell function might be impaired in chronic hepatitis C virus (HCV) infection. Circulating NK cell frequency and cytolytic activity were examined freshly ex vivo in HCV-infected and uninfected subjects. Surprisingly, the intrinsic cytolytic activity of peripheral blood NK-enriched cells was similar between HCV-infected and uninfected groups (P = .91). Although the percentage of circulating CD3- CD16/56+ NK cells was 30% lower in HCV-infected compared with uninfected subjects (P = .02) paralleled by a decrease of CD56(dim) cytolytic NK cells (P = .02), overall K562 cytolysis by unfractionated peripheral blood mononuclear cells was not affected (P = .29). Analysis of the relationships between NK cytolytic activity and other clinical information revealed an inverse association with liver fibrosis stage (P = .035). In conclusion, NK cell cytolytic function does not appear to be impaired in chronic hepatitis C, but higher levels of NK cell cytolysis are associated with less liver fibrosis.