Chromium Release Cytotoxicity Assays Research Articles

TLR7 agonist, DSP-0509, with radiation combination therapy enhances anti-tumor activity and modulates T cell dependent immune activation

BackgroundTLR7 is a key player in the antiviral immunity. TLR7 signaling activates antigen-presenting cells including DCs and macrophages. This activation results in the adaptive immunity including T cells and B cells. Therefore, TLR7 is an important molecule of the immune system. Based on these observations, TLR7 agonists considered to become a therapy weaponize the immune system against cancer. Radiation therapy (RT) is one of the standard cancer therapies and is reported to modulate the tumor immune response. In this study, we aimed to investigate the anti-tumor activity in combination of TLR7 agonist, DSP-0509, with RT and underlying mechanism.ResultWe showed that anti-tumor activity is enhanced by combining RT with the TLR7 agonist DSP-0509 in the CT26, LM8, and 4T1 inoculated mice models. We found that once- weekly (q1w) dosing of DSP-0509 rather than biweekly (q2w) dosing is needed to achieve superior anti-tumor activities in CT26 model. Spleen cells from the mice in RT/DSP-0509 combination treatment group showed increased tumor lytic activity, inversely correlated with tumor volume, as measured by the chromium-release cytotoxicity assay. We also found the level of cytotoxic T lymphocytes (CTLs) increased in the spleens of completely cured mice. When the mice completely cured by combination therapy were re-challenged with CT26 cells, all mice rejected CT26 cells but accepted Renca cells. This rejection was not observed with CD8 depletion. Furthermore, levels of splenic effector memory CD8 T cells were increased in the combination therapy group. To explore the factors responsible for complete cure by combination therapy, we analyzed peripheral blood leukocytes (PBLs) mRNA from completely cured mice. We found that Havcr2low, Cd274low, Cd80high, and Il6low were a predictive signature for the complete response to combination therapy. An analysis of tumor-derived mRNA showed that combination of RT and DSP-0509 strongly increased the expression of anti-tumor effector molecules including Gzmb and Il12.ConclusionThese data suggest that TLR7 agonist, DSP-0509, can be a promising concomitant when used in combination with RT by upregulating CTLs activity and gene expression of effector molecules. This combination can be an expecting new radio-immunotherapeutic strategy in clinical trials.

Cryopreservation impairs 3-D migration and cytotoxicity of natural killer cells

Natural killer (NK) cells are important effector cells in the immune response to cancer. Clinical trials on adoptively transferred NK cells in patients with solid tumors, however, have thus far been unsuccessful. As NK cells need to pass stringent safety evaluation tests before clinical use, the cells are cryopreserved to bridge the necessary evaluation time. Standard degranulation and chromium release cytotoxicity assays confirm the ability of cryopreserved NK cells to kill target cells. Here, we report that tumor cells embedded in a 3-dimensional collagen gel, however, are killed by cryopreserved NK cells at a 5.6-fold lower rate compared to fresh NK cells. This difference is mainly caused by a 6-fold decrease in the fraction of motile NK cells after cryopreservation. These findings may explain the persistent failure of NK cell therapy in patients with solid tumors and highlight the crucial role of a 3-D environment for testing NK cell function.

Rapid flow cytometry-based assay for the evaluation of γδ T cell-mediated cytotoxicity.

The effector function of natural killer, lymphokine-activated killer cells and T lymphocytes is commonly evaluated by radioactive chromium-release cytotoxicity assays. In addition to this indirect method, fluorescence assays have been described for the assessment of in vitro cell-mediated cytotoxicity. In the present study, target cells were stained with 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE), which is a stable integrated fluorescent probe that allows target and effector cells to be distinguished from one another. Staining of target THP-1 cells with 8 µM CFSE revealed high and stable loading of fluorescence and no effect of the viability of cells. After 4 h of in vitro co-culture between γδ T cells and CFSE-labeled infected or uninfected THP-1 cells, staining with propidium iodide (PI) was performed to distinguish between vital and dead cells. During sample acquisition, target cells were gated on the CFSE positivity and examined for cell death based on the uptake of PI. CFSE and PI double positive cells were recognized as the dead target cells. The percentage of cytotoxicity in the CFSE-gated cell population was calculated by subtracting the value obtained for non-specific PI-positive target cells, which was measured in a control group that did not contain effector cells. The present study describes a simple and convenient assay that is based on the direct quantitative and qualitative analysis of cell damage at a single cell level utilizing a two-color flow cytometric assay. In conclusion, the flow cytometric-based assay described in the current study is a simple, sensitive and reliable tool to determine the cytolytic activity of γδ T lymphocytes against mycobacteria. Therefore, the present study may provide valuable information concerning the methods employed to investigate the function of γδ T cells and potentially other lymphocyte subsets.

Abstract 3151: T cells engineered to express a chimeric antigen receptor targeting chondroitin sulfate proteoglycan 4 (CSPG4) specifically kill medulloblastoma and produce inflammatory cytokines

Abstract Human T-cells expressing chimeric antigen receptors (CARs) recognizing common cancer antigens have shown great promise in the clinic with the potential for significant anti-tumor responses. CSPG4 is a chondroitin sulfate proteoglycan previously shown to be overexpressed in melanoma and breast cancer, as well as glioblastoma stem cells. CARs directed against CSPG4 have recently shown promising pre-clinical efficacy against these tumors. We evaluated the feasibility of targeting CSPG4 using CAR T cell therapy in medulloblastoma and neuroblastoma. Response to standard therapies for advanced neuroblastoma and medulloblastoma is poor and carries significant toxicities, which may be ameliorated by immunotherapy. We found that multiple medulloblastoma and neuroblastoma cell lines (DAOY, D283 Med, D425 Med, UW-228, and SKNAS) express CSPG4 at high levels, as does 143b osteosarcoma as a positive control. By contrast, CSPG4 in normal tissue is expressed only on pericytes and in low levels on small bowel as previously assessed by protein array [Beard et al, J Immunother Cancer 2014]. To evaluate therapeutic efficacy, a second generation CAR with a CD3ζ signaling domain and CD28 co-stimulatory domain containing the TP41.2 anti-CSPG4 scFv was transduced using a γ-retrovirus into T cells. CAR T cells were found to specifically kill 20% of DAOY, 50% of D283 Med, and 50% of 143b cancer cells at a 20:1 effector-to-target ratio in a 4-hour chromium release cytotoxicity assay, while not affecting CSPG4 negative control SY5Y (neuroblastoma) and DND41 (T cell leukemia) cells. Both CD4 and CD8 CAR T cells were also shown to produce IFNγ after exposure to 143b cells. Cytokine production in response to other CSPG4+ lines is ongoing as are cytotoxicity studies with neuroblastomas. For our in vivo model, we have begun to evaluate the efficacy of CAR T cells delivered either intravenously or via intratumoral injection against DAOY and D283 Med cells that have been orthotopically implanted in the cerebellum of immunodeficient NOD/SCID/IL2Rγ-/- mice. These results, if successful, will inform the development of a new clinical protocol for the treatment of children and young adults with medulloblastoma and neuroblastoma. Citation Format: Christopher M. Rota, Nicholas Tschernia, Steven Feldman, Crystal Mackall, Daniel W. Lee. T cells engineered to express a chimeric antigen receptor targeting chondroitin sulfate proteoglycan 4 (CSPG4) specifically kill medulloblastoma and produce inflammatory cytokines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3151. doi:10.1158/1538-7445.AM2015-3151

449. Multipathogen-Specific T Cells for Immune Reconstitution – A Decade of Manufacturing Development and Clinical Use

Since 2003 we have treated 101 patients with antigen specific T cells for treatment or prevention of opportunistic infections in the context of haemopoietic progenitor cell (HPC) and solid organ transplant. Over this time, we have modified laboratory procedures to progressively incorporate T cell therapy into standard transplant practice. We have increased the number of pathogen targets, utilised HPC products and streamlined culture techniques. We are now able to produce a multipathogen product within 3 weeks of HPC harvest in a manner consistent with good manufacturing practice and within the scope of systems established for HPC processing. Our approach could be used by most accredited transplant laboratories.We have developed robust, simple and inexpensive methods that use marrow or G-CSF mobilised HPC products as starting material for T cell expansion. This has major practical importance in minimising repeated donor screening, blood draws and apheresis. Our current procedure uses monocyte derived dendritic cells (mo-DC) isolated using a modified CD14 selection method that requires minimal quantities of CD14 selection reagent. Maturation of mo-DCs utilises GM-CSF, IL-4, PG-E2, IL-6 and IL-1β. Multiple antigens are presented to T cells by mo-DCs including peptide mixes for cytomegalovirus (CMV), Epstein Barr virus (EBV), BK virus and adenovirus, vaccines (Varicella Zoster and Influenza virus) and mycelial lysate (Aspergillus). Activated antigen specific T cells are expanded using IL-2. We use no T cell selection/depletion procedures, feeder layers, cytokine, tetramer capture or clinical grade sorting methodologies. The overall cost of manufacture of each product, including reagents, staffing and ongoing infrastructure costs is approximately AU$2400 per dose.The phenotypic composition of multipathogen products is mainly CD3 (mean 97%, range 90-97), with variable CD4:8 ratio, minimal contaminating B cells (mean 0.1%), monocytes (mean 0.3%) and NK cells (mean 3%). We have demonstrated antigen specificity using intracellular cytokine staining, MHC multimer analysis (CMV and EBV), interferon-γ Elispot and chromium (Cr51) release cytotoxicity assay. Products do not show alloreactivity by Cr51 release assay (Mean 0.8% specific lysis at E:T ratio of 20:1, range 0-5.3).Clinical results of the first 50 patients treated with CMV specific T cells have previously been reported and showed infusion to be safe and to reduce the need for CMV-directed antiviral therapy. Clinical outcomes of the remaining patients treated with multi-pathogen T cells specific for up to 8 pathogens will be reported when data is mature.Future areas of development will involve extension of the range of infectious pathogens targeted, addition of cells targeting malignant antigens and combination of these therapies with CD34+ stem cell selection. Developing progressively greater selectivity through graft engineering and immunotherapy will improve outcomes of transplantation in the future.

Regulatory T Cells Expanded from HIV-1-Infected Individuals Maintain Phenotype, TCR Repertoire and Suppressive Capacity

While modulation of regulatory T cell (Treg) function and adoptive Treg transfer are being explored as therapeutic modalities in the context of autoimmune diseases, transplantation and cancer, their role in HIV-1 pathogenesis remains less well defined. Controversy persists regarding their beneficial or detrimental effects in HIV-1 disease, which warrants further detailed exploration. Our objectives were to investigate if functional CD4+ Tregs can be isolated and expanded from HIV-1-infected individuals for experimental or potential future therapeutic use and to determine phenotype and suppressive capacity of expanded Tregs from HIV-1 positive blood and tissue. Tregs and conventional T cell controls were isolated from blood and gut-associated lymphoid tissue of individuals with HIV-1 infection and healthy donors using flow-based cell-sorting. The phenotype of expanded Tregs was assessed by flow-cytometry and quantitative PCR. T-cell receptor ß-chain (TCR-β) repertoire diversity was investigated by deep sequencing. Flow-based T-cell proliferation and chromium release cytotoxicity assays were used to determine Treg suppressive function. Tregs from HIV-1 positive individuals, including infants, were successfully expanded from PBMC and GALT. Expanded Tregs expressed high levels of FOXP3, CTLA4, CD39 and HELIOS and exhibited a highly demethylated TSDR (Treg-specific demethylated region), characteristic of Treg lineage. The TCRß repertoire was maintained following Treg expansion and expanded Tregs remained highly suppressive in vitro. Our data demonstrate that Tregs can be expanded from blood and tissue compartments of HIV-1+ donors with preservation of Treg phenotype, function and TCR repertoire. These results are highly relevant for the investigation of potential future therapeutic use, as currently investigated for other disease states and hold great promise for detailed studies on the role of Tregs in HIV-1 infection.

Myeloma Can Modulate Expanded Natural Killer Cell Function Through Multiple Mechanisms

Abstract 4020We previously showed that highly activated natural killer cells can be generated in large numbers from the peripheral blood mononuclear cells (PBMC) of healthy donors and multiple myeloma (MM) patients by co-culturing with K562 stimulator cells modified to express 41BBL and membrane bound IL15 (Garg et al. Haematologica, 2012; in press). We are utilizing these expanded NK (ENK) cells in a clinical trial to treat genomically-defined high-risk relapsed MM. In this study we investigated possible mechanisms of MM escape from ENK cell control. First we determined whether it is feasible to induce ENK cell resistance by co-culturing 11 MM cell lines with ENK cells from the same donor in 6 well-plates at E:T ratios of 10:1. Fresh ENK cells were added every 3–4 weeks. The MM cell lines were tested for emergence of resistance at regular intervals using standard chromium release cytotoxicity assays. In seven of 11 cell lines tested all MM cells died upon adding ENK cells. However, from 4 MM cell lines (OPM2, JJN3, ANBL-6, INA-6), subpopulations emerged which were resistant to ENK cell-mediated lysis (Figure 1A). These cell lines retained resistance even when passaged for ≥3 months in the absence of ENK cells. Flow cytometry showed down regulation of Tumor Necrosis Factor-Related Apoptosis Inducing Ligand-Receptors (TRAIL-R) –R1 and -R2 on all ENK resistant cell lines. Further experiments with OPM2 showed that the resistant cell line was less sensitive to recombinant TRAIL-induced apoptosis. Bortezomib treatment of both ENK-resistant and-sensitive OPM2 cell lines increased TRAIL-R1 and –R2 expression and susceptibility to recombinant TRAIL. Interestingly, gene expression profiling of CD138 positive MM patient cells failed to demonstrate a statistically significant difference in the level of TRAIL-R1 and-R2 expression among molecular subgroups, high- versus low-risk 70 gene score, diagnosis versus relapse, or baseline versus 48 hours post bortezomib. However, we also observed that TRAIL-R1 gene expression was down regulated in only 2 of the 4 resistant cell lines suggesting that there might not always be a tight correlation for TRAIL-R expression between GEP and flow cytometry. Next, we demonstrated that supernatants derived from both ENK cell-sensitive and -resistant MM cell lines could down modulate cell surface molecules either by ligand-shedding or reducing expression of receptors critical for activation such as CD54, NKp30, NKp80, NKG2D, CD26, CD69, CD70 and DNAM-1. Furthermore, the cytolytic activity of ENK cells was adversely affected, suggesting an additional NK cell suppressive effect mediated by soluble factors. The latter could be reversed by re-incubation of ENK cells in fresh medium with IL2 (Figure 1B). In summary, down regulation of the TRAIL-R deserves further exploration as a potential mechanism of resistance to ENK cell mediated lysis of MM cells and could be examined pre-and post-ENK cell therapy. MM cells may also evade ENK cell-mediated lysis via the production of suppressive factors. However, both mechanisms of resistance may be surmountable by NK cell sensitizing agents such as bortezomib or by generating the optimal cytokine milieu for infused ENK cells. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

160P - Glucose and NKG2D Ligand Expression: A Link Between Cellular Immunogenicity and Warburg Metabolism

ABSTRACT Natural killer group 2D (NKG2D) is an activating receptor constitutively expressed on the surface of human natural killer cells and CD8+ T-lymphocytes. The ligands for NKG2D, including MICA, are up-regulated in several pathophysiological settings, including cancer, and viral infection. In these contexts, glucose metabolism is often perturbed (the Warburg effect) and glucose levels can be low in the tumour microenvironment. We hypothesized that low glucose availability would reduce cell surface NKG2D ligand expression and lower the immunogenicity of malignant cells. Cell lines cultured in different glucose concentrations displayed a direct correlation between glucose level and cell surface expression of MICA, a key activating ligand of NKG2D. Real-time RT-PCR demonstrated an increase in MICA mRNA transcript levels with increasing glucose concentration. Chromium-release cytotoxicity assays demonstrated an NKG2D-dependent change in NK cell-mediated killing of target cells, in response to changing glucose availability. This suggests that extracellular glucose concentration may alter the susceptibility of malignant cells to immune-mediated clearance. We demonstrate that the contribution of glucose to nucleotide synthesis is necessary for the observed increase in MICA expression. In a primary cell-viral infection model of NKG2D ligand induction, we demonstrate that glucose availability determines the level of NKG2D ligand expression in the transition from healthy to infected cells. As with cell line models, reduced glucose availability in this primary cell model limits the up-regulation of NKG2D ligands observed in virus-infected cells. These results suggest that Warburg metabolism is important to support NKG2D ligand expression in these models. The reduction in NKG2D ligand expression in a low glucose environment may represent a novel immune-evasion mechanism in aberrantly vascularized tumour microenvironments. Further understanding of the downstream signaling pathways could lead to the development of novel therapeutic agents. Disclosure All authors have declared no conflicts of interest.

Regulatory T cells can be expanded from HIV-1 infected individuals with preservation of suppressive function and TCR repertoire (170.26)

Abstract While natural regulatory T cells (Tregs) are being explored as therapeutic modalities in the context of autoimmune diseases and transplantation, their role in HIV-1 pathogenesis is less well defined and to date it is unknown if Tregs can be isolated and expanded from HIV-1 infected individuals. Tregs and conventional T cells (Tconvs) were isolated from the peripheral blood of HIV-1 infected and healthy donors using flow-based cell sorting, stimulated and cultured. Expanded Tregs were phenotyped by flow cytometry and qPCR. TCR repertoire diversity was investigated by high-throughput sequencing. Suppressive function was assessed using flow-based proliferation and chromium release cytotoxicity assays. Infectability of expanded Tregs was investigated using VSV-G pseudotyped HIV-GFP. Tregs from HIV-1+ individuals, including infants, were successfully expanded with a mean fold change of 29 at day 7. Expanded Tregs expressed high levels of FOXP3, CTLA4 and HELIOS and a demethylated TSDR (Treg-specific demethylated region). The TCR repertoire of expanded Tregs was not skewed and they maintained their suppressive function in vitro. Expanded Tregs were readily infectable with HIV-GFP. Our data show that Tregs from HIV-1 positive individuals can be successfully isolated and expanded ex vivo. This approach holds great potential for detailed studies on the role of regulatory T cells in the setting of HIV-1 infection and has implications for potential future therapeutic use of Tregs.

Natural killer cell lines preferentially kill clonogenic multiple myeloma cells and decrease myeloma engraftment in a bioluminescent xenograft mouse model

Novel therapies capable of targeting drug resistant clonogenic MM cells are required for more effective treatment of multiple myeloma. This study investigates the cytotoxicity of natural killer cell lines against bulk and clonogenic multiple myeloma and evaluates the tumor burden after NK cell therapy in a bioluminescent xenograft mouse model. The cytotoxicity of natural killer cell lines was evaluated against bulk multiple myeloma cell lines using chromium release and flow cytometry cytotoxicity assays. Selected activating receptors on natural killer cells were blocked to determine their role in multiple myeloma recognition. Growth inhibition of clonogenic multiple myeloma cells was assessed in a methylcellulose clonogenic assay in combination with secondary replating to evaluate the self-renewal of residual progenitors after natural killer cell treatment. A bioluminescent mouse model was developed using the human U266 cell line transduced to express green fluorescent protein and luciferase (U266eGFPluc) to monitor disease progression in vivo and assess bone marrow engraftment after intravenous NK-92 cell therapy. Three multiple myeloma cell lines were sensitive to NK-92 and KHYG-1 cytotoxicity mediated by NKp30, NKp46, NKG2D and DNAM-1 activating receptors. NK-92 and KHYG-1 demonstrated 2- to 3-fold greater inhibition of clonogenic multiple myeloma growth, compared with killing of the bulk tumor population. In addition, the residual colonies after treatment formed significantly fewer colonies compared to the control in a secondary replating for a cumulative clonogenic inhibition of 89-99% at the 20:1 effector to target ratio. Multiple myeloma tumor burden was reduced by NK-92 in a xenograft mouse model as measured by bioluminescence imaging and reduction in bone marrow engraftment of U266eGFPluc cells by flow cytometry. This study demonstrates that NK-92 and KHYG-1 are capable of killing clonogenic and bulk multiple myeloma cells. In addition, multiple myeloma tumor burden in a xenograft mouse model was reduced by intravenous NK-92 cell therapy. Since multiple myeloma colony frequency correlates with survival, our observations have important clinical implications and suggest that clinical studies of NK cell lines to treat MM are warranted.

Pluripotent Stem Cell-Derived Human Natural Killer Cells with Potent Anti-Multiple Myeloma Activity,

Abstract 4034Natural Killer (NK) cells are lymphocytes of the innate immune system with anti-viral and anti-cancer activity. Over the past decade, they have gained interest as a promising cellular source for use in adoptive immunotherapy for the treatment of cancer. Most notably, NK cells play an important role in the graft-vs-tumor effect seen in allogeneic hematopoietic stem cell transplantation (allo-HSCT), and a better understanding of NK cell biology has translated into improved transplant outcomes in acute myelogenous leukemia (AML). Small studies have demonstrated a role for NK cell activity in multiple myeloma (MM) patients receiving allo-HSCT. Investigators have also utilized haplo-identical killer immunoglobulin-like receptor (KIR) mismatched NK cells for adoptive immunotherapy in patients with multiple myeloma (MM). Our group has focused on the development of NK cells from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) as a novel starting source of lymphocytes for immunotherapy. We have previously demonstrated potent anti-tumor activity of hESC-derived NK cells in vitro and in vivo against a variety of different targets. We have also shown that iPSC-derived NK cells from a variety of different somatic cell starting sources posses potent anti-tumor and anti-viral activity. Here, we demonstrate hESC- and iPSC-derived NK cell development in a completely defined, feeder-free system that is amenable to clinical scale-up. These cultures contain a pure population of mature NK cells devoid of any T or B cell contamination, which are common adverse bystanders of cellular products isolated and enriched from peripheral blood. Our cultures are homogenous for their expression of CD56 and express high levels of effector molecules known to be important in anti-MM activity, including KIR, CD16, NKG2D, NKp46, NKp44, FasL and TRAIL. We have now tested the activity of hESC- and iPSC-derived NK cells against MM tumor cells in order to provide a universal source of lymphocytes for adoptive immunotherapy in patients with treatment refractory disease. We find that similar to peripheral blood NK cells (PB-NK), hESC- and iPSC-derived NK cells are cytotoxic against 3 distinct MM cell lines in a standard chromium release cytotoxicity assay. Specifically, activated PB-NK cells killed 48.5% of targets at 10 to 1 effector to target ratios, whereas hESC (46.3%) and iPSC (42.4%) derived NK cells also demonstrated significant anti-MM activity. Also, hESC- and iPSC-derived NK cells secrete cytokines (IFNγ and TNFα) and degranulate as demonstrated by CD107a surface expression in response to MM target cell stimulation. When tested against freshly isolated samples from MM patients, hESC- and IPSC-derived NK cells respond at a similar level as activated PB-NK cells, the current source of NK cells used in adoptive immunotherapy trials. These MM targets (both cell lines and primary tumor cells) are known to express defined ligands (MICA/B, DR4/5, ULBP-1, BAT3) for receptors expressed on NK cells as well as a number of undefined ligands for natural cytotoxicity receptors (NCRs) and KIR. As these receptor-ligand interactions drive the anti-MM activity of NK cells, we are currently evaluating expression of each of these molecules on the surface of both the effector and target cell populations. Not only do hESC- and iPSC-derived NK cells provide a unique, homogenous cell population to study these interactions, they also provide a genetically tractable source of lymphocytes for improvement of the graft-vs-myeloma effect and could be tailored on a patient specific basis using banks of hESC-or iPSC-derived NK cells with defined KIR genotypes for use as allogeneic or autologous effector cells. Disclosures:No relevant conflicts of interest to declare.

Chemotherapy-resistant osteosarcoma is highly susceptible to IL-15-activated allogeneic and autologous NK cells

High-grade osteosarcoma occurs predominantly in adolescents and young adults and has an overall survival rate of about 60%, despite chemotherapy and surgery. Therefore, novel treatment modalities are needed to prevent or treat recurrent disease. Natural killer (NK) cells are lymphocytes with cytotoxic activity toward virus-infected or malignant cells. We explored the feasibility of autologous and allogeneic NK cell–mediated therapies for chemotherapy-resistant and chemotherapy-sensitive high-grade osteosarcoma. The expression by osteosarcoma cells of ligands for activating NK cell receptors was studied in vitro and in vivo, and their contribution to NK cell–mediated cytolysis was studied by specific antibody blockade. Chromium release cytotoxicity assays revealed chemotherapy-sensitive and chemotherapy-resistant osteosarcoma cell lines and osteosarcoma primary cultures to be sensitive to NK cell–mediated cytolysis. Cytolytic activity was strongly enhanced by IL-15 activation and was dependent on DNAM-1 and NKG2D pathways. Autologous and allogeneic activated NK cells lysed osteosarcoma primary cultures equally well. Osteosarcoma patient–derived NK cells were functionally and phenotypically unimpaired. In conclusion, osteosarcoma cells, including chemoresistant variants, are highly susceptible to lysis by IL-15-induced NK cells from both allogeneic and autologous origin. Our data support the exploitation of NK cells or NK cell–activating agents in patients with high-grade osteosarcoma.Electronic supplementary materialThe online version of this article (doi:10.1007/s00262-010-0965-3) contains supplementary material, which is available to authorized users.

NK-92 Preferentially Targets Acute Myeloid Leukemia Stem Cells

AbstractAbstract 4300 Introduction:Outcome of patients with acute myeloid leukemia (AML) remains poor because many relapse from residual leukemic stem cells (LSCs) enriched in the CD34+CD38- fraction of AML blasts. NK-92 is a human permanent natural killer (NK) cell line in phase I clinical trials for relapsed and refractory malignancies. We recently showed that NK-92 targets LSCs preferentially over bulk leukemia in the cell line KG1 (Cytotherapy. 2010 Mar 15). Here, we evaluate the action of NK-92 and another NK cell line, KHYG-1, against five AML cell lines and five primary AML samples by the chromium release and methylcellulose cytotoxicity assays to determine the mechanism of recognition and killing and the effect on leukemic stem cells. Results:Using a 4 hour chromium release assay (CRA), the highest effector:target (E:T) ratio tested (25:1) for NK-92 and KHYG-1 against cell line targets revealed % lysis as follows: K562: 81.2+/−5.4%; 82.2 +/−3.9%; KG1: 41.0+/−7.2%; 37.3% +/−3.6; OCI/AML2: 37.3+/−22.7%; 33.0+/−13.9%; OCI/AML3: 33.8+/−4.5%; 51.0+/− 6.8% and OCI/AML5: 99.0+/−4.9%; 52.9 +/− 5.6%, respectively. Killing by NK-92 and KHYG-1 was completely inhibited by calcium chelation using 4 mM EGTA for all cell lines tested. Blockade of class I HLA on target cells using 10 ug/ml of anti-class I monoclonal antibody did not affect killing by NK-92 and KHYG-1 except for a decrease in killing of OCI/AML5 by both NK-92 and KHYG-1 from 99.0 +/− 4.9% to 39.0 +/−3.2% and 52.9+/−5.6% to 19.6 +/− 6.25%, respectively. Blockade of NKG2D using 10 ug/ml of anti-NKG2D monoclonal antibody did not significantly affect killing of AML cell lines by NK-92 or KHYG-1. Five primary AML blast samples treated with NK-92 at 25:1 E:T yielded only slight to moderate degrees of cytotoxicity by CRA: 15.6 +/−12.7%, 42.3+/−3.6%, 29.8 +/−3.6%, 43.9 +/− 1.47%, 42.6 +/− 0.1%lysis. KHYG-1 at 25:1 E:T had minimal killing of this panel of AML blasts by CRA: 1.27 +/−21.9%, 9.8+/−2.2%, 5.2+/−2.5%, 17.1+/−2.8%, 8.5+/−3.3% lysis. Blockade of class I HLA did not affect killing of primary AML blasts by NK-92, but the fourth sample only was rendered more sensitive to killing by KHYG-1 from 17.1 +/−2.8% to 35.8 +/−1.2% lysis. Blockade of NKG2D with 10 ug/ml anti-NKG2D monoclonal antibody did not significantly affect killing of primary AML samples by NK-92 or KHYG-1. To further assess killing by NK-92 of LSCs from primary AML, we used a methylcellulose cytotoxicity assay (MCA) established previously by our lab. The MCA showed that NK-92 at 25:1 E:T eliminated clonogenic growth of 3/5 primary AML blast samples with minimal colony growth in 2/5 with % cytotoxicity of 100 +/−0%, 86.3 +/− 2.3%, 98.4 +/−2.8%, 100 +/− 0% and 100 +/−0%, demonstrating higher toxicity than obtained with the CRA. Primary AML derived CD34+CD38- sorted LSCs were more sensitive to killing than CD34+CD38+ blasts by NK-92 in a 4 hour CRA at 1:1 E:T: 58.9+/−11.5%, 20.3 +/−1.71%; 5:1: 78.3 +/−9.7%, 43.5+/−11.1% and 10:1: 72.9+/−5.6%, 38.5 +/−2.4% lysis. Summary and conclusion:We demonstrate cytotoxicity of NK-92 and KHYG-1 against a range of AML targets and show greater killing of primary AML blasts by NK-92. The mechanism of cytotoxicity for both cell lines is primarily by granule exocytosis and is NKG2D independent. Unexpectedly, blockade of class I HLA resulted in reduced killing of OCI/AML5 by both NK-92 and KHYG-1, suggesting the presence of an activating KIR receptor common to both cell lines. One of five primary AML samples had increased killing by KHYG-1, suggesting KIR mediated inhibition. Further assessment of NK-92 against LSCs by the MCA demonstrated greater cytotoxicity than with the CRA and indicated preferential killing of LSCs. This finding was confirmed using the CRA with sorted immunophenotypically defined CD34+CD38- LSCs and CD34+CD38+ blasts. Our findings support the use of NK-92 in the treatment of AML. Disclosures:No relevant conflicts of interest to declare.

Clonogenic assays measure leukemia stem cell killing not detectable by chromium release and flow cytometric cytotoxicity assays

NK-92, a permanent natural killer (NK) cell line, shows cytotoxicity against a variety of tumors and has been tested in a phase I trial. We tested the toxicity of NK-92 and chemotherapy drugs against the stem cell capacity of the acute leukemia cell line, KG1. While the chromium-release assay is the most common method for assessing cytotoxicity of immune effectors, and flow cytometry is increasingly used, the relationship of either assay to clonogenic readouts remains unknown. KG1 was assessed for stem cell frequency by serial dilution, single-cell sorting and colony growth in methylcellulose. KG1 was sorted into CD34(+) CD38(+) and CD34(+) CD38⁻ populations and recultured in liquid medium or methylcellulose to determine the proliferative capacity of each fraction. Cytotoxicity of NK-92, daunorubicin and cytarabine against KG1 was measured using the chromium-release assay, flow cytometry and clonogenic assays. The culture-initiating cell frequency of whole KG1 was between 1 in 100 to 1000 by serial dilution and single-cell sorting. Although a rare (1-3%) CD34(+) CD38⁻ population could be demonstrated in KG1, both fractions had equivalent proliferative capacity. The cumulative flow cytotoxicity assay was more sensitive than the chromium-release assay in detecting target cell killing. At a 10:1 ratio NK-92 eliminated the clonogenic capacity of KG1, which was not predicted by the chromium-release assay. Clonogenic assays provide a more sensitive means of assessing the effect of a cytotoxic agent against putative cancer stem cells within cell lines, provided that they grow well in liquid culture medium or methylcellulose.

Potential therapeutic activity of adecatumumab (MT201), a fully human monoclonal antibody, against epithelial cell adhesion molecule (EpCAM) in uterine serous papillary carcinoma

e16502 Background: Uterine serous papillary carcinoma (USPC) represents a variant of endometrial cancer characterized by a highly aggressive biologic behavior and inborn resistance to chemotherapy. MT201 is a fully human monoclonal antibody recently developed against epithelial cell adhesion molecule (EpCAM). In this study we have evaluated the potential of MT201 as a novel therapeutic strategy against USPC. Methods: EpCAM expression was evaluated by real time-PCR and immunohistochemistry (IHC) in a total of 54 USPC fresh-frozen biopsies and paraffin embedded tissues. EpCAM surface expression was evaluated by flow cytometry in 6 freshly established USPC cell lines derived from advanced stage USPC patients. Sensitivity to MT201 antibody dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) was tested in standard 4-hour chromium (51Cr) release cytotoxicity assays. Results: EpCAM transcript was significantly overexpressed in fresh frozen USPC when compared to fresh-frozen normal endometrial cells (NEC) [i.e., mean (minimum-maximum) copy number of 515.4 (31.5–1568.3) in tumor samples versus 8.1 (1.0–25.5) in flash-frozen NEC (p < 0.001)]. By IHC, EpCAM protein expression was found in 96% (26 out of 27) USPC samples and this expression was significantly higher when compared to normal endometrial cells (p < 0.01). High surface expression of EpCAM by flow cytometry was found in 83% (5 out of 6) of the primary USPC cell lines available to this study. Importantly, while primary USPC cell lines were highly resistant to natural killer dependent cytotoxicity in vitro, EpCAM positive cell lines were found highly sensitive to MT201-mediated ADCC. Human serum IgG did inhibit MT201-mediated cytotoxicity against USPC in vitro. Conclusions: These results demonstrate for the first time high EpCAM expression in uterine serous carcinoma at mRNA and protein levels and high sensitivity to MT201-mediated cytotoxicity in vitro by primary USPC cell lines. MT201 might be a novel and attractive therapeutic strategy in patients harboring advanced, recurrent or metastatic USPC refractory to standard treatment modalities. No significant financial relationships to disclose.

HapMap Scanning of Novel Human Minor Histocompatibility Antigens

Minor histocompatibility antigens (mHags) are the molecular targets of allo-immunity associated with major anti-tumor activities in hematopoietic stem cell transplantation (HSCT), but are also involved in the pathogenesis of graft-versus-host disease (GVHD). They are typically defined by the host's SNPs that are not shared by the donor and immunologically recognized by cytotoxic T-cells isolated from the post-HSCT patients. However, despite their critical importance in transplantation medicine, fewer than 20 mHags have been identified during the past 20 years due to the lack of an efficient method for their isolation.Here we developed a novel method in which the large data set from the International HapMap Project can be directly used for genetic mapping of novel mHags. Concretely, immortalized B lymphoblastoid cell lines (LCLs) from a HapMap panel are grouped into mHag positive (mHag+) and negative (mHag−) subpanels according to their susceptibility to a cytotoxic T-cells (CTL) clone as determined by conventional chromium release cytotoxicity assays (CRAs), and the target mHag locus could be directly identified by association scan (indicated by χ2 statistic) using the highly qualified HapMap data set having over 3,000,000 SNP markers. The major concern about this approach arises from the risk of overfitting observed phenotypes to one or more incidental SNPs from this large number of the HapMap SNPs. To address this problem, we first estimated the maximum sizes of the test statistics under the null hypothesis (i.e., no associated SNPs within the HapMap set) empirically by simulating 10,000 case-control HapMap panels in different experimental conditions, and compared them with the expected size of test statistic values from the marker SNPs associated with the target SNP, assuming different linkage disequilibrium (LD), or values in between. Except for those mHags having very low minor allele frequencies (MAF) below ~0.05, the possibility of overfitting is progressively reduced as the number of LCLs increases, allowing for unique identification of the target locus in a broad range of values.To demonstrate the feasibility of this method, we tried to map the locus for HA-1H mHag, by actually immunophenotyping 58 LCLs from the JPT+CHB HapMap panel with CRAs using HLA-A*0206-restricted LCL (CTL-4B1). As expected, the genome-wide scan clearly indicated a unique association within the HMHA1 gene, showing a peak χ2 statistic of 52.8 (not reached in 100,000 permutations) at rs10421359. Next, we applied this method to mapping novel mHags recognized by HLA-B*4002-restricted CTL-3B6 and HLA-A*0206-restricted CTL-1B2, both of whose target mHags had not been identified. The peak in chromosome 19q13.3 for the CTL-3B6 set showed the theoretically maximum χ2 value of 50 (not reached in 100,000 permutations) at rs3027952, which was mapped within a small LD block of ~182kb containing a single gene, SLC1A5, as a candidate mHag gene. In fact, when expressed in HEK293T with HLA-B*4002 transgene, recipient-derived, but not donor-derived, SLC1A5 cDNA was able to stimulate interferon-γ secretion from CTL-3B6, indicating that SLC1A5 encodes the target mHag recognized by CTL-3B6. Conventional epitope mapping finally identified an undecameric peptide, AEATANGGLAL, which was further confirmed by epitope reconstitution assays. The target mHag locus for CTL-1B2 was identified at the peak (max χ2 = 44, not reached in 100,000 permutations) within a 598 kb block on chromosome 4q13.1, and coincides with the locus for a previously reported mHag, UGT2B17. Our epitope mapping by using UGT2B17 cDNA deletion mutants, prediction of candidate epitopes by HLA-binding algorithms and epitope reconstitution assays successfully identified a novel nonameric peptide, CVATMIFMI.Our results demonstrate how effectively the HapMap resources could be used for genetic mapping of clinically relevant human traits. This method may be also applied to disclosing other relevant human variations, if an accurate bioassay is applied to discriminate them. We anticipate our method based on the HapMap scan greatly accelerates isolation of novel mHags, which could be used for the development of selective allo-immune therapies to intractable blood cancers, circumventing potentially life-threatening GVHD, while harnessing its anti-tumor effects. Such knowledge on mHags should also promote our understanding of allo-immunity.

Pilot study of anti-angiogenic vaccine using fixed whole endothelium in patients with progressive malignancy after failure of conventional therapy

Vaccines targeting tumour angiogenesis were recently shown to inhibit tumour growth in animal models. However, there is still a lack of information about the clinical utility of anti-angiogenic vaccination. Therefore, here, we aimed to test the clinical effects of a vaccine using glutaraldehyde-fixed human umbilical vein endothelial cells (HUVECs). Six patients with recurrent malignant brain tumours and three patients with metastatic colorectal cancer received intradermal injections of 5 × 10 7 HUVECs/dose (in total 230 vaccinations). ELISA and flow cytometry revealed immunoglobulin response against HUVECs’ membrane antigens. ELISPOT and chromium-release cytotoxicity assay revealed a specific cellular immune response against HUVECs, which were lysed in an effectors:targets ratio-dependent manner. Gadolinium-contrasted MRI showed partial or complete tumour responses in three malignant brain tumour patients. Except for a DTH-like skin reaction at the injection site, no adverse effect of vaccination could be observed. Our results suggest that the endothelial vaccine can overcome peripheral tolerance of self-angiogenic antigens in clinical settings, and therefore should be useful for adjuvant immunotherapy of cancer.

Investigating the Combination of Trastuzumab and HER2/neu Peptide Vaccines for the Treatment of Breast Cancer

Trastuzumab, an anti-HER2/neu monoclonal antibody, is thought to promote HER2/neu receptor internalization and/or turnover. This study was designed to investigate the kinetics of trastuzumab treatment on tumor cells with varying levels of HER2/neu expression and to determine the effect of trastuzumab on HER2/neu-specific cytotoxic T lymphocyte-mediated lysis. Three cell lines with varying levels of HER2/neu expression were incubated with varying doses of trastuzumab at multiple time points. Trastuzumab binding and HER2/neu expression were determined. Peripheral blood mononuclear cells from three HLA-A2(+) healthy donors and four E75 peptide-vaccinated patients were stimulated with HER2/neu-derived peptides and tested in standard chromium release cytotoxicity assays with HER2/neu(+) tumor cells pretreated with trastuzumab. Treatment of tumor cells with 10 microg/mL of trastuzumab in an overnight incubation resulted in saturation of cell-surface HER2/neu receptors. At higher doses, trastuzumab staining and HER2/neu expression decreased in a time-dependent manner. Pretreatment of tumor cells with trastuzumab resulted in increases in specific cytotoxicity by peptide-stimulated cytotoxic T lymphocytes from HLA-A2(+) donors over untreated cells by an average of 5.6% and 15.3% (P = .0002) for doses of 10 and 50 microg/mL, respectively. In similar experiments involving peripheral blood mononuclear cells obtained from immunized patients, the average specific cytotoxicity for untreated cells was 34.2% +/- 1.3% vs. 40.6% +/- 2.5% (P = .035) and 40.7% +/- 1.6% (P = .0005) for those treated with 10 and 50 microg/mL, respectively. Our data suggest that pretreatment of breast cancer cells with trastuzumab induces turnover of the HER2/neu protein and enhanced killing by HER2/neu peptide-stimulated CTLs. This increased lysis occurs regardless of the degree of HER2/neu expression and seems more pronounced in vaccinated patients. These findings support further investigation into the use of combination immunotherapy with trastuzumab and HER2/neu peptide-based vaccines.

Pathogenic Role for Virus-Specific CD4 T Cells in Mice with Coronavirus-Induced Acute Encephalitis

Acute viral encephalitis is believed to result from direct virus destruction of infected cells and from virus-induced host immune response, but the relative contribution of each remains largely unknown. For example, C57BL/6 (B6) mice infected with mouse hepatitis virus (JHM strain, JHMV) develop severe encephalitis, with death occurring within 7 days. Here, we show that the host response to a single JHMV-specific immunodominant CD4 T-cell epitope is critical for severe disease. We engineered a recombinant JHMV with mutations in the immunodominant CD4 T-cell epitope (rJ.MY135Q). Infection of naïve B6 mice with this virus resulted in mild disease with no mortality. However, introduction of a CD4 T-cell epitope from Listeria monocytogenes into rJ.MY135Q generated a highly virulent virus. The decrease in disease severity was not due to a switch from Th1 to Th2 predominance in rJ.MY135Q-infected mice, an effect on CD8 T-cell function, or differential expression of tumor necrosis factor-α by JHMV-specific CD4 T cells. These results show that the response to a single virus-specific CD4 T-cell epitope may contribute to a pathogenic host response in the setting of acute viral disease and that abrogation of this response ameliorates clinical disease without diminishing virus clearance.

Reply to ‘NK cell subsets and CD107a mobilization assay’ by Zimmer et al

In the extensive comment by Zimmer et al on our article three points of criticism are expressed: (1) a simultaneous publication dealing with the same topic has not been cited, (2) the reported results are not specific for CD56 NK-cells because CD3 NKT-cells were not excluded from the analysis and (3) the degranulation of CD56CD16 in response to tumor targets does not fit to previous studies reporting cytotoxic effects after co-incubation with CD56CD16 NK cells. First, although we have not cited the online version of the most recent paper of Alter et al, we have cited the earlier work by this group on the assessment of NK-cell activity by CD107a surface expression in the introduction of our article. Second, the analysis of CD3 CD56 NK-T cells (after staining with anti-CD3 FITC mAb, anti-CD56 PE and antiCD107a PE-Cy5) revealed that ca 2% of these cells degranulated in response to K562 cells. Since the finding that a part of the CD56CD16 cells are NK-T cells, which to some extent mediate natural cytotoxicity towards tumor targets, is not new we have not discussed it explicitly in our paper. Nevertheless (and most importantly), our observation that neither CD56 nor CD56/CD16 NK cells degranulated in response to tumor targets is independent from the presence or absence of CD56CD3 cells. Third, the fact that the surface expression of CD107a in response to tumor targets is restricted to the CD56/CD16 subpopulation has been reproducibly and unambiguously shown under different experimental conditions. This finding and the close correlation of degranulation and cytotoxicity can indeed not easily be linked to the results of previous studies showing that CD56/CD16 NK cells are able to kill K562 target cells, as indicated in chromium release cytotoxicity assays. We apologize that we have not further discussed this incongruity by ourselves and so far cannot provide a convincing explanation for these discrepant but clear results. However, there are many examples for scientific progress through results that are somehow discrepant to previous and well-established assumptions. We hope that our reply has helped to clarify methodological aspects and that our findings may have stimulated the discussion in this field.