Target Cell Lysis Research Articles

Abstract LB-A07: Using Impedance-Based Approaches for Measuring Cell-Mediated Cytotoxicity and Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC)

Abstract The most common method for measuring cell mediated cytotoxicity is the release assay based on the loss of target cell membrane integrity. Within up to 4 hours following effector cell addition resulting in target cell lysis, either the radioactive release from target cells pre-labeled with Chromium-51 or Indium-111 is measured, or the release of naturally occurring substances, such as lactate dehydrogenase (LDH), into the culture medium is assayed. Release of these substances thus serves as an indirect measure of the extent of cell damage due to effector cell-mediated target cell lysis. Alternative endpoint methods also include flow cytometry, enzyme-linked immunosorbent assay-based granzyme measurement, and morphometric analyses by microscopy. Here we describe an impedance-based real-time label-free method that can automated capture the kinetics of the cell mediated or antibody-dependent cell mediated cytolysis (ADCC) of the target cancer cells. To determine if cell-mediated cytotoxicity, and specifically ADCC, can be investigated using an impedance-based xCELLigence system, the response of tumor cells (as target cells) to natural killer (NK) cell activity (as effector cells), in the presence or absence of immunoglobulin G isotype-specific antibody, was measured. Importantly, it is shown that the addition of NK cells in suspension, over a monolayer of adherent tumor cells, does not produce impedance changes, because the NK cells do not come in contact with the electronic sensor. However, the secretion of perforins and granzymes by these non-adherent NK cells does activate caspases inducing tumor cell apoptosis. Dysfunctional and dying tumor cells detach from the sensor electrode, reducing the number of viable and adhering cells on the electrode surface. Overall, our results show that the impedance-base technology is a label-free alternative to the traditional end-point assays. The automated readouts provide direct, sensitive and specific measurement of target cell changes both short-term (hrs) and long-term (days). It allows the easy quantification of the cell-mediated cytotoxicity and evaluation of the potencies for specific antibodies. Citation Format: Leyna Zhao, Yama Abassi. Using Impedance-Based Approaches for Measuring Cell-Mediated Cytotoxicity and Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-A07.

A Promoter Polymorphism in the CD59 Complement Regulatory Protein Gene in Donor Lungs Correlates With a Higher Risk for Chronic Rejection After Lung Transplantation.

Complement activation leads primarily to membrane attack complex formation and subsequent target cell lysis. Protection against self-damage is regulated by complement regulatory proteins, including CD46, CD55, and CD59. Within their promoter regions, single-nucleotide polymorphisms (SNPs) are present that could influence transcription. We analyzed these SNPs and investigated their influence on protein expression levels. A single SNP configuration in the promoter region of CD59 was found correlating with lower CD59 expression on lung endothelial cells (p = 0.016) and monocytes (p = 0.013). Lung endothelial cells with this SNP configuration secreted more profibrotic cytokine IL-6 (p = 0.047) and fibroblast growth factor β (p = 0.036) on exposure to sublytic complement activation than cells with the opposing configuration, whereas monocytes were more susceptible to antibody-mediated complement lysis (p < 0.0001). Analysis of 137 lung transplant donors indicated that this CD59 SNP configuration correlates with impaired long-term survival (p = 0.094) and a significantly higher incidence of bronchiolitis obliterans syndrome (p = 0.046) in the recipient. These findings support a role for complement in the pathogenesis of this posttransplant complication and are the first to show a deleterious association of a donor CD59 promoter polymorphism in lung transplantation.

A Novel Method for Assessment of Natural Killer Cell Cytotoxicity Using Image Cytometry

Natural killer (NK) cells belong to the innate arm of the immune system and though activated NK cells can modulate immune responses through the secretion of cytokines, their primary effector function is through target cell lysis. Accordingly, cytotoxicity assays are central to studying NK cell function. The 51Chromium release assay, is the “gold standard” for cytotoxicity assay, however, due to concerns over toxicity associated with the use and disposal of radioactive compounds there is a significant interest in non-radioactive methods. We have previously used the calcein release assay as a non-radioactive alternative for studying NK cell cytotoxicity. In this study, we show that the calcein release assay varies in its dynamic range for different tumor targets, and that the entrapped calcein could remain unreleased within apoptotic bodies of lysed tumor targets or incompletely released resulting in underestimation of percent specific lysis. To overcome these limitations, we developed a novel cytotoxicity assay using the Cellometer Vision Image Cytometer and compared this method to standard calcein release assay for measuring NK cell cytotoxicity. Using tumor lines K562, 721.221, and Jurkat, we demonstrate here that image cytometry shows significantly higher percent specific lysis of the target cells compared to the standard calcein release assay within the same experimental setup. Image cytometry is able to accurately analyze live target cells by excluding dimmer cells and smaller apoptotic bodies from viable target cell counts. The image cytometry-based cytotoxicity assay is a simple, direct and sensitive method and is an appealing option for routine cytotoxicity assay.

Redirection of CD4+ and CD8+ T lymphocytes via an anti-CD3 × anti-CD19 bi-specific antibody combined with cytosine arabinoside and the efficient lysis of patient-derived B-ALL cells

BackgroundB-acute lymphoblastic leukemia (B-ALL) is derived from B cell progenitors. Recently, the development of appropriate combinations of chemotherapy and immunotherapy represents a promising approach for eliminating cancer. We previously constructed an anti-CD3 × anti-CD19 bi-specific antibody in a diabody configuration and its disulfide-stabilized format (ds-diabody). The combination of the diabody or ds-diabody and Ara-C was highly effective in enhancing the cytotoxicity of T cells against the CD19+ human leukemia cell-line, Nalm-6, both in vitro and in vivo. This study verified whether B-ALL patient-derived cells were sensitive to the diabody or ds-diabody and low-dosage Ara-C combination.MethodsThis study aimed to detect the B7 family members B7.1 (CD80) and B7.2 (CD86) that were expressed in B-ALL patient-derived cells pre-treated by Ara-C (0.25 μM) and to determine the targeted killing ability of T cell subtypes induced by the diabody or ds-diabody combination with Ara-C both in vitro and in vivo. We also determined the levels of the cytokines that were released by activated CD4+ or CD8+ T cells during therapy.ResultLow-dose Ara-C enhanced CD80 and CD86 expression in nearly 50 % of specimens of B-ALL patient-derived cells. A combination of diabody or ds-diabody and Ara-C enhanced T cell against B-ALL cells in vitro and in vivo. Both CD8+ and CD4+ T cells were potently activated. Expression of CD25 and CD69 was augmented equally by CD4+ or CD8+ T cells. However, CD8+ T cells made the major contribution by redirecting target cell lysis in a granzyme B and perforin-dependent mechanism. CD4+ T cells played an important immunomodulatory role by secreting IL2. Consequently, IL3, IL6, TNFα, and IFNγ were also released by CD4+ or CD8+ T cells following diabody-mediated T cell activation.ConclusionT cell therapy induced by diabody or ds-diabody combined with low dose of Ara-C was effective against cancer cell-lines and in clinical trials. In vivo, the ds-diabody was more efficient than its parent diabody due to its enhanced stability.

A DNA vaccine encoding mutated HPV58 mE6E7-Fc-GPI fusion antigen and GM-CSF and B7.1.

BackgroundPersistent infection with high-risk human papillomavirus (HPV) is a predominant cause of cervical cancer, and HPV58 is the third most common virus detected in the patients with cervical cancer in Asia. E6 and E7 are the viral oncogenes which are constitutively expressed in HPV-associated tumor cells and can be used as target antigens for related immunotherapy. In this study, we modified the HPV58 E6 and E7 oncogenes to eliminate their oncogenic potential and constructed a recombinant DNA vaccine that coexpresses the sig-HPV58 mE6E7-Fc-GPI fusion antigen in addition to granulocyte-macrophage colony-stimulating factor (GM-CSF) and B7.1 as molecular adjuvants (PVAX1-HPV58 mE6E7FcGB) for the treatment of HPV58 (+) cancer.MethodsPVAX1-HPV58 mE6E7FcGB recombinant DNA vaccine was constructed to express a fusion protein containing a signal peptide, a modified HPV58 mE6E7 gene, and human IgG Fc and glycosylphosphatidylinositol (GPI)-anchoring sequences using the modified DNA vaccine vector PVAX1-IRES-GM/B7.1 that coexpresses GM-CSF, and B7.1. C57BL/6 mice were challenged by HPV58 E6E7-expressing B16-HPV58 E6E7 cells, followed by immunization by PVAX1-HPV58 mE6E7FcGB vaccine on days 7, 14, 21 after tumor challenge. The cellular immune responses in immunized mice were assessed by measuring IFN-γ production in splenocytes upon stimulation by HPV58 E6E7-GST protein and the lysis of B16-HPV58 E6E7 target cells by splenocytes after restimulation with HPV58 E6E7-GST protein. The antitumor efficacy was evaluated by monitoring the growth of the tumor.ResultsPVAX1-HPV58 mE6E7FcGB elicited varying levels of IFN-lsgdB58onn T-cell immune responses and lysis of target cell in mice in response to the recombinant antigen HPV58 E6E7-GST. Furthermore, the vaccine also induced antitumor responses in the HPV58 (+) B16-HPV58 E6E7 tumor challenge model as evidenced by delayed tumor development.ConclusionThe recombinant DNA vaccine PVAX1-HPV58 mE6E7FcGB efficiently generates cellular immunity and antitumor efficacy in immunized mice. These data provide a basis for the further study of this recombinant vaccine as a potential candidate vaccine.

Abstract B57: NK cell-mediated cytotoxicity detection can be miniaturized by direct imaging in assay wells using a Terasaki plate format using only 100,000 effector cells (PBMC)

Abstract Introduction: Natural Killer (NK) Cell - and Antibody Dependent (ADCC) Cell - Cytotoxicity has traditionally been assessed by the release of radioactive Chromium from target cells following lysis. This assay is laborious, and requires substantial quantities of patient blood to detect minor changes in cell lysis due to the inherent background noise (spontaneous release). We have developed an assay that can visualize individual target cells to detect cytolytic activity without involving radioactivity, via high-throughput imaging in microtiter well format. We also developed a miniaturized version of this assay using Terasaki plates to measure NK activity with one tenth of the blood needed for a classic Chromium Release assay, and with less labor. Methods: The assay we developed, Target cell Visualization Assay (TVA™) images individual fluorescence-labeled target cells. Labelled K562 tumor cells were used as targets, and peripheral blood mononuclear cells (PBMC) as effector cells. In an assay setup similar to Chromium Release Assays (CRA), constant number of labelled target cells was incubated with serially diluted effector cells. Four hours later, the cells were transferred to flat bottom microtiter plates and the number of viable tumor cells was quantitated using a plate reader; CTL ImmunoSpot® Analyzer, S6ULT-00-9000. For the miniaturized version, one tenth of effector and target cells were plated in a Terasaki plate format and imaged directly in the assay wells. Results: Only viable target cells retain the fluorescent dye; it is lost upon cell death. When effector and target cells are mixed in various ratios, the % of target cell lysis is inversely proportional to the number of effector cells in the well. The TVA™ and CRA in a 96-well format provided equivalent results. However, the TVA™ is a non-radioactive system and can be performed with much less labor. The TVA™ assay exhibited high intermediate precision as well as inter-assay repeatability. The assay results obtained in Terasaki plates, miniaturizing the assay tenfold, paralleled those of the 96-well format. Conclusion: The miniaturized TVA™ uses only 100,000 effector cells for eight serial dilutions, for Effector:Target ratios starting from 100:1. We have demonstrated the feasibility of detecting and assessing NK function in a non-radioactive, high-throughput capable system using a fraction of the blood/effector cells as is required by traditional CRA. This is of particular value when access to PBMC is limited, such as pediatric, geriatric and immune deficient populations. The assay readout and analysis is automated by the CTL ImmunoSpot® software providing audit trails for each test condition. Citation Format: Srividya Sundararaman, Kinga Karacsony, Diana Roen, Jaya Ghosh, Paul V. Lehmann. NK cell-mediated cytotoxicity detection can be miniaturized by direct imaging in assay wells using a Terasaki plate format using only 100,000 effector cells (PBMC). [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B57.

Deficient Natural Killer Dendritic Cell Responses Underlay the Induction of Theiler's Virus-Induced Autoimmunity.

ABSTRACTThe initiating events in autoimmune disease remain to be completely understood, but it is thought that genetic predisposition synergizes with “environmental” factors, including viral infection, leading to disease. One elegant animal model used to study the pathogenesis of multiple sclerosis that perfectly blends genetics and environmental components in the context of virus-induced autoimmunity is Theiler’s murine encephalitis virus-induced demyelinating disease (TMEV-IDD). TMEV-infected disease-susceptible SJL/J mice develop a persistent central nervous system (CNS) infection and later develop autoimmune demyelination, while disease-resistant C57BL/6 (B6) mice rapidly clear the infection and develop no autoimmune pathology. Mice of the (B6 × SJL/J)F1 cross between these two mouse strains are classified as intermediately susceptible. We employed this model to investigate if rapid virus clearance in B6 versus SJL/J mice was perhaps related to differences in the innate immune response in the CNS of the two strains in the first few days following intracerebral virus inoculation. Here we show that SJL/J mice lack, in addition to NK cells, a novel innate immune subset known as natural killer dendritic cells (NKDCs), which express phenotypic markers (CD11cint NK1.1+) and functional activity of both NK cells and DCs. These NKDCs are activated in the periphery and migrate into the infected CNS in a very late antigen 4 (VLA-4)-dependent fashion. Most significantly, NKDCs are critical for CNS clearance of TMEV, as transfer of NKDCs purified from B6 mice into TMEV-IDD-susceptible (B6 × SJL/J)F1 mice promotes viral clearance. Together the findings of this work show for the first time a link between NKDCs, viral infection, and CNS autoimmunity.

Abstract 1303: Quantification of natural killer cell-mediated cytotoxicity using Celigo imaging cytometry

Abstract Cytotoxicity assays play a central role in studying the function of immune effector cells such as cytolytic T lymphocytes (CTL) and natural killer (NK) cells. Traditionally, cytotoxicity assays have been performed using 51Chromium (51Cr) and Calcein release assays. The assays involve labeling tumor cells (target) with radioisotope or fluorescent dyes, when the target cells are subjected to cytolysis by CTLs or NK cells (effector), they releases the entrapped labels into the media upon lysis. The amount of labels in the media is measured to determine the level of cytotoxicity the effectors have induced. These traditional methods may generate inconsistent results due to low sensitivity caused by poor loading efficiency and high spontaneous release of the reagents. In this work, we demonstrate a novel cytotoxicity assay using the Celigo imaging cytometry method. Utilizing imaging cytometry, direct cell counting of live fluorescent target cells can be performed, which is a direct method for assessment of cytotoxicity. Human NK cells from one healthy donor were used as effectors, and K562 (suspension) and IMR32 (adherent) were used as the target cells. Both target cells were first stained with Calcein AM, and seeded at 10,000 cells/well in a standard 96-well microplate. The donor NK cells were then added to each well at Effector-to-Target (E:T) ratios 10:1, 5:1, 2.5:1, 1.25:1, 0.625:1, and 0.3125:1. The 96 well plate was then scanned and analyzed using Celigo imaging cytometer at t = 1, 2, 3, and 4 h to measure the% lysis of target cells. The results showed increasing% lysis as incubation time and E:T ratio increased. The propose Celigo imaging cytometry is an accurate and simple method for direct quantification of cytotoxicity. Citation Format: Leo Li-Ying Chan, Kelsey Rosbach, Srinivas S. Somanchi, Dean Lee. Quantification of natural killer cell-mediated cytotoxicity using Celigo imaging cytometry. [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 1303. doi:10.1158/1538-7445.AM2015-1303

Abstract 2509: Human anti-MUC1 antibodies elicited by a prophylactic cancer vaccine for mAb and CAR-modified T cell immunotherapies

Abstract MUC1 is a tumor-associated protein that is abnormally expressed on human adenocarcinomas. In our recent clinical trial, individuals at-risk for colonic adenocarcinoma received a prophylactic MUC1 cancer vaccine. Many individuals responded to the vaccine by producing high titers of anti-MUC1 IgG antibodies (Kimura, 2013). We isolated a panel of these antibodies and are seeking to develop them as antibody and chimeric antigen receptor (CAR) T cell cancer immunotherapies. To this end we used a novel method combining proteomics and next-gen DNA sequencing to identify and recombinantly produce 12 high-affinity anti-MUC1 antibodies from the sera of vaccinated individuals. By ELISA we determined that the antibodies bind with high-affinity to diverse epitopes within the MUC1 vaccine peptide. We also found that several of the antibodies can stain MUC1+ pancreas, colon, and breast cancer cell lines, but not a cell line expressing normal MUC1. We then determined that the antibodies could stain MUC1+ human pancreas and colon tumor samples, but not normal tissues. Having established tumor-specificity we have begun assessing the antibodies in a series of functional assays. In preliminary in vitro testing we determined that several of the antibodies can be internalized by MUC1+ cells making them potential candidates for toxin delivery as antibody drug conjugates (ADCs). Additionally, we found that two of the antibodies are capable of efficiently performing complement-dependent cytotoxicity (CDC) on MUC1+ cells. Finally, we have constructed CAR lentiviral expression constructs using the variable regions of several of the antibodies as targeting domains and are testing their ability to mediate MUC1-specific T cell activation, cytokine production, and target cell lysis. As the antibodies are human-derived, indication of efficacy in pre-clinical testing has the potential to directly enable clinical trials and hopefully approval of these reagents for therapy of cancer patients. Citation Format: Jason J. Lohmueller, Shuji Sato, Wan Cheung Cheung, Isabel Chu, Lana Popova, Christopher A. Manning, Katherine Crosby, Christopher Grange, James D. Ham, Roberto D. Polakiewicz, Olivera J. Finn. Human anti-MUC1 antibodies elicited by a prophylactic cancer vaccine for mAb and CAR-modified T cell immunotherapies. [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 2509. doi:10.1158/1538-7445.AM2015-2509

Abstract 2735: Tributyltin alters the production and secretion of inflammatory cytokines from human and mouse immune cells

Abstract Tributyltin (TBT) is an environmental contaminant that has been used for several industrial, agricultural and household purposes. Despite limited bans on the usage of Tributyltin, the stability and toxicity of the compound may prove to have negative effects on immune function in terrestrial mammals. Studies show that there is an increase in the incidences of tumors and decreased immune cell function in TBT-exposed mammals. TBT interferes with the ability of human natural killer (NK) cells to lyse target cells; it also alters the secretion of the pro-inflammatory cytokines Interferon gamma (IFNγ) and Tumor necrosis factor alpha (TNFα) from human immune cells ex vivo. Any dysregulation of the cytokine communication network may have detrimental effects on immune function, as cytokines play a key role in the regulation of immune responsiveness. There have been no in vivo studies on the effects of exposure to TBT on the cytokine communication network. The effects of 24 hour exposure to TBT on the production of IFNγ and TNFα from human immune cells were analyzed in an ex vivo system using western blotting. The serum of TBT-exposed mice was analyzed for changes in the levels of cytokine secretion and production using a Milliplex mouse cytokine/chemokine magnetic bead premixed 32 plex kit and western blotting; the effects of TBT-exposure was assessed using a time curve of 6h, 12h, 24h, 48h and control. TBT increased the levels of the cytokines IFNγ, TNFα, IL1β, IL5, IL7, IL12βp40, IL13, IL15, MIP1β, MIP2 and RANTES in the serum at a minimum of one time point. IFNγ and TNFα secretion and production from human cells also increased, showing striking agreement in the response to TBT between the human and mouse systems. Supported by: NIH grant 5U54CA163066-03 Citation Format: Shanieek T. Lawrence, Margaret Whalen, Samuel Pellom, Anil Shanker. Tributyltin alters the production and secretion of inflammatory cytokines from human and mouse immune cells. [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 2735. doi:10.1158/1538-7445.AM2015-2735

Design Parameters for Granzyme-Mediated Cytotoxic Lymphocyte Target-Cell Killing and Specificity

Cytotoxic lymphocytes are key elements of the immune system that are primarily responsible for targeting cells infected with intracellular pathogens, or cells that have become malignantly transformed. Target cells are killed mainly via lymphocyte exocytosis of specialized lysosomes containing perforin, a pore-forming protein, and granzymes, which are proteases that induce apoptosis. Due to its central role in lymphocyte biology, as well as its implication in a host of pathologies from cancer to autoimmunity, the granzyme-perforin pathway has been the subject of extensive investigation. Nevertheless, the details of exactly how granzyme and perforin cooperate to induce target-cell death remain controversial. To further investigate this system, we developed a biophysical model of the immunological synapse between a cytotoxic lymphocyte and a target cell using a spatial stochastic simulation algorithm. We used this model to calculate the spatiotemporal evolution of granzyme B and perforin from the time of their exocytosis to granzyme internalization by the target cell. We used a metric of granzyme internalization to delineate which biological processes were critical for successful target-cell lysis. We found that the high aspect ratio of the immunological synapse was insufficient in this regard, and that molecular crowding within the synapse is critical to preserve sufficient concentrations of perforin and granzyme for consistent pore formation and granzyme transfer to target cells. However, even when pore formation occurs in our model, a large amount of both granzyme and perforin still escape from the synapse. We argue that a tight seal between the cytotoxic lymphocyte and its target cell is not required to avoid bystander killing. Instead, we propose that the requirement for spatiotemporal colocalization of granzyme and perforin acts as an effective bimolecular filter to ensure target specificity.

Human Cytomegalovirus variant peptides adapt by decreasing their total coordination upon binding to a T cell receptor.

The tertiary structure of the native Cytomegalovirus peptide (NLV) presented by HLA-A2 and bound to the RA14 T cell receptor was used as a reference for the calculation of atomic coordination differences of both the NLV as well as of a number of singly substituted NLV variants in the absence of TCR. Among the pMHC complexes, the native peptide was found to exhibit the highest total coordination difference in respect to the reference structure, suggesting that it experienced the widest structural adaptation upon recognition by the TCR. In addition, the peptide on the isolated NLV-MHC complex was over-coordinated as compared to the rest of the variants. Moreover, the trend was found to account for a set of measured dissociation constants and critical concentrations for target-cell lysis for all variants in complexation with RA14: functionally, all variant peptides were established to be either weak agonists or null peptides, while, at the same time, our current study established that they were also under-coordinated in respect to NLV. It could, thus, be argued that the most ‘efficient’ structural adaptation upon pMHC recognition by the TCR requires of the peptide to undergo the widest under-coordination possible. The main structural characteristic which differentiated the NLV in respect to the variants was a the presence of 16 oxygen atoms (waters) in the former׳s second coordination shell which accounted for over-coordination of roughly 100% and 30% in the O–O and C–O partials respectively. In fact, in the absence of second shell oxygens, the NLV peptide was decidedly under-coordinated in respect to all of the variants, as also suggested by the C–C partial.

Targeting of MYCN by means of DNA vaccination is effective against neuroblastoma in mice.

The MYCN oncogene is a strong genetic marker associated with poor prognosis in neuroblastoma (NB). Therefore, MYCN gene amplification and subsequent overexpression provide a possible target for new treatment approaches in NB. We first identified an inverse correlation of MYCN expression with CD45 mRNA in 101 NB tumor samples. KEGG mapping further revealed that MYCN expression was associated with immune-suppressive pathways characterized by a down-regulation of T cell activation and up-regulation of T cell inhibitory gene transcripts. We then aimed to investigate whether DNA vaccination against MYCN is effective to induce an antigen-specific and T cell-mediated immune response. For this purpose, we generated a MYCN-expressing syngeneic mouse model by MYCN gene transfer to NXS2 cells. MYCN-DNA vaccines were engineered based on the pCMV-F3Ub plasmid backbone to drive ubiquitinated full-length MYCN-cDNA and minigene expression. Vaccines were delivered orally with attenuated S. typhimurium strain SL7207 as a carrier. Immunization with both MYCN-DNA vaccines significantly reduced primary tumor growth of MYCN-expressing NB cells in contrast to negative controls. The immune response was mediated by tumor-infiltrating T cells in vivo, which revealed MYCN-specific and MHC class I-restricted lysis of inducible MYCN-expressing NB target cells in vitro. Finally, these antigen-specific T cells also killed MYCN-negative mammary carcinoma cells pulsed with MYCN peptides in contrast to controls. In summary, we demonstrate proof of concept that MYCN can be targeted by DNA vaccination, which may provide an approach to overcoming MYCN immune-suppressive activities in patients with MYCN-amplified disease.

TGF-βdownregulates KLRG1 expression in mouse and human CD8(+) T cells.

The inhibitory receptor killer cell lectin-like receptor G1 (KLRG1) and the integrin αE (CD103) are expressed by CD8(+) T cells and both are specific for E-cadherin. However, KLRG1 ligation by E-cadherin inhibits effector T-cell function, whereas binding of CD103 to E-cadherin enhances cell-cell interaction and promotes target cell lysis. Here, we demonstrate that KLRG1 and CD103 expression in CD8(+) T cells from untreated and virus-infected mice are mutually exclusive. Inverse correlation of KLRG1 and CD103 expression was also found in human CD8(+) T cells-infiltrating hepatocellular carcinomas. As TGF-β is known to induce CD103 expression in CD8(+) T cells, we examined whether this cytokine also regulates KLRG1 expression. Indeed, our data further reveal that TGF-β signaling in mouse as well as in human CD8(+) T cells downregulates KLRG1 expression. This finding provides a rationale for the reciprocal expression of KLRG1 and CD103 in different CD8(+) T-cell subsets. In addition, it points to the limitation of KLRG1 as a marker for terminally differentiated CD8(+) T cells if lymphocytes from tissues expressing high levels of TGF-β are analyzed.

Cytomegalovirus-specific cytokine-induced killer cells: concurrent targeting of leukemia and cytomegalovirus

Background aimsHuman cytomegalovirus (CMV) infection and reactivation is a leading complication of allogeneic hematopoietic stem cell transplantation (HSCT). In addition to drug treatment, the adoptive transfer of virus-specific T cells to restore cellular immunity has become a standard therapy after allogeneic HSCT. We recently demonstrated potent anti-leukemic activity of interleukin (IL)-15–activated cytokine-induced killer (CIK) cells. With the use of the same expansion protocol, we asked whether concurrent CMV antigen-pulsing might generate CIK cells with anti-leukemic and anti-CMV activity. MethodsCIK cells expanded in the presence of interferon-γ, IL-2, IL-15 and anti-CD3 antibody were pulsed once with CMVpp65 peptide pool. CMV-specific CIK (CIKpp65) and conventional CIK cells were phenotypically and functionally characterized according to their cytokine secretion pattern, degranulation capacity and T-cell receptor (TCR)-mediated and NKG2D-mediated cytotoxicity. ResultsWe demonstrated that among CIK cells generated from CMV-seropositive donors, a single stimulation with CMVpp65 protein co-expanded cytotoxic CMV-specific cells without sacrificing anti-tumor reactivity. Cells generated in this fashion lysed CMVpp65-loaded target cells and CMV-infected fibroblasts but also leukemic cells. Meanwhile, the alloreactive potential of CIKpp65 cells remained low. Interestingly, CMV reactivity was TCR-mediated and CMV-specific cells could be found in CD3+CD8+CD56+/− cytotoxic T-cell subpopulations. ConclusionsWe provide an efficient method to generate CIKpp65 cells that may represent a useful cell therapy approach for preemptive immunotherapy in patients who have both an apparent risk of CMV and impending leukemic relapse after allogeneic stem cell transplantation.

Induction of apoptosis-like cell death by coelomocyte extracts from Eisenia andrei earthworms.

Earthworm's innate immunity is maintained by cellular and humoral components. Our objective was to characterize the cytotoxicity leading to target cell death caused by earthworm coelomocytes. Coelomocyte lysates induced strong cytotoxicity in tumor cell lines. Transmission electron microscopy revealed cell membrane and intracellular damage in cells treated with coelomocyte lysates. Using TUNEL-assay, within 5 min of incubation we detected DNA fragmentation. Moreover, we found phosphatidylserine translocation in target cell-membranes. Furthermore, we detected dose-dependent Ca(2+) influx and decrease of mitochondrial membrane potential in coelomocyte lysate-treated cells. Interestingly, caspase 3/8 activation was undetectable in exposed tumor cells. One such cytotoxic molecule, lysenin identified in earthworms binds to sphingomyelin and causes target cell lysis in vertebrates. Pretreatment with our anti-lysenin monoclonal antibody rescued the majority but not all target cells from coelomocyte induced death. These data suggest that, not only lysenin but also other factors participate in the caspase-independent apoptosis induced by coelomocytes.

Cytokine-Mediated Activation of NK Cells during Viral Infection.

Natural killer (NK) cells provide a first line of defense against infection via the production of antiviral cytokines and direct lysis of target cells. Cytokines such as interleukin 12 (IL-12) and IL-18 are critical regulators of NK cell activation, but much remains to be learned about how cytokines interact to regulate NK cell function. Here, we have examined cytokine-mediated activation of NK cells during infection with two natural mouse pathogens, lymphocytic choriomeningitis virus (LCMV) and murine cytomegalovirus (MCMV). Using a systematic screen of 1,849 cytokine pairs, we identified the most potent combinations capable of eliciting gamma interferon (IFN-γ) production in NK cells. We observed that NK cell responses to cytokine stimulation were reduced 8 days after acute LCMV infection but recovered to preinfection levels by 60 days postinfection. In contrast, during MCMV infection, NK cell responses to cytokines remained robust at all time points examined. Ly49H-positive (Ly49H+) NK cells recognizing viral ligand m157 showed preferential proliferation during early MCMV infection. A population of these cells was still detected beyond 60 days postinfection, but these divided cells did not demonstrate enhanced IFN-γ production in response to innate cytokine stimulation. Instead, the maturation state of the NK cells (as determined by CD11b or CD27 surface phenotype) was predictive of responsiveness to cytokines, regardless of Ly49H expression. These results help define cytokine interactions that regulate NK cell activation and highlight variations in NK cell function during two unrelated viral infections. Natural killer cells play an important role in immunity to many viral infections. From an initial screen of 1,849 cytokine pairs, we identified the most stimulatory cytokine combinations capable of inducing IFN-γ production by NK cells. Ly49H+ NK cells, which can be directly activated by MCMV protein m157, preferentially proliferated during MCMV infection but did not show enhanced IFN-γ production following direct ex vivo cytokine stimulation. Instead, mature CD11b+ and/or CD27+ NK cells responded similarly to innate cytokine stimulation regardless of Ly49H expression. Collectively, our data provide a better foundation for understanding cytokine-mediated NK cell activation during viral infection.

CD137 co-stimulation and blocking PD-1 enhances NK cell-mediated target cell lysis by CD30/CD16A TandAb AFM13.

3050 Background: The CD30/CD16A bispecific tetravalent TandAb antibody AFM13 recruits and activates NK cells by specific binding to CD16A for targeted lysis of CD30+ tumor cells. Given promising cl...

High-throughput direct cell counting-based natural killer cell-mediated cytotoxicity assay using Celigo Imaging Cytometry (TUM2P.1027)

Abstract Cytotoxicity assays play a central role in studying the function of immune effector cells such as cytolytic T lymphocytes (CTL) and natural killer (NK) cells. Traditionally, cytotoxicity assays have been performed using 51Chromium (51Cr) and Calcein release assays. The assays involve labeling tumor cells (target) with radioisotope or fluorescent dyes, when the target cells are subjected to cytolysis by CTLs or NK cells, they releases the entrapped labels into the media upon lysis. These traditional methods may generate inconsistent results due to low sensitivity caused by poor loading efficiency and high spontaneous release of the reagents. In this work, we demonstrate a novel high-throughput direct cell counting method for cytotoxicity assay using the Celigo imaging cytometry method. Human NK cells from one healthy donor were used as effectors, and K562 and IMR32 were used as the target cells. Both target cells were first stained with Calcein AM, and seeded at in a 96-well microplate. NK cells were then added to each well at various Effector-to-Target (E:T) ratios. The 96 well plate was then scanned and analyzed using the Celigo to measure the % lysis of target cells at different time points. The results showed increasing % lysis as incubation time and E:T ratio increased. The propose Celigo imaging cytometry is an accurate and simple method for direct quantification of cytotoxicity, which can be an attractive method for both academic and clinical research.

Type 1 interferon primes naïve CD8 T cells to respond to lower concentrations and affinities of cognate ligands (VIR6P.1178)

Abstract During an acute virus infection, antigen-specific CD8 T cells undergo clonal expansion and differentiation into effector cells in order to control the infection. Studies have shown that CD8 T cells need to divide at least once before they acquire specific effector functions, including the ability to directly lyse target cells and produce certain cytokines. However, recently we have shown that pre-exposure of the host to type I interferon (IFN) sensitizes CD8 T cells to rapidly acquire effector function without the need to divide. The IFN-primed naïve CD8 T cells produced IFNγ, enhanced TNF and degranulated upon stimulation with high affinity cognate antigen. The threshold required for IFN-induced sensitized CD8 T cells to become activated and produce effector functions was reduced, as primed CD8 T cells produced cytokines in response to lower affinity ligands and lower concentrations of cognate ligands. In addition, an in vivo cytotoxicity assay showed that primed naïve CD8 T cells specifically lysed target cells. The reduced requirement of naïve CD8 T cells to produce effector functions if pre-exposed to IFN-induced environments may increase the overall number of naïve T cells able to respond to any given antigen. IFN-primed increased numbers and response of naïve T cells may help to control antigen load during an acute infection.