Killer Cell Activity Research Articles

Qi and Blood Deficiency Contributes to Peripheral T-Cell Dysfunction and Reduced Natural Killer Cell Activity in Gastric Cancer

Gastric Cancer (GC), a major cause of cancer-related mortality, is susceptible to invasion and metastasis, which triggers dismal prognosis. Traditional Chinese medicine (TCM) exerts a pivotal role in various cancers, including GC. Correct syndrome differentiation is the most important principle guiding the treatment strategy. Here in, the present study aims to explore the relationship of different TCM syndrome types of GC patients and the cellular immune function, which was evaluated by observing peripheral T-cell subsets and activity of Natural Killer (NK) cells. GC patients and healthy controls were enrolled in this study, with venous blood during fasting collected. Patients with deficiency syndromes exhibited decreased number of CD4+ T-cells, ratio of CD4+/CD8+ T-cells and the activity of NK cells as compared with those with sufficiency syndromes. Then, the relationship between TCM syndrome types and TNM classification was analyzed, which revealed that TCM syndrome types were associated with TNM classification. Finally, we evaluated the number of T-cell subsets and NK cell activity in peripheral blood by means of flow cytometry and Lactate Dehydrogenase (LDH) release assay. GC patients with qi and blood deficiency showed reduced CD4+ T-cells, CD4+/CD8+ T-cells and NK cell activity and increased CD8+ T-cells when compared with other syndrome types, which indicated significantly impaired cellular immune function. Taken together, we concluded that peripheral T-cell subsets and NK cell activity, representing the cellular immune function, were associated with different TCM syndrome types of GC. Notably, qi and blood deficiency led to the most severe cellular immune dysfunction of the GC patients.

The killing effect of Tanshinol on breast cancer cells: insight into the reversion of TGF-𝜷1-mediated suppression of NK cell functions

Background: Natural killer (NK) cells play an indispensable role in anti-tumor immunity. TGF-β1 is the main accomplice of tumor immune escape, inhibiting tumor immunity mediated by NK cells. It is reported that Salvia miltiorrhiza can promote the immune killing effect of NK cells. In this study, Tanshinol, a water-soluble active component of Salvia miltiorrhiza, was used to investigate its effect on the inhibition of NK cell functions mediated by TGF-β1 in breast cancer. Methods: We constructed a mouse model of breast cancer by tail vein injection, H&E staining and ELISA were used to verify the role of TGF-β1 and the effects of Tanshinol on breast cancer and NK cells. In vitro, we used CCK8 and cytotoxicity assays to preliminarily evaluate the effect of Tanshinol on the anti-tumor effect of NK cells intervention by TGF-β1. We explored the killing activity of NK cells and related signal pathways by immunofluorescence imaging technology, RT-PCR, ELISA and flow cytometry. Also, Western blot, RT-PCR and immunofluorescence experiments were applied to investigate the expression level of the natural killer group 2 member D (NKG2D)-NKG2D ligands (NKG2DL) signal axis, and combined with immunoprecipitation, to detect the formation of NKG2D-DNAX-activating protein of 10 kD (DAP10) complex. Results: TGF-β1 played a role in promoting lung metastasis of breast cancer and inhibiting the secretion of cytotoxic mediators from NK cells, but Tanshinol could reverse it. High-dose Tanshinol also significantly optimized the survival rate of tumor-bearing mice. TGF-β1 could destroy the NKG2D-NKG2DL axis, down-regulate the expression and nuclear accumulation of p-smad2/3. Moreover, TGF-β1 inhibited the activation of PI3K-ERK1/2-PLCγ2 signaling pathway that is related to the degranulation of NK cells, and diminished the expression of degranulation marker CD107a and the release of anti-tumor cytotoxic killing medium of NK cells. However, Tanshinol was able to interfere with the negative regulation of TGF-β1 on the functions of NK cells, mainly through promoting the expression of NKG2D and its molecular chaperone DAP10, thereby propelling the formation of NKG2D-DAP10 complex. Conclusions: Collectively, Tanshinol enables NK cells to activate and release multiple killing mediators to carry out immune attacks on tumor cells.

A Sensitive and Simple Method to Assess NK Cell Activity by RT-qPCR for Granzyme B Using Spleen and Blood

A new assay method for natural killer (NK) cell activity was established using quantitative RT-PCR (RT-qPCR) to determine the gene expression of granzyme B (GzmB) and perforin 1 (Prf1). The RT-qPCR method was compared to a conventional cytotoxic assay. Upregulated expression of GzmB and Prf1 mRNA and enhanced cytotoxic activity were observed in splenocytes from lipopolysaccharide (LPS)-treated rats. A high correlation, R2 = 0.71, was observed between the gene expression of GzmB and the cytotoxic activity of splenocytes from these rats, indicating that GzmB RT-qPCR is a reliable alternative method to assess NK cell activity/activation. Remarkably, 12.6- to 59.7-fold upregulation of GzmB mRNA expression was observed in leukocytes, the spleen, and splenocytes from LPS-injected rats. Its upregulation appeared to be dose-dependent on the LPS concentration in the range of 0.01 - 0.1 mg/kg. Whereas, only 1.3- to 1.9-fold increase of cytotoxic activity was detected in splenocytes from the rats treated with LPS in the same range. From these, it is evident that, to assess NK cell activity/activation, the GzmB RT-qPCR method is highly sensitive compared with the conventional cytological assay. Furthermore, this GzmB RT-qPCR method is advantageous, as it does not require freshly prepared splenocytes and cell culture procedures. The convenience of GzmB RT-qPCR enables the use of whole blood, leukocytes, the spleen, and/or their frozen samples to evaluate NK cell activity/activation.

Selectively and Efficiently Expanding Natural Killer Cells in Vitro

Natural killer (NK) cells are the main innate immune cells, and have powerful antitumor activity in vitro, but NK cells based immunotherapy does not often get satisfactory results in clinical practice due to insufficient number and poor activity of NK cells.. Therefore, how to improve expansion efficiency and killing activity of NK cells in vitro is critical. Here we report an in vitro culture system culturing peripheral blood mononuclear cells (PBMC) with irradiated LCL as feeder cells in 10%FBS/RPMI1640 culture medium in the presence of 100U/ml IL-2 and 5ng/ml IL-15 for 14 days could selectively expand NK cells. With this culture system, the purity of NK cells increased from 11.5%± 7.01% to 75.3±10.9% (P< 0.01), and the expansion efficiency was about 625±63 times than the original PBMC. Compared with the fresh NK cells isolated from PBMC, there was no significant change in the expression of inhibitory receptors on the surface of expanded NK cells, while the expression of activating receptors was significantly increased (p<0.05). The cytotoxicity of expanded NK cells against leukemia cell line K562 was significantly higher than that of freshly isolated NK cells (72± 5.6% vs 8.7±2.3%, E: T=1, P<0.01), expanded NK cells also showed stronger cytotoxicity to LCL (22±3.6%, E: T=1) and primary leukemia cells (42±5.2%, E:T=1). In summary, we could selectively expand NK cells with this in vitro culture system, the expanded NK cells gained higher expression of NCRs and NKG2D receptors, and displayed greater cytotoxicity against tumor cells, therefore could be readily applied to anti-tumor treatment in clinical treatment. Figure Disclosures No relevant conflicts of interest to declare.

Abstract 3309: Cardioprotective paraoxonase 2 enhances the tumoricidal activity of natural killer cells in a paracrine manner

Abstract Background: Immune system can recognize the neoplastic cells and target them for destruction through tumoricidal process. Further, immune cell dysfunction leads to escape of cancer cells from immune recognition, lead to the initiation and progression of tumor formation. Clinical shreds of evidence suggest that the presence of intratumoral immune cells correlates with improved progression free and overall survival among patients with ovarian carcinoma and provide evidence of activation of antitumor mechanisms. Hence identification of drugs that target the cancer cells as well as activate the immune cells is a sound strategy for cancer therapy. Paraoxonase 2 (PON2) is a membrane-associated lactonase with lipid transport, anti-oxidant and phagocytosis/efferocytosis properties. PON2 deficiency aggravates systemic inflammation, and causes the metabolic associated diseases. Recently we have reported that overexpression of PON2 reduces the ovarian cells proliferation and tumor formation by reducing IGF-1 production and its signaling pathway. Further, using microarray based experiment, we have identified several pathways that modulate the immune cells function such as activation of Natural Killer (NK) cells, dendritic cell maturation, B cells activation, and communication between innate and an adaptive cells signaling pathway were regulated by PON2. Hypothesis: PON2 enhances tumoricidal activity in a paracrine fashion. Methods and Results: NK cells were isolated from control C57BL/6 and purity was assessed with flow cytometry using FITC anti mouse CD49B and results revealed that 95 % of NK cells population. NK cells treated with condition medium from ID8hPON2 (human PON2 overexpressed in mouse ovarian cancer cell line) show an increase in tumoricidal activity compared to condition medium from ID8EV. Neither ID8hPON2 nor ID8EV condition medium affects the viability of NK cells. Moreover, there is no difference in CD49B positive staining in the tumor microenvironment between ID8EV and ID8hPON2 cells receiving mice. Condition medium obtained from ID8hPON2 ID8EV group shows increases the interleukin 18 level and neutralizing antibody against IL18 fail to induce the tumoricidal activity indicating mechanistically PON2 enhances the tumoricidal activity via IL18 dependent manner. Furthermore, IL18 levels are increased in tumor micro environment. PON2 increase the IL18 through transactivation of c-Myc gene that are independent of mitochondrial function. Conclusion: Taken together, our study for the first time showed that PON2 enhances the tumerocidal activity via paracrine manner by upregulating IL18 and PON2 could be used as conventional as well as immunotherapy for ovarian cancer. Citation Format: Asokan Devarajan, Dawoud Sulaiman, Feng Su, Ekambaram Ganapathy, Robin Farias-Eisner, Srinivasa Reddy. Cardioprotective paraoxonase 2 enhances the tumoricidal activity of natural killer cells in a paracrine manner [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3309.

Abstract CT053: Use of CRISPR knock-out screen to identify genes that regulate tumor cell sensitivity to NK cell-based immunotherapy

Abstract Natural killer (NK) cells play a key role in tumor immune surveillance by their ability to recognize and kill both hematological and solid tumor cells. NK cell-based immunotherapy is emerging as a powerful treatment against certain malignancies. However, intrinsic and extrinsic mechanisms can lead tumor cells to develop resistance mechanisms to escape NK cell-mediated killing, including up or down-regulation of genes involved in the interaction between tumor and immune cells. Here, we use a “two cell type” (TCT) screening system to identify novel molecular targets regulating NK cell-mediated killing of glioblastoma stem cells (GSC), meningioma cells, as well as head and neck squamous cells carcinomas (HNSCC). By using a whole genome-scale CRISPR-Cas9 library, we induced mutations in tumor cells to mimic loss of function mutations involved in resistance or sensitivity to NK cells. After a round of selection with human peripheral blood NK cells, we profiled the genes that impaired the killing activity of NK cells or increased the sensitivity of tumor cells to NK cells. The most common genes lost in GSC and meningioma cells that lead to increased sensitivity to NK cells are involved in the ER-phagosome pathway, antigen presentation and cellular localization. We then used GSC to validate our top genetic hits identified by the screening. One gene of interest, CHMP2A is a component of ESCRT-III, a complex involved in multi vesicular bodies and exosomes formation known to be involved in intercellular communications, mediating numerous physiological processes among which immunology and tumor biology. We confirmed that impaired expression of CHMP2A in GSC lines increases their sensitivity to NK cell-mediated killing. RNA sequencing demonstrates knock-out (KO) of CHMP2A leads to increased expression of immune response genes, including certain chemokines. Indeed, NK cells demonstrated increased migration towards KO GSC corresponding to higher concentrations of CXCL10, CXCL12 and IL-8 in GSC-KO conditioned media. Since HNSCC are among the most highly immune-infiltrated cancer types, including a high degree of infiltrating NK cells, we next tested CHMP2A-KO-Cal27 cells as a model of HNSCC. Again, we observed an increased sensitivity to NK cells, higher levels of CXCL10 and increased migration of NK cells, demonstrating these mechanisms regulate NK cell-mediated killing of diverse tumor types. Additionally, in vivo studies using a Cal27 xenograft model and adoptive transfer of human NK cells in immunodeficient mice demonstrate increased NK cell-mediated killing of CHMP2A-KO-Cal27 cells compared to wild type Cal27 cells. Together, these studies demonstrate the utility of a TCT screen to identify new mechanisms that mediate resistance of diverse tumor types to NK cell-mediated killing and allow us to now identify novel strategies to further improve this immunotherapeutic approach. Citation Format: Davide Bernareggi, Qi Xie, Briana Prager, Li Xiqing, Silvio Gutkind, Dan S. Kaufman. Use of CRISPR knock-out screen to identify genes that regulate tumor cell sensitivity to NK cell-based immunotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT053.

Abstract 3144: Using artificial intelligence and flow cytometry to confirm the need for a prostate biopsy in at-risk men

Abstract The goal of this study was to create a blood based, liquid biopsy test for confirming the need for a prostate biopsy by objectively analyzing flow cytometry-based immunophenotyping data using machine learning (ML) by evaluating whether a patient is at-risk for having intermediate-/high-grade prostate cancer PCa [Gleason score (GS) ≥ 7]. Currently, there is a lack of blood-based biomarkers that can accurately predict high-risk PCa in at-risk men making it difficult for clinicians to assess disease status. Furthermore, a large percentage of men undergo unnecessary prostate biopsy procedures each year since the majority of biopsies return either benign pathologies, such as benign prostatic hyperplasia (BPH), or low-grade PCa [Gleason score (GS) = 6). Flow cytometry-based immunophenotyping of peripheral blood is an accessible and non-invasive technology, but as more biomarkers are included, new methods must be developed for the efficient analysis and utilization of these large datasets for clinical applications. Here, we used ML to create pattern recognition neural networks (PRNNs) to identify patients with intermediate-/high-grade PCa from those that have either low-grade PCa or BPH based upon the immunophenotyping of 27 different myeloid and lymphoid cell populations. A key population of interest are myeloid-derived suppressor cells (MDSCs) since they are known to be key contributors in supporting tumor progression and escape through their ability to suppress anti-tumor responses mediated through T cell and natural killer (NK) cell activity. We used standard multiparametric flow cytometry to measure MDSCs and other immune cell populations found in the peripheral blood of 114 biopsy-confirmed PCa and 58 biopsy-confirmed BPH patients that were collected in Streck Cyto-Chex® BCT. PRNNs were trained with raw flow cytometry from two data sets: 37 patients with PCa GS ≥ 7 and 40 patients with BPH/PCa GS6. Predictions were evaluated using the performance of the trained PRNNs on known PCa GS ≥ 7 and BPH/PCa GS6 not used in the PRNN training set (holdout samples). In the holdout samples, we identified 21 out of 23 patients who were identified as PCa GS ≥ 7 (Sens = 91.3%, 95%CI = 72.0% to 98.9%; PPV = 33.3%, 95%CI = 28.4% to 38.7%) while 30 out of 72 patients were identified as BPH/PCa GS6 (Spec = 41.7%, 95%CI = 30.2% to 53.9%; NPV = 93.8%, 95%CI = 79.5% to 98.3%) for a combined accuracy of 53.7% (95%CI = 43.2% to 64.0%). The PRNN classified 17 out of 33 BPH, 13 out of 26 GS6 PCa, 13 out of 14 GS7 PCa, and 8 out of 9 > GS7 PCa samples correctly. For this study, out of the 112 patients who were ultimately diagnosed with BPH or low-grade PCa, approximately 47 procedures could have been avoided or delayed. In a clinical setting, we believe that this technology, in use with other known clinical factors, would allow for clinicians to have a more informed decision when recommending their patients for a prostate biopsy procedure while reducing the number of unnecessary biopsy procedures performed each year. Citation Format: George A. Dominguez, John Roop, Anthony J. Campisi, Thomas Schlumpberger, Amit Kumar. Using artificial intelligence and flow cytometry to confirm the need for a prostate biopsy in at-risk men [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3144.

Abstract 7: Gross thymic extract inhibits tumor growth by modulating the immune response in mice bearing Ehrlich ascites carcinoma cells

Abstract Purpose. Gross thymic extract (GTE) obtained from pathogen-free pigs, a product also known as Thymax has previously been shown to boost various parameters of the immune system in aged mice, as well as activating human dentritic cells and the DC-directed T-cell response in vitro. The current study examines the immunomodulatory and chemopreventive effects of GTE in mice inoculated with Ehrlich ascites carcinoma (EAC) cells. Materials and Methods. Female Swiss albino mice were inoculated with EAC cells in the thigh to develop solid tumor. Oral treatment with GTE (5.45mg/kg body weight) was started either 14 days prior to tumor cell inoculation or 9 days post inoculation for mice bearing palpable tumors. Body weight and tumor growth were monitored until day 30 post-inoculation. The percentage of infiltrated CD4+, and CD8+T cells, natural killer (NK) cell activity and TNF-α expression in tumor cells were analyzed by flow cytometry and RT-PCR. Tumor histopathology was also examined, and blood lymphocytes and safety parameters were evaluated. GTE (Thymax) was kindly provided by YS Nature Company, Tokyo, Japan. Results. Treatment with GTE prior tumor cell inoculation showed tumor incidence in 61.1 % of the animals. Pre-treatment and post-treatment with GTE, markedly delayed tumor development and resulted in tumor weight reduction by -76.7% and -48.7% of untreated control, respectively. GTE, increased blood lymphocytes count by 60% and 53%, elevated the percentage of infiltrating CD4+ cells by144%, 94% and CD8+ T cells by 146%, 53%, for pre- and post-treatment, respectively. Oral intake of GTE, profoundly augmented NK cell cytolytic activity by 5.8 and 3.1 fold and TNF-α expression by 5.5 and 3.2 fold relative to the untreated control for pre-and post-treatment, respectively. Mice treated with GTE maintained normal body, spleen, liver and kidney weights and normal values for liver function enzymes including Aspartate aminotransferase (AST), Alanine aminotransferase (ALT) and Gamma-glutamyl transferase (GGT). Histopathological studies showed tumor tissue obtained from GTE -treated groups had a marked decrease in tumor cell density with increased vacuolation of their cytoplasm, marked increase of apoptotic cells and expansion of the degenerative areas. Conclusion: GTE is a safe, non-toxic agent, that exhibits a chemopreventive effect in mice bearing solid Ehrlich carcinoma via its ability to induce potent immunomodulation. This may suggest that GTE could be used for the treatment of solid tumors in humans. Citation Format: Azza I. Othman, Nariman K. Badr El-Din, Maggie E. Amer, Mamdooh Ghoneum. Gross thymic extract inhibits tumor growth by modulating the immune response in mice bearing Ehrlich ascites carcinoma cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 7.

The effect of Esberitox on the functional activity of 2D-positive natural killer cells (NKG2D) and interferon status in patients with recurrence of herpes simplex virus infection

The aim of the study was to assess changes in the functional activity of 2D-positive natural killer cells (NKG2D) and interferon status in patients with recurrence of herpes virus infection under the influence of Esberitox.Materials and methods. We studied 30 patients aged 22-45 years old with frequent relapsing labial herpes, previously verified by PCR. The frequency ofexacerbations at the time of the study was 6 (5.3 ± 1.4) and more than once a year. Patients were initially examined within 24 hours of the appearance of rashes.The expression of CD314 on the surface of CD3-CD56 lymphocytes was determined by flow cytometry(EPICS XL, Beckman Coulter). The content of cytokines (α-and γ-IFN) in supernatants was determined by enzyme immunoassay (IBL, Germany). Patients took Esberitox tablets, 2 tablets x 3 times a day, and daily1-gram tablets of Valacyclovir for 7 days. The control group consisted of 10 patients receiving only tablet valacyclovir.Results. As a result of the study, it was found that the final leveling of symptoms with combined treatment with Esberitox was noted by the end of 5-6 days, with basic treatment - by 9-10 days. There was a decrease in the relapse rate (3 patients) during the observation period within 2 months after the end of the course of treatment.In patients receiving basic Valacyclovir therapy, relapses during the observation period were recorded in more than half of the examined individuals (6 patients). The positive dynamics of the increase in the level of γ-IFN and α-IFN in patients of the main group and itsalmost absence in the control group were established.The dynamics of surface expression of the NKG2D receptor on peripheral blood NK cells showed an increase parameters under the influence of the Esberitox drug compared with Valacyclovir monotherapy, with no significant changes in other lymphocyte subpopulations. Conclusions. The administration of Esberitox in case of frequently recurring herpes virus infection is a promising method of combined immuno-adaptive therapy, which requires further careful study.

Effects of Porphyra tenera Supplementation on the Immune System: A Randomized, Double-Blind, and Placebo-Controlled Clinical Trial.

Objective: The purpose of this study was to determine if Porphyra tenera extract (PTE) has immune-enhancing effects and is safe in healthy adults. Methods: Subjects who met the inclusion criteria (3 × 103 ≤ peripheral blood leukocyte level ≥ 8 × 103 cells/µL) were recruited for this study. Enrolled subjects (n = 120) were randomly assigned to either the PTE group (n = 60) and were given 2.5 g/day of PTE (as PTE) in capsule form or the placebo group (n = 60) and were given crystal cellulose capsules with the identical appearance, weight, and flavor as the PTE capsules for 8 weeks. Outcomes were assessed based on measuring natural killer (NK) cell activity, cytokines level, and upper respiratory infection (URI), and safety parameters were assessed at baseline and 8 weeks. Results: Compared with baseline, NK cell activity (%) increased for all effector cell-to-target cell ratios in the PTE group after 8 weeks; however, changes were not observed in the placebo group (p < 0.10). Subgroup analysis of 101 subjects without URI showed that NK cell activity in the PTE group tended to increase for all effector cell/target cell (E:T) ratios (E:T = 12.5:1 p = 0.068; E:T = 25:1 p = 0.036; E:T = 50:1 p = 0.081) compared with the placebo group. A significant difference between the two groups was observed for the E:T = 25:1 ratio, which increased from 20.3 ± 12.0% at baseline to 23.2 ± 12.4% after 8 weeks in the PTE group (p = 0.036). A significant difference was not observed in cytokine between the two groups. Conclusion: PTE supplementation appears to enhance immune function by improving NK cell activity without adverse effects in healthy adults.

Abstract C038: MHC class I polypeptide related sequence A (MICA) race-related differential expression in prostate cancer

Abstract Prostate cancer (PCa) is the second most common cancer in American men, with higher incidence and death rates in African Americans (AA) relative to Caucasian Americans (CA); nevertheless, these disparities are still poorly understood. MHC class I polypeptide related sequence A (MICA) is a cell surface protein able to promote activation of natural killer cells. Cleavage of MICA and generation of its soluble form (sMICA) has been described as an immunoevasion mechanism presented by different types of aggressive tumors. In agreement with the role of MICA on tumor immunoevasion, recent studies correlated better prognosis for patients expressing higher levels of MICA in different types of cancer. We speculated that MICA expression would be different between AA and CA PCa tumors. To access MICA expression in PCa patients, we stained a TMA containing 30 AA and 27 CA tumor cores. Our results showed that CA PCa tumor cores had higher expression of MICA than the AA (p=0.0019). In order to establish an in vitro system to study the biologic effects of MICA in the context of disparity in PCa, we assessed the expression of surface MICA and release rate of sMICA in different PCa cell lines: MDA-PCa-2b and E006AA-hT, derived from AA, and DU-145 and LNCaP, derived from CA. Flow cytometry analysis showed baseline cell fraction expression positive for surface MICA of 3% in MDA-PCa-2b, 18% in E006AA-hT, 54% in DU-145, and 67% in LNCaP, with significant difference between each cell line (p&amp;lt;0.0001). Baseline release rate of sMICA was nondetectable for MDA-PCa-2b, 5pg/ml/106 cells in E006AA-hT, 120pg/ml/106 cells in DU-145, and 54pg/ml/106 cells for LNCaP cells. Overall, our results show that MICA expression is race related. Further studies are necessary to address the biologic function of the expression and the impact in the immune system. Citation Format: Charles R. Pound, Marcelo J. Sakiyama, Amit Reddy, Ingrid Espinoza, Eldrin Bhanat, Krista Syrigos, Roya Gordji, Deepak Kumar, Jack Lewin, Xinchun Zhou, Jesus Monico, Christian Gomez. MHC class I polypeptide related sequence A (MICA) race-related differential expression in prostate cancer [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr C038.

Abstract PR03: Use of CRISPR knockout screen to identify genes that regulate tumor cell sensitivity to NK cell-based immunotherapy

Abstract Natural killer (NK) cells play a key role in tumor immune surveillance by their ability to recognize and kill both hematologic and solid tumor cells. NK cell-based immunotherapy is emerging as a powerful treatment against certain malignancies. However, intrinsic and extrinsic mechanisms can lead tumor cells to develop resistance mechanisms to escape NK cell-mediated killing, including up- or downregulation of genes involved in the interaction between tumor and immune cells. Here, we use a “two cell type” (TCT) screening system to identify novel molecular targets regulating NK cell-mediated killing of glioblastoma stem cells (GSC), meningioma cells, as well as head and neck squamous cell carcinomas (HNSCC). By using a whole genome-scale CRISPR-Cas9 library, we induced mutations in tumor cells to mimic loss of function mutations involved in resistance or sensitivity to NK cells. After a round of selection with human peripheral blood NK cells, we profiled the genes that impaired the killing activity of NK cells or increased the sensitivity of tumor cells to NK cells. The most common genes lost in GSC and meningioma cells that lead to increased sensitivity to NK cells are involved in the ER-phagosome pathway, antigen presentation, and cellular localization. We then used GSC to validate our top genetic hits identified by the screening. One gene of interest, CHMP2A, is a component of ESCRT-III, a complex involved in multivesicular bodies and exosomes formation known to be involved in intercellular communications, mediating numerous physiologic processes among which are immunology and tumor biology. We confirmed that impaired expression of CHMP2A in GSC lines increases their sensitivity to NK cell-mediated killing. RNA sequencing demonstrates knockout (KO) of CHMP2A leads to increased expression of immune response genes, including certain chemokines. Indeed, NK cells demonstrated increased migration towards KO GSC corresponding to higher concentrations of CXCL10, CXCL12, and IL-8 in GSC-KO conditioned media. Since HNSCC are among the most highly immune-infiltrated cancer types, including a high degree of infiltrating NK cells, we next tested CHMP2A-KO-Cal27 cells as a model of HNSCC. Again, we observed an increased sensitivity to NK cells, higher levels of CXCL10, and increased migration of NK cells, demonstrating these mechanisms regulate NK cell-mediated killing of diverse tumor types. Additionally, in vivo studies using a Cal27 xenograft model and adoptive transfer of human NK cells in immunodeficient mice demonstrate increased NK cell-mediated killing of CHMP2A-KO-Cal27 cells compared to wild-type Cal27 cells. Together, these studies demonstrate the utility of a TCT screen to identify new mechanisms that mediate resistance of diverse tumor types to NK cell-mediated killing and allow us to now identify novel strategies to further improve this immunotherapeutic approach. This abstract is also being presented as Poster A25. Citation Format: Davide Bernareggi, Qi Xie, Briana Prager, Li Xiqing, Silvio Gutkind, Dan S. Kaufman. Use of CRISPR knockout screen to identify genes that regulate tumor cell sensitivity to NK cell-based immunotherapy [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr PR03.

Natural killer cells and their activity as a potential biomarker for predicting response to checkpoint inhibitors in non-small cell lung cancer.

e15559 Background: Despite the increased promise of checkpoint inhibitors in the treatment of non-small cell lung cancer, the majority of patients do not respond and the potential for severe toxicity is significant. Unfortunately, the predictive value of PD-L1 expression has not always proven to be definitive and the overall need to find better biomarkers that will optimize the selection of patients who will best respond to therapy remains. Recent evidence has pointed to Natural Killer (NK) cells as an essential mediator in overall response to checkpoint inhibitors. We explore whether NK cell populations and/or their activity have any correlation with response. Methods: Peripheral blood mononuclear cells (PBMCs) from nine patients with Stage IV non-small cell lung cancer (four adenocarcinoma, five squamous cell carcinoma) were collected pre and post six-weeks of checkpoint immunotherapy (six received pembrolizumab, and three received nivolumab). Overall, four of nine (44.5%) were diagnosed as SD or PR via RECIST 1.1. The immune cell composition of the PBMCs of all nine patients was analyzed using multi-variated single-cell analysis using mass cytometry (CyTOF) with an optimized 32-marker panel. Natural Killer (NK) cell activity was measured with the NKVue kit which detects NK-secreted IFN-γ levels using a quantitative sandwich ELISA from NK cells exposed to a specific recombinant cytokine. Results: The overall percentages of NK cell populations found in the immune cells of PBMCs were prominently elevated in patients who responded (SD or PR) to checkpoint therapy compared to those who had progressive disease (non-responders). While there were no significant differences in the population of other immune cells between these two groups, the overall NK cell activity in patients who responded was highly elevated compared to the NK cell activity in non-responders. From the analysis of NK subsets, there were no differences in the population of early NK cells between the two groups, but the functionally differentiated late NK cells were prominently high in the responder group. Conclusions: Natural Killer (NK) cells have been recently implicated to play a key role in anti-tumor response to checkpoint inhibitors and our results suggest that the overall number of NK cells and their activity could prove to be an independent and reliable predictive tool/biomarker in patients with NSCLC.

Targeting non-engrafting IL-2 activated intentionally mismatched donor lymphocytes with monoclonal or bispecific antibodies for treatment of otherwise incurable cancer.

e15033 Background: Cure of certain resistant cancers by donor lymphocyte infusion (DLI) following allogeneic stem cell transplantation (SCT) suggests that alloreactive lymphocytes can eliminate otherwise resistant cancer. First patient with resistant relapse following supra-lethal chemoradiotherapy treated with DLI is alive &amp; well &gt; 34 years; &gt; 400 patients with hematological malignancies confirmed DLI efficacy for prevention &amp; treatment of relapse. Using murine leukemia (BCL1) &amp; metastatic breast cancer (4T1), IL-2 activated mismatched lymphocytes with no prior SCT eliminated otherwise lethal minimal residual disease (MRD). Using anti-EpCAM x anti-CD3 bispecific antibody (BSA) lethal melanoma was eliminated, also resulted in long-lasting immunity against lethal tumor challenge. Graft-vs-host disease (GVHD) was prevented by consistent rejection of mismatched lymphocytes. Accordingly, our goal was to develop effective immunotherapy of cancer using non-engrafting intentionally mismatched Allogeneic Targeted Activated Cancer Killer cells (ATACK) using relevant monoclonal antibodies (MoAb) or BSA. Methods: Patients with multiple myeloma &amp; lymphoma and advanced solid tumors were treated with haploidentical or unrelated donor lymphocytes pre-activated 5 days with IL-2, following non-myeloablative conditioning with endoxan or fludarabine and IL-2 injection x5 days for in vivo lymphocyte activation. ATACK with no MoAb was investigated in 40 patients with different cancers. Safety of ATACK with MoAb was investigated in 16 patients with resistant NHL, metastatic breast, lung &amp; colorectal cancer, using commercial MoAbs against CD20, Her-2, EGFR or VEGF, respectively, or using anti-EpCAM BSA. Results: Protocols were well-tolerated with mild transient toxicity following ATACK and BSA. No patient developed GVHD. Disease-free survival &gt; 15 years was documented in 2/2 patient with multiple myeloma and 2/2 patients with lymphoma. Longterm progression-free survival was observed in 5/31 evaluable patients with advanced metastatic solid tumors treated with no MoAbs. Minor toxicity in targeted ATACK recipients was manageable in outpatient clinic. Conclusions: Intentionally mismatched IL-2 activated killer cells guided by MoAbs or BSA represents a promissing approach for treatment, possibly even cure, of cancer patients with MRD while avoiding GVHD.

Genotype characteristics and immunological indicator evaluation of 311 hemophagocytic lymphohistiocytosis cases in China

BackgroundPrimary hemophagocytic lymphohistiocytosis (pHLH) is a genetic disorder that is classically diagnosed by genetic testing. Secondary HLH (sHLH) is usually caused by infections, malignancies, or autoimmune disorders, but may display some mutations or polymorphisms. Rapid immunological assays examining natural killer (NK) cell activity, degranulation function (CD107a), and protein expression related to genetic deficiencies have been recommended for early pHLH identification.MethodsA retrospective analysis of 311 HLH patients from a Chinese population was performed to evaluate the potential correlations between genetic testing and rapid immunological assays; genotyping characteristics, age of onset, and etiology were examined.ResultsAmong the 128 (128/311) patients who were positive in the genetic screening, the most frequently detected mutant gene was UNC13D (29%), followed by LYST (21%), PRF1 (17%), and STXBP2 (10%). Among pHLH patients (n = 39), the majority (67%) had PRF1 and UNC13D defects. FHL-2 was predominant (12/27, 44%) in patients aged under 18, while FHL-3 was the most common (6/12, 50%) in adults. Differences in genetic variant types and etiological components were noted in HLH patients based on the age of onset. NK cell activity and CD107a were observed to show a consistent trend (Ptrend < 0.001) when grouping patients according to the severity of the genetic variant type. Moreover, NK cell activity was generally consistent within a certain range of ΔCD107a values (Ptrend < 0.001). The PPV for bi-allelic degranulation gene mutations in patients with CD107a < 5% was 38.9% (7/18), while the PPV in patients with CD107a ≤10% was 16.7% (13/78). The PPV for pHLH was 41.4% (29/70) with NK cell activity ≤13%. To further evaluate the diagnostic efficacy of NK cell activity assay in pHLH, a receiver operating characteristic (ROC) curve was generated and showed an area under the curve (AUC) of 0.872, and the optimal cutoff value was determined to be 13.425% with a sensitivity of 84.21% and specificity of 80.67% when the corresponding Youden index was maximized. Flow cytometry screening for deficient proteins, including perforin, SAP, and XIAP, showed a relatively high sensitivity (83.33–93.33%). The positive predictive values (PPVs) of perforin and XIAP were relatively low (20.83–26.92%), but the negative predictive values (NPVs) for all three were excellent (all > 98%).ConclusionsVarious immunological indicators have different clinical prediction and application values for the diagnosis of pHLH. The degree of reduction of immunological indicators also needs attention, and choosing appropriate cutoff value may be of important significance in guiding clinical judgment for pHLH.

Sesamolin promotes cytolysis and migration activity of natural killer cells via dendritic cells.

The defense mechanism of the immune system is based on the interaction of many kinds of leukocytes. Among them, dendritic cells (DCs) control most immune responses. In our previous study, sesamolin was shown to create an optimal environment for natural killer (NK) cells to kill cancer cells. Here we attempted to demonstrate how sesamolin influences DCs to promote the killing and migration activity of NK cells. We co-cultured DCs and NK cells and analyzed the communication between them. NK cells co-cultured with 5 µg/ml sesamolin-treated mature dendritic cells (mDCs) had better cytolytic activity than did NK cells or mDCs co-cultured NK cells. Moreover, the migration of NK cells toward mDCs was enhanced compared to immature dendritic cells (iDCs). The migration of NK cells stimulated by mDCs was stronger after sesamolin activation of the mDCs. Altogether, this study demonstrated that sesamolin activated NK cells by modulating the differentiation and activation of DCs.

Abstract B43: Positron emission tomography (PET) imaging of the natural killer (NK) cell activation receptor NKp30

Abstract Natural killer (NK) cells are essential defenders against viral and cancer threats. Therapeutic strategies utilizing NK cells include both adoptive cell transfer and modulating NK response in situ. Therapeutic response is gauged using CT/MRI or analysis of circulating NK cells via flow cytometry. Measuring tumor size via CT/MRI has limited prognostic value for immunotherapies and circulating NK cell activity is a poor surrogate for NK cell engagement and activation at desired tumor sites. New approaches are needed to noninvasively monitor NK cell localization and activation to better predict therapy responses, determine causes of therapy failure, and facilitate the clinical translation of emerging NK cell cancer therapies. To address this challenge, positron emission tomography (PET) tracers specific for NKp30 (NCR3) were developed and validated in vivo. NKp30 is expressed almost exclusively on NK cells along with rare ILC1 subtypes and highly upregulated in activated NK cells. PET imaging allows quantitative, serial, non-invasive imaging. A variety of PET radionuclides are used, depending on the targeting probes’ pharmacokinetics. An NKp30-specific antibody was radiolabeled with either copper-64 (64Cu, t1/2 = 12.7h) or zirconium-89 (89Zr, t1/2 = 78.4h) to determine which probe offers superior contrast. 64Cu and 89Zr allow in vivo imaging out to 48 hours and 7 days post-injection, respectively. The antibody was conjugated with the chelators DOTA or DFO followed by 64Cu or 89Zr radiolabeling. 64Cu-NKp30 had a radiochemical yield of 70-75% and specific activity of 7.5-10 uCi/ug, whereas 89Zr-NKp30 had a radiochemical yield of &amp;gt;85% and a specific activity of 4-5 uCi/ug. HPLC results demonstrated radiochemical purity of &amp;gt;98% and no antibody aggregation or fragmentation. To evaluate the binding specificity of these tracers in vivo, HeLa cells stably expressing NKp30 were subcutaneously implanted into flanks of nu/nu mice, along with HeLa wild-type (WT) cells in the opposite flank. 15 ug of 64Cu-NKp30 or 89Zr-NKp30 was injected, and PET/CT imaging completed at 24 and 48h for 64Cu-NKp30 and every 24h to 144h for 89Zr-NKp30. 64Cu-NKp30 had 14.4±4.4 %Id/g uptake in NKp30 xenografts, compared to 5.8±1.4 %Id/g for WT cells (p= 0.042, n=3) at 48h. 89Zr-NKp30 had 22.9±1.9 %Id/g uptake in NKp30 xenografts compared to 7.3±0.9 %Id/g for WT cells (p=0.0027, n=3) at 144h. Tumor/blood ratios were 1.6 for 64Cu-NKp30 vs 4.7 89Zr-NKp30 at 48 and 144 hours, respectively. These results demonstrate specific uptake in cells expressing NKp30 by both probes, with 89Zr-NKp30 at 144h demonstrating superior contrast. We have developed NKp30-specific PET tracers for imaging activated NK cell distribution with the potential for monitoring of NK cell immunotherapy in vivo. Citation Format: Travis M. Shaffer, Amin Aalipour, Sanjiv S. Gambhir. Positron emission tomography (PET) imaging of the natural killer (NK) cell activation receptor NKp30 [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B43.

Effects of propofol and sevoflurane on tumor killing activity of peripheral blood natural killer cells in patients with gastric cancer.

ObjectiveThis study aimed to investigate the effects of propofol and sevoflurane on cytotoxicity of natural killer (NK) cells in patients with gastric cancer.MethodsPatients with gastric cancer were anesthetized by propofol or sevoflurane. Peripheral blood NK cells were isolated and co-cultured with BGC-823 gastric cancer cell culture supernatant, and the rate of apoptosis and effector molecules were analyzed by flow cytometry. Effects of propofol and sevoflurane on NK cell function and SMAD4 protein expression were investigated.ResultsCytotoxicity of NK cells in patients with gastric cancer was inhibited before surgery, but it was enhanced in patients who were anesthetized by propofol compared with those who had sevoflurane. In vitro co-culture with BGC-823 cells significantly inhibited the cytotoxicity of NK cells, which was abolished by treatment of propofol or transforming growth factor (TGF)-β1. SMAD4 protein expression in the NK cell nucleus was significantly downregulated by TGF-β1 treatment and BGC-823 supernatant co-culture, and this expression could be restored by propofol.ConclusionsCytotoxicity of NK cells in patients with gastric cancer is low, but it can be promoted by propofol. Propofol regulates cytotoxicity in NK cells by promoting SMAD4, thereby affecting cellular function.

Abstract P3-09-18: Immunization of breast cancer patients in combination with standard-of-care chemotherapy: Results of a phase Ib clinical trial

Abstract We have developed a peptide mimotope-based cancer vaccine, P10s-PADRE, targeting tumor associated carbohydrate antigens (TACAs). We have previously shown safety, feasibility and induction of immune response in a phase I clinical trial in stage IV breast cancer patients. In the current clinical trial, immunization with P10s-PADRE vaccine is combined with the standard-of-care chemotherapy in neo-adjuvant setting in subjects diagnosed with early stage ER-positive breast cancer. Various schedules (timing of vaccination doses relative to chemo) for combination therapy were tested. Immunological endpoint variable include antibody response and activation of natural killer (NK) cells as tested in peripheral blood mononuclear cells. Tumor size before treatment and after surgery was measured for each subject. The secondary endpoint was pathologic complete response (pCR). We observed induction of antibody response and activation of NK cells independent of each other. Vaccination affected the expression of several NK markers including CD94 and NKp46 as we observed a significant upregulation of NKp46 (median = 1.79, P&amp;lt;0.0001), and CD94 (median = 1.50, P&amp;lt;0.0001) in response to immunization. The data indicate a significant increase in serum IFN-γ (Median=1.74, P=0.0016) upon vaccination. Increase in IFN-γ was positively correlated with increase in both CD69 (r = 0.46, P=0.0321) and NKp46 (r = 0.39, P=0.0763) on CD56-dim NK cells. Tumor shrinkage was associated with an increase in NK cells, upregulation of NKp46 and CD94 molecules, and increase in frequency of NKp46-positive and CD94-positive CD56-dim NK cells. Among 25 subjects enrolled in 5 different immunization schedules, 5 experienced complete eradication of their primary tumors that was associated with vaccine induced increase in the frequency of CD94-positive CD56-dim NK cells (p= 0.0473). The results are intriguing and warrant expansion of the immunologically effective combination schedule to further establish the clinical efficacy along with potential biomarkers of response. Citation Format: Issam Makhoul, Fariba Jousheghany, Saddam Mohammed, Eric Siegel, Angela Pennisi, J Thaddeus Beck, Laura Hutchins, Thomas Kieber-Emmons, Behjatolah Monzavi-Karbassi. Immunization of breast cancer patients in combination with standard-of-care chemotherapy: Results of a phase Ib clinical trial [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-09-18.

Development of a protein-based system for transient epigenetic repression of immune checkpoint molecule and enhancement of antitumour activity of natural killer cells

BackgroundImmune checkpoint blockade (ICB) therapy improved the prognosis of cancer patients, but general administration of ICBs occasionally induces side effects that include immune-related adverse events and tumour hyper-progression. Here, we established a protein-based system, by which endogenous expression of IC molecule in natural killer (NK) cells was transiently repressed on enhancement of their antitumour activity.MethodsA protein-based genome modulator (GM) system is composed of a transcription activator-like effector (TALE), DNA methyltransferase and a newly identified potent cell-penetrating peptide with nuclear-trafficking property named NTP. TALE was designed to target the promoter region of the programmed cell death-1 (PD-1) gene. After culturing human NK cells in the presence of NTP-GM protein, we examined endogenous PD-1 expression and antitumour activity of the treated cells.ResultsNTP-GM protein efficiently downregulated PD-1 expression in NK cells with increased CpG DNA methylation in the promoter region. The antitumour activity of the treated NK cells was enhanced, and repeated intraperitoneal administrations of the treated NK cells attenuated tumour growth of programmed death-ligand 1-positive tumour cells in vivo.ConclusionsBecause the incorporated NTP-GM protein was quickly degraded and negligible in the administered NK cells, the NTP-GM system could be an alternative option of an ICB without side effects.