Natural Killer Cell Migration Research Articles

Activin A, a Novel Chemokine, Induces Mouse NK Cell Migration via AKT and Calcium Signaling.

Natural killer (NK) cells can migrate quickly to the tumor site to exert cytotoxic effects on tumors, and some chemokines, including CXCL8, CXCL10 or and CXCL12, can regulate the migration of NK cells. Activin A, a member of the transforming growth factor β (TGF-β) superfamily, is highly expressed in tumor tissues and involved in tumor development and immune cell activation. In this study, we focus on the effects of activin A on NK cell migration. In vitro, activin A induced NK cell migration and invasion, promoted cell polarization and inhibited cell adhesion. Moreover, activin A increased Ca2+, p-SMAD3 and p-AKT levels in NK cells. An AKT inhibitor and Ca2+ chelator partially blocked activin A-induced NK cell migration. In vivo, exogenous activin A increased tumor-infiltrating NK cells in NS-1 cell solid tumors and inhibited tumor growth, and blocking endogenous activin A with anti-activin A antibody reduced tumor-infiltrating NK cells in 4T-1 cell solid tumors. These results suggest that activin A induces NK cell migration through AKT signaling and calcium signaling and may enhance the antitumor effect of NK cells by increasing tumor-infiltrating NK cells.

CD56/NCAM mediates cell migration of human NK cells by promoting integrin-mediated adhesion turnover.

Natural killer (NK) cells patrol tissue to mediate lysis of virally infected and tumorigenic cells. Human NK cells are typically identified by their expression of neural cell adhesion molecule (NCAM, CD56), yet, despite its ubiquitous expression on NK cells, CD56 remains a poorly understood protein on immune cells. CD56 has been previously demonstrated to play roles in NK cell cytotoxic function and cell migration. Specifically, CD56-deficient NK cells have impaired cell migration on stromal cells and CD56 is localized to the uropod of NK cells migrating on stroma. Here, we show that CD56 is required for NK cell migration on ICAM-1 and is required for the establishment of persistent cell polarity and unidirectional actin flow. The intracellular domain of CD56 (NCAM-140) is required for its function and the loss of CD56 leads to enlarged actin foci and sequestration of phosphorylated Pyk2 accompanied by increased size and frequency of activated LFA-1 clusters. Together, these data identify a role for CD56 in regulating human NK cell migration through modulation of actin dynamics and integrin turnover. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text].

Real-time ex vivo monitoring of NK cell migration toward obesity-associated oesophageal adenocarcinoma following modulation of CX3CR1

Oesophagogastric adenocarcinomas (OAC) are poor prognosis, obesity-associated cancers which may benefit from natural killer (NK) cell-based immunotherapies. Cellular immunotherapies encounter two key challenges to their success in OAC, namely recruitment to extratumoural tissues such as the omentum at the expense of the tumour and an immunosuppressive tumour microenvironment (TME) which can hamper NK cell function. Herein, we examined approaches to overcome the detrimental impact of obesity on NK cells and NK cell-based immunotherapies. We have demonstrated that NK cells migrate preferentially to the chemotactic signals of OAC patient-derived omentum over tumour in an ex vivo model of immune cell migration. We have identified CX3CR1 modulation and/or tumour chemokine profile remodelling as approaches to skew NK cell migration towards tumour. We also report targetable immunosuppressive facets of the obese OAC TME which dampen NK cell function, in particular cytotoxic capabilities. These data provide insights into approaches to therapeutically overcome key challenges presented by obesity and will inform superior design of NK cell-based immunotherapies for OAC.

Interleukin-10 gene intervention ameliorates liver fibrosis by enhancing the immune function of natural killer cells in liver tissue

Background and aimsInterleukin 10 (IL-10) and natural killer (NK) cells have the potential to combat liver fibrosis. However, whether NK cells play an important role in the anti-fibrotic effects of IL-10 is not sufficiently elucidated. In this study, we investigated the regulatory effects of IL-10 on NK cells during liver fibrosis. MethodsFibrotic mice induced with carbon tetrachloride were treated with or without IL-10 in the presence or absence of NK cells. Liver damage and fibrosis were assessed using hematoxylin and eosin and Sirius Red staining and serum transaminase and liver hydroxyproline assays, respectively. NK cell distribution, quantity, activation, cytotoxicity, development, and origin were analyzed using immunohistochemistry, immunofluorescence, and flow cytometry. Enzyme-linked immunosorbent assay was used to determine chemokine levels. ResultsIn the presence of NK cells, IL-10 gene intervention improved liver fibrosis and enhanced NK cell accumulation and function in the liver, as evidenced by increased NKG2D, interferon-γ, and CD107a expression. Furthermore, IL-10 promoted the migration of circulating NK cells to the fibrotic liver and elevated C-C motif ligand 5 levels. However, depletion of NK cells exacerbated liver fibrosis and impaired the anti-fibrotic effect of IL-10. ConclusionsThe anti-fibrotic effect of IL-10 relies on the enhancement of NK cell immune function, including activation, cytotoxicity, development, and migration. These results provide valuable insights into the mechanisms through which IL-10 regulates NK cells to limit the progression of liver fibrosis.

C–C chemokine receptor 5 is essential for conventional NK cell trafficking and liver injury in a murine hepatitis virus-induced fulminant hepatic failure model

BackgroundPrevious studies have demonstrated that natural killer (NK) cells migrated into the liver from peripheral organs and exerted cytotoxic effects on hepatocytes in virus-induced liver failure.AimThis study aimed to investigate the potential therapeutic role of chemokine receptors in the migration of NK cells in a murine hepatitis  virus strain 3 (MHV-3)-induced fulminant hepatic failure (MHV-3-FHF) model and its mechanism.ResultsBy gene array analysis, chemokine (C–C motif) receptor 5 (CCR5) was found to have remarkably elevated expression levels in hepatic NK cells after MHV-3 infection. The number of hepatic CCR5+ conventional NK (cNK) cells increased and peaked at 48 h after MHV-3 infection, while the number of hepatic resident NK (rNK) cells steadily declined. Moreover, the expression of CCR5-related chemokines, including macrophage inflammatory protein (MIP)-1α, MIP-1β and regulated on activation, normal T-cell expressed and secreted (RANTES) was significantly upregulated in MHV-3-infected hepatocytes. In an in vitro Transwell migration assay, CCR5-blocked splenic cNK cells showed decreased migration towards MHV-3-infected hepatocytes, and inhibition of MIP-1β or RANTES but not MIP-1α decreased cNK cell migration. Moreover, CCR5 knockout (KO) mice displayed reduced infiltration of hepatic cNK cells after MHV-3 infection, accompanied by attenuated liver injury and improved mouse survival time. Adoptive transfer of cNK cells from wild-type mice into CCR5 KO mice resulted in the abundant accumulation of hepatic cNK cells and aggravated liver injury. Moreover, pharmacological inhibition of CCR5 by maraviroc reduced cNK cell infiltration in the liver and liver injury in the MHV-3-FHF model.ConclusionThe CCR5-MIP-1β/RANTES axis played a critical role in the recruitment of cNK cells to the liver during MHV-3-induced liver injury. Targeted inhibition of CCR5 provides a therapeutic approach to ameliorate liver damage during virus-induced acute liver injury.

Aconite aqueous extract inhibits the growth of hepatocellular carcinoma through CCL2-dependent enhancement of natural killer cell infiltration

ObjectiveAconite is a traditional Chinese herbal medicine that has been found to inhibit the development of liver cancer; however, its exact molecular mechanisms in this process remain unclear. This study explores how aconite aqueous extract (AAE) inhibits hepatocellular carcinoma (HCC). MethodsAn in vivo mouse model of subcutaneous liver cancer was established. After AAE treatment, immunohistochemistry (IHC) was used to determine the effect of AAE on natural killer (NK) cells. Subsequently, C57BL/6 mice were used to establish the subcutaneous tumor model, and a group of these mice were treated with anti-PK163 antibody to remove NK cells, which was verified by flow cytometry and IHC. The effect of AAE on the proliferation of HCC cells in vitro was determined using cell counting kit-8. The effect of AAE on chemokine production in HCC cells was measured using real-time quantitative polymerase chain reaction and an enzyme-linked immunosorbent assay. The effect of AAE on the migration of NK cells was determined using a transwell assay. Finally, the molecular mechanism was investigated using the Western blotting method. ResultsWe demonstrated that the ability of AAE to induce overexpression of the cytokine C–C motif chemokine ligand 2 (CCL2) in HCC cells is fundamental to the infiltration of NK cells into the tumor bed. Mechanistically, we found that the upregulation of CCL2 was achieved by the activation of c-Jun N-terminal kinase but not extracellular regulated protein kinase or p38. ConclusionOur findings suggest that AAE can be used as an effective immune adjuvant to enhance antitumor immunity by increasing NK cell infiltration into tumors, which could help to improve the efficacy of HCC treatments.Please cite this article as: Yang KD, Zhang X, Shao MC, Wang LN. Aconite aqueous extract inhibits the growth of hepatocellular carcinoma through CCL2-dependent enhancement of natural killer cell infiltration. J Integr Med. 2023; 21(6): 575–583.

Mapping the chemotactic landscape in NK cells reveals subset-specific synergistic migratory responses to dual chemokine receptor ligation

Natural killer (NK) cells have a unique capability of spontaneous cytotoxicity against malignant cells and hold promise for off-the-shelf cell therapy against cancer. One of the key challenges in the field is to improve NK cell homing to solid tumors. To gain a deeper understanding of the cellular mechanisms regulating trafficking of NK cells into the tumor, we used high-dimensional flow cytometry, mass cytometry, and single-cell RNA-sequencing combined with functional assays, creating a comprehensive map of human NK cell migration phenotypes. We found that the chemokine receptor repertoire of peripheral blood NK cells changes in a coordinated manner becoming progressively more diversified during NK cell differentiation and correlating tightly with the migratory response of the distinct NK cell subsets. Simultaneous ligation of CXCR1/2 and CX3CR1, synergistically potentiated the migratory response of NK cells. Analysis of 9471 solid cancers from publicly available TCGA/TARGET repositories revealed dominant chemokine patterns that varied across tumor types but with no tumor group expressing ligands for more than one chemokine receptor present on mature NK cells. The finding that chemokine stimulation can elicit a synergistic migratory response in NK cells combined with the identified lack of naturally occurring pairs of chemokines-chemokine receptors in human cancers may explain the systematic exclusion of NK cells from the tumor microenvironment and provides a basis for engineering next-generation NK cell therapies against malignancies. The Polish Ministry of Science and Higher Education, the National Science Centre, Poland, The Norwegian Cancer Society, the Norwegian Research Council, the South-Eastern Norway Regional Health Authority, The Swedish Cancer Society, the Swedish Children's Cancer Foundation, The Swedish Research Council, The Center of Excellence: Precision Immunotherapy Alliance, Knut and Alice Wallenberg Foundation and National Cancer Institute.

High-Content Screening Assay for the Identification of Antibody-Dependent Cellular Cytotoxicity Modifying Compounds.

Immunotherapy with antigen-specific antibodies or immune checkpoint inhibitors has revolutionized the therapy of breast cancer. Breast cancer cells expressing the epidermal growth factor receptor HER2 can be targeted by the anti-HER-2 antibody trastuzumab. Antibody-dependent cellular cytotoxicity (ADCC) is an important mechanism implicated in the antitumor action of HER-2. Trastuzumab bound to cancer cells can be recognized by the Fc receptors of ADCC effector cells (e.g., natural killer (NK) cells, macrophages, and granulocytes), triggering the cytotoxic activity of these immune cells leading to cancer cell death. We set out to develop an image-based assay for the quantification of ADCC to identify novel ADCC modulator compounds by high-content screening. In the assay, HER2 overexpressing JIMT-1 breast cancer cells are co-cultured with NK-92 cells in the presence of trastuzumab, and target cell death is quantified by automated microscopy and quantitative image analysis. Target cells are distinguished from effector cells based on their EGFP fluorescence. We show how compound libraries can be tested in the assay to identify ADCC modulator drugs. For this purpose, a compound library test plate was set up using randomly selected fine chemicals off the lab shelf. Three microtubule destabilizing compounds (colchicine, vincristine, podophyllotoxin) expected to interfere with NK cell migration and degranulation were also included in the test library. The test screen identified all three positive control compounds as hits proving the suitability of the method to identify ADCC-modifying drugs in a chemical library. With this assay, compound library screens can be performed to identify ADCC-enhancing compounds that could be used as adjuvant therapeutic agents for the treatment of patients receiving anticancer immunotherapies. In addition, the method can also be used to identify any undesirable ADCC-inhibiting side effects of therapeutic drugs taken by cancer patients for different indications.

The RIG-I agonist M8 triggers cell death and natural killer cell activation in human papillomavirus-associated cancer and potentiates cisplatin cytotoxicity.

Although the activation of innate immunity to treat a wide variety of cancers is gaining increasing attention, it has been poorly investigated in human papillomavirus (HPV)-associated malignancies. Because these tumors harbor a severely impaired cGAS-STING axis, but they still retain a largely functional RIG-I pathway, another critical mediator of adaptive and innate immune responses, we asked whether RIG-I activation by the 5'ppp-RNA RIG-I agonist M8 would represent a therapeutically viable option to treat HPV+ cancers. Here, we show that M8 transfection of two cervical carcinoma-derived cell lines, CaSki and HeLa, both expressing a functional RIG-I, triggers intrinsic apoptotic cell death, which is significantly reduced in RIG-I KO cells. We also demonstrate that M8 stimulation potentiates cisplatin-mediated cell killing of HPV+ cells in a RIG-I dependent manner. This combination treatment is equally effective in reducing tumor growth in a syngeneic pre-clinical mouse model of HPV16-driven cancer, where enhanced expression of lymphocyte-recruiting chemokines and cytokines correlated with an increased number of activated natural killer (NK) cells in the tumor microenvironment. Consistent with a role of RIG-I signaling in immunogenic cell killing, stimulation of NK cells with conditioned medium from M8-transfected CaSki boosted NK cell proliferation, activation, and migration in a RIG-I-dependent tumor cell-intrinsic manner. Given the highly conserved molecular mechanisms of carcinogenesis and genomic features of HPV-driven cancers and the remarkably improved prognosis for HPV+ oropharyngeal cancer, targeting RIG-I may represent an effective immunotherapeutic strategy in this setting, favoring the development of de-escalating strategies.

Abstract 2950: CD16A shedding facilitates repetitive targeting of tumor cells by AFM13-armed NK cells

Abstract Antibody-Dependent Cellular Cytotoxicity (ADCC) is a powerful mechanism of Natural Killer (NK) cells to kill antibody-opsonized target cells. However, ADCC mediated by conventional antibodies has its limitations in killing of tumor cells commonly being characterized by low tumor antigen expression. Several strategies have been implemented to boost ADCC including shedding inhibition of CD16A, a key ADCC-mediating Fc receptor on the plasma membrane of NK cells. However, previous results have shown that genetic engineering of the cleavage site of CD16A leads to decreased serial engagement of target cells. Another approach to boost ADCC is provided by tetravalent-bispecific innate cell engagers (ICE®) cross-linking CD16A and tumor antigens triggering anti-tumoral cytotoxicity. In the current study, we have investigated whether ICE molecules can induce effective ADCC while maintaining the natural function of CD16A with a particular focus on preserved CD16A shedding. To study cytotoxicity and contact dynamics, we have used an in-house developed live-cell microchip screening with single cell resolution together with a microcontact printing-based assay combined with an ICE®. Single cell analysis using microchip screening revealed that the CD16A/CD30 targeting ICE® AFM13 induces stronger ADCC of NK cells towards CD30+ target cells when compared to anti-CD30 antibodies. This stronger response was reached through increasing both the overall number of cytotoxic NK cells and the fraction of NK serial killers i.e., NK cells performing three or more kills in sequence. Interestingly, the differences were most prominent towards target cells expressing low levels of CD30. Combination of AFM13 and inhibition of CD16 shedding increased NK cell conjugation time with target cells, which could potentially limit targeting of additional tumor cells located at a distance from the primary target. To investigate this phenomenon further, we used a microcontact printing assay. Here, a grid of spatially distributed protein patches consisting of AFM13 and anti-LFA-1 was printed on glass surfaces enabling the formation of “artificial immune synapses” when probed by NK cells. On these “artificial immune synapses” we investigated the influence of shedding inhibition on NK cell migration and interaction dynamics. In conclusion, we show that AFM13 increases both the fraction of tumor-target responsive NK cells and the fraction of serial killing NK cells compared to conventional monoclonal antibodies. Based on our data we hypothesize that CD16A shedding facilitates AFM13 induced ADCC potential of NK cells by allowing potent migration to distantly located tumor cells and serial killing. Especially in the context of AFM13 primary indications, Hodgkin and peripheral T cell lymphoma, migration of NK cells might have a particularly strong impact when treating cancer patients due to the disseminated nature of the disease. Citation Format: Chiara Zambarda, Karolin Guldevall, Damien Toullec, Susanne Wingert, Christian Breunig, Sheena Pinto, Jacopo Fontana, Joachim Koch, Björn Önfelt. CD16A shedding facilitates repetitive targeting of tumor cells by AFM13-armed NK cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2950.

Predictive biomarkers for the responsiveness of recurrent glioblastomas to activated killer cell immunotherapy

BackgroundRecurrent glioblastoma multiforme (GBM) is a highly aggressive primary malignant brain tumor that is resistant to existing treatments. Recently, we reported that activated autologous natural killer (NK) cell therapeutics induced a marked increase in survival of some patients with recurrent GBM.MethodsTo identify biomarkers that predict responsiveness to NK cell therapeutics, we examined immune profiles in tumor tissues using NanoString nCounter analysis and compared the profiles between 5 responders and 7 non-responders. Through a three-step data analysis, we identified three candidate biomarkers (TNFRSF18, TNFSF4, and IL12RB2) and performed validation with qRT-PCR. We also performed immunohistochemistry and a NK cell migration assay to assess the function of these genes.ResultsResponders had higher expression of many immune-signaling genes compared with non-responders, which suggests an immune-active tumor microenvironment in responders. The random forest model that identified TNFRSF18, TNFSF4, and IL12RB2 showed a 100% accuracy (95% CI 73.5–100%) for predicting the response to NK cell therapeutics. The expression levels of these three genes by qRT-PCR were highly correlated with the NanoString levels, with high Pearson’s correlation coefficients (0.419 (TNFRSF18), 0.700 (TNFSF4), and 0.502 (IL12RB2)); their prediction performance also showed 100% accuracy (95% CI 73.54–100%) by logistic regression modeling. We also demonstrated that these genes were related to cytotoxic T cell infiltration and NK cell migration in the tumor microenvironment.ConclusionWe identified TNFRSF18, TNFSF4, and IL12RB2 as biomarkers that predict response to NK cell therapeutics in recurrent GBM, which might provide a new treatment strategy for this highly aggressive tumor.

Autophagy-mediated NKG2D internalization impairs NK cell function and exacerbates radiation pneumonitis.

Radiation pneumonitis is a critical complication that constrains the use of radiation therapy for thoracic malignancies, leading to substantial morbidity via respiratory distress and lung function impairment. The role of Natural killer (NK) cells in inflammatory diseases is well-documented; however, their involvement in radiation pneumonitis is not fully understood. To explore the involvement of NK cells in radiation pneumonitis, we analyzed tissue samples for NK cell presence and function. The study utilized immunofluorescence staining, western blotting, and immunoprecipitation to investigate CXCL10 and ROS levels, autophagy activity, and NKG2D receptor dynamics in NK cells derived from patients and animal models subjected to radiation. In this study, we observed an augmented infiltration of NK cells in tissues affected by radiation pneumonitis, although their function was markedly diminished. In animal models, enhancing NK cell activity appeared to decelerate the disease progression. Concomitant with the disease course, there was a notable upsurge in CXCL10 and ROS levels. CXCL10 was found to facilitate NK cell migration through CXCR3 receptor activation. Furthermore, evidence of excessive autophagy in patient NK cells was linked to ROS accumulation, as indicated by immunofluorescence and Western blot analyses. The association between the NKG2D receptor and its adaptor proteins (AP2 subunits AP2A1 and AP2M1), LC3, and lysosomes was intensified after radiation exposure, as demonstrated by immunoprecipitation. This interaction led to NKG2D receptor endocytosis and subsequent lysosomal degradation. Our findings delineate a mechanism by which radiation-induced lung injury may suppress NK cell function through an autophagy-dependent pathway. The dysregulation observed suggests potential therapeutic targets; hence, modulating autophagy and enhancing NK cell activity could represent novel strategies for mitigating radiation pneumonitis.

Cell Surface Engineering to Improve Migration of Natural Killer Cells Toward Bladder Cancer

Natural killer (NK) cells therapy for bladder cancer has shown a promise in clinical studies. However, insufficient of NK cells to the bladder cancer represents an important reason for the poor clinic trials against bladder cancer. How to improve the homing of infused NK cells is an important challenge. It is well known that chemokine receptor 3 which induces NK cells migration toward bladder cancer, expressed on NK cells. We hypothesized that CXCR3-modified NK cells could improving anti-tumor effect by enhancing homing of infused NK cells to the bladder cancer area. In this study, to provide a good mean to improve the homing of NK cells, we studied a surface modification method to incorporate CXCR3 on the surface of NK cells. In the results, this modification method shows a good biocompatibility for NK cells, and the results show that the migration of NK cells toward and against bladder cancer was enhanced. These preclinical findings suggest that CXCR3 modified NK cells may be a promising therapy for targeting bladder cancer and other tumors.

The role of CCL2, CCL7, ICAM-1, and VCAM-1 in interaction of endothelial cells and natural killer cells

Natural killer (NK) cell–based therapy has been studied for the treatment of patients with cancers, but the inadequate infiltration of NK cells into solid tumors remains a big challenge to its clinical application. In this study, we examined the interaction between NK cells and endothelial cells, which might play a major role in NK cell homing to solid tumors. We found that endothelial cells were activated by TNF-α and IL-1β, which were produced by tumor-associated CD11b+ cells, which included F4/80+ macrophages. TNF-α-treated endothelial cells increased NK cell migration by producing CCL2 and CCL7, which was proved by transwell and imaging assays. TNF-α-treated endothelial cells adhered well to NK cells, which was due to a TNF-α-induced increase in ICAM-1 and VCAM-1 expression on endothelial cells. Imaging data confirmed that TNF-α-treated endothelial cells transfected with ICAM-1 or VCAM-1 siRNAs did not establish stable contacts with NK cells. Taken together, our data suggest that CCL2, CCL7, ICAM-1, and VCAM-1 expressed by endothelial cells will be potential targets to guide adequate interaction with NK cells, which is a crucial step for NK cell homing to the tumor microenvironment.

Abstract SY24-03: Modulating cellular senescence to reinstate natural killer cell immunity for pancreatic cancer immunotherapy

Abstract Background: Cancer immunotherapies can lead to durable and sometimes curative responses in patients with otherwise treatment-refractory disease, and as such have revolutionized our approach to cancer therapy. Still, there are many roadblocks that must be overcome to make immunotherapies effective across a broader spectrum of patients and cancer types. Immune checkpoint blockade (ICB) therapies that are effective in some malignancies through reactivation of cytotoxic T cell immunity have failed in immunologically “cold” tumors such as pancreatic ductal adenocarcinoma (PDAC) due to poor T cell infiltration and weak antigen presentation necessary for effective anti-tumor T cell responses. We and others have demonstrated that induction of cellular senescence and the senescence associated secretory phenotype (SASP), which leads to production of immunomodulatory cytokines, chemokines, and growth factors, can be a powerful way to not only enhance T cell infiltration into tumors but also reactivate a different type of innate, antigen-independent Natural Killer (NK) cell immunity that can mediate tumor control. Recently, we found that a combination of two RAS pathway targeting therapies- the MEK inhibitor trametinib and CDK4/6 inhibitor palbociclib (T/P)- could induce senescence in genetically engineered mouse models of KRAS mutant lung adenocarcinoma and PDAC (Ruscetti*, Leibold*, Bott* et al. Science 2018; Ruscetti*, Morris*, Mezzadra* et al. Cell 2020). Strikingly, whereas therapy-induced senescence led to NK cell-mediated tumor regressions and long-term survival benefit in KRAS mutant lung cancer, it had little effect on NK cell immunity and tumor progression in PDAC. Here we set out to understand how the pancreas tumor microenvironment (TME) suppresses NK immunity and develop strategies to harness NK cell surveillance for immunotherapy in PDAC. Results and Discussion: We took advantage of genetically similar Kras mutant, p53 altered PDAC and lung cancer cell lines that could be transplanted into different organs of C57BL/6 mice to study whether and how the pancreas TME may impact senescence-driven immune responses. Syngeneic KRAS mutant lung and PDAC tumor cells transplanted into the pancreas of C57BL/6 mice displayed transcriptional repression and reduced chromatin accessibility of SASP transcriptional activators (NF-KB, IRFs) and factors necessary for NK cell activity (IL-15, IL-18) and chemotaxis (CCL2, CCL7, CCL8, CXCL9, CXCL10) following T/P-induced senescence, and did not undergo anti-tumor NK immune surveillance as compared to tumors propagated in the lungs and liver. Transcriptomic analysis identified induction of EZH2 and epigenetic repression of its targets, including key SASP factors and regulators, specifically in tumor cells grown in the pancreas TME. Genetic or pharmacological inhibition of EZH2 enhanced proinflammatory SASP signaling and resulted in NK cell migration and anti-tumor cytotoxicity in murine and human PDAC lines and transplanted and genetically engineered PDAC mouse models. Remarkably, tumor-intrinsic EZH2 knockdown in combination with T/P treatment led to complete tumor regressions in some PDAC-bearing mice that were reversed following NK cell depletion or suppression of key SASP factors. Pharmacological inhibition of EZH2 methyltransferase activity also enhanced NK cell infiltration and tumor control following therapy-induced senescence in transplanted and genetically engineered PDAC mouse models. Importantly, we found that an EZH2 repression signature was associated with enhanced NK cell infiltration and SASP induction in human PDAC patient samples. These results demonstrate that EZH2 mediates transcriptional repression of the proinflammatory SASP in the pancreas TME, and that EZH2 blockade in combination with senescence-inducing therapies could be a powerful means to reactivate absent NK cell surveillance in PDAC to achieve immune-mediated tumor responses. In the future we plan to test these therapeutic interventions in combination with anti-PD-1/PD-L1 ICB therapy as a means to further activate both NK and T cell immunity to fully eradicate pancreatic cancer. This work highlights the importance of understanding the impact of the resident tumor microenvironment on anti-tumor immune responses to come up with new paradigms to potentiate immunotherapy in this and other immunologically “cold” tumor settings that are historically unresponsive to ICB therapy. Citation Format: Marcus Ruscetti. Modulating cellular senescence to reinstate natural killer cell immunity for pancreatic cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr SY24-03.

Abstract LB116: CXCL8/Interleukin-8 orchestrates radiation-induced immune responses in pancreatic adenocarcinoma

Abstract The role of radiotherapy in the treatment of pancreatic adenocarcinoma (PDAC) is controversial. In many other tumor types, radiotherapy is a mainstay therapy whose anti-tumor effects have partially been ascribed to T cell responses. The role of Natural Killer (NK) cells in radiotherapy tumor responses remains unclear. Here, we focused on the role of NK cells in radiotherapy of pancreatic cancer. In a randomized controlled pancreatic cancer trial, we found that CXCL8 serum levels increased under radiotherapy and NK cells positively correlated with prolonged overall patient survival. Accordingly, NK cells preferentially infiltrated irradiated tumors and exhibited CD56dim like cytotoxic transcriptomic states. In preclinical experimental systems in mice and tumor cells, NF-κB and mTOR signaling prompted radiation-induced CXCL8 secretion from senescent tumor cells causing directional migration of CD56dim NK cells into tumors. Thus, we link senescence-associated CXCL8 release to innate immune surveillance of human tumors. Moreover, combined radiotherapy and adoptive NK cell transfer improved tumor control over monotherapies in xenografted mice suggesting NK cells combined with radiotherapy as a rational cancer treatment strategy which may be integrated into PDAC treatment algorithms. Citation Format: Thomas Walle, Joscha A. Kraske, Boyu Liao, Carmen Timke, Benedicte Lenoir, Niels Halama, Peter E. Huber. CXCL8/Interleukin-8 orchestrates radiation-induced immune responses in pancreatic adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB116.

Activation of Tumor-Cell STING Primes NK-Cell Therapy.

Activation of the stimulator of interferon genes (STING) pathway promotes antitumor immunity but STING agonists have yet to achieve clinical success. Increased understanding of the mechanism of action of STING agonists in human tumors is key to developing therapeutic combinations that activate effective innate antitumor immunity. Here, we report that malignant pleural mesothelioma cells robustly express STING and are responsive to STING agonist treatment ex vivo. Using dynamic single-cell RNA sequencing of explants treated with a STING agonist, we observed CXCR3 chemokine activation primarily in tumor cells and cancer-associated fibroblasts, as well as T-cell cytotoxicity. In contrast, primary natural killer (NK) cells resisted STING agonist-induced cytotoxicity. STING agonists enhanced migration and killing of NK cells and mesothelin-targeted chimeric antigen receptor (CAR)-NK cells, improving therapeutic activity in patient-derived organotypic tumor spheroids. These studies reveal the fundamental importance of using human tumor samples to assess innate and cellular immune therapies. By functionally profiling mesothelioma tumor explants with elevated STING expression in tumor cells, we uncovered distinct consequences of STING agonist treatment in humans that support testing combining STING agonists with NK and CAR-NK cell therapies.

CHMP2A regulates tumor sensitivity to natural killer cell-mediated cytotoxicity

Natural killer (NK) cells are known to mediate killing of various cancer types, but tumor cells can develop resistance mechanisms to escape NK cell-mediated killing. Here, we use a “two cell type” whole genome CRISPR-Cas9 screening system to discover key regulators of tumor sensitivity and resistance to NK cell-mediated cytotoxicity in human glioblastoma stem cells (GSC). We identify CHMP2A as a regulator of GSC resistance to NK cell-mediated cytotoxicity and we confirm these findings in a head and neck squamous cells carcinoma (HNSCC) model. We show that deletion of CHMP2A activates NF-κB in tumor cells to mediate increased chemokine secretion that promotes NK cell migration towards tumor cells. In the HNSCC model we demonstrate that CHMP2A mediates tumor resistance to NK cells via secretion of extracellular vesicles (EVs) that express MICA/B and TRAIL. These secreted ligands induce apoptosis of NK cells to inhibit their antitumor activity. To confirm these in vitro studies, we demonstrate that deletion of CHMP2A in CAL27 HNSCC cells leads to increased NK cell-mediated killing in a xenograft immunodeficient mouse model. These findings illustrate a mechanism of tumor immune escape through EVs secretion and identify inhibition of CHMP2A and related targets as opportunities to improve NK cell-mediated immunotherapy.

R848/TLR7-Mediated Stronger CD8+ T Immunity Is Dependent on DC-NK Cell Interactions

Background: Toll-like receptor (TLR) 7 agonists are effective candidates for Th1 immune adjuvants, which compensate for the insufficient Th1 immune responses induced by traditional adjuvants. This effect is currently dependent on TLR7-mediated induction of dendritic cell (DC) maturation and increased IL-12 production. Methods: In vivo, we intraperitoneally injected TLR agonists with OVA, and LNs were collected for detection. In vitro, Activated DCs, natural killer (NK) cells, and CD8⁺ T cells were tested using flow cytometry for surface expression and enzyme-linked immunosorbent assay for cytokine production. NK cell migration was evaluated using transwell system. All experiments were performed in both C57BL/6 and BALB/C backgrounds. Results: Our findings revealed that the enhanced CD8⁺ T immunity characterized by CD8⁺ T accumulation, proliferation, and IFN-γ⁺CD8⁺ T induction induced by R848 was attributed to DC-dependent NK cell migration and DC-NK interactions. Our results demonstrated that R848 induced CD8⁺ T cell accumulation and IFN-γ⁺CD8⁺ T cells in lymph nodes (LNs) to a greater degree in vivo than TLR4 agonists (lipopolysaccharide) and TLR9 agonists (Class C CPG). R848-activated DCs enhanced CD8⁺ T cell proliferation and increased IFN-γ⁺CD8⁺ T cells with the assistance of NK cells. In contrast, depletion of NK cell decreased IFN-γ⁺CD8⁺ T cell production. Greater NK cell migration to LNs occurred in R848-immunized mice. A similar effect of R848 on NK cell migration was observed in an in vitro transwell study. When co-cultured, NK cells plus R848 could promote DCs maturation, and in turn, DCs in combination of R848 augmented NK cells activation. Further studies demonstrated that among several TLR agonists, R848 produced the largest amount of the chemokine CXCL9 from activated DCs, which is relevant to NK cell migration. CXCL9 blockade reduced the number of migrated NK cells, and the addition of CXCL9 increased the number of NK cells. Discussion: Taken together, R848-mediated stronger CD8⁺ T cell immunity does not depend on DC activation alone, rather that NK cells must also be considered. By increasing our immunological understanding of the effect of R848/TLR7, these findings provide a new perspective for applying R848 in future clinical studies.

Quantifying Human Natural Killer Cell Migration by Imaging and Image Analysis.

Migration is an important function for natural killer cells. Cell motility has implications in development, tissue infiltration, and cytotoxicity, and measuring the properties of natural killer (NK) cell migration using in vitro assays can be highly informative. Many researchers have an interest in studying properties of NK cell migration in the context of genetic mutation, disease, or in specific tissues and microenvironments. Motility assays can also provide information on the localization of proteins during different phases of cell migration. These assays can be performed on different surfaces for migration or coupled with chemoattractants and/or target cells to test functional outcomes or characterize cell migration speeds and phenotypes. NK cells undergo migration during differentiation in tissue, and these conditions can be modeled by culturing NK cells on a confluent bed of stromal cells on glass and imaging cell migration. Alternatively, fibronectin- or ICAM-1-coated surfaces promote NK cell migration and can be used as substrates. Here, we will describe techniques for the experimental setup and analysis of NK cell motility assays by confocal microscopy or in-incubator imaging using commercially available systems. Finally, we describe open-source software for analyzing cell migration using manual tracking or automated approaches and discuss considerations for the implementation of each of these methods.