Priming Of Natural Killer Cells Research Articles

Abstract 1240: Enhancing NK cell function in the ‘cold’ tumor microenvironment of prostate cancer with a novel tri-specific killer engager

Abstract Introduction: Natural killer (NK) cells are associated with good prognosis in patients with metastatic castration-resistant prostate cancer (mCRPC). However, NK cell function is hindered in the immunosuppressive and hypoxic tumor microenvironment (TME) of mCRPC. To improve NK cell-mediated responses against mCRPC, we designed a novel Tri-specific Killer Engager (TriKE®) that engages with NK cell activating receptor CD16, binds to prostate-specific membrane antigen (PSMA) that is highly and specifically expressed on mCRPC, and has an interleukin (IL)-15 moiety that is essential for NK cell survival, proliferation, and priming. Methods: PSMA TriKE was produced using the mammalian expression system and tested using healthy donor and prostate cancer patient-derived NK cells. Flow cytometry-based functional and dye dilution proliferation assays were used to compare activation and proliferation of NK cells treated with PSMA TriKE or IL-15. NK cell cytolytic capacity against C4-2 cells, a PSMA-expressing prostate cancer line, was measured using IncuCyte live cell imaging. In various assays, hypoxic (1% oxygen) culture condition and cytokine-induced myeloid-derived suppressor cells (MDSC) were incorporated to better examine PSMA TriKE function in the physiological setting of mCRPC. In vivo testing of the PSMA TriKE was performed using C4-2 xenograft model and expanded NK cells in NSG mice. Results: PSMA TriKE significantly enhanced expansion of peripheral blood NK cells derived from healthy donors up to 8-fold. Additionally, PSMA TriKE induced significantly higher NK cell degranulation and intracellular IFNγ and TNFα buildup, when compared to IL-15 treatment, after incubation with C4-2 cells. Responses of NK cells derived from prostate cancer patients were equivalent to healthy controls. Although NK cell activation was not observed against PSMA knockout (KO) C4-2, indicating specificity of PSMA TriKE treatment, bystander killing of PSMA-KO C4-2 was achieved with PSMA TriKE treatment when wildtype C4-2 cells were cocultured. This suggests potential PSMA TriKE benefit in controlling tumor antigen escape, through natural cytotoxicity once primed. In settings that mimic the TME of mCRPC, NK cells treated with PSMA TriKE in prolonged exposure to hypoxia showed retention of cytotoxicity against C4-2 while IL-15 treated NK cells showed greatly impaired cytotoxicity. Similarly, MDSC suppressed NK cell cytotoxicity in the presence of IL-15, but PSMA TriKE treatment abrogated MDSC-induced suppression. Finally, PSMA TriKE enhanced tumor control and improved survival of mice as compared to IL-15 and no treatment groups in vivo. Conclusion: PSMA TriKE demonstrates potential in overcoming suppression of NK cells in the TME of mCRPC and is a promising candidate for advanced prostate cancer therapy. Citation Format: Shee Kwan Phung, Yvette Soignier, Nicholas Zorko, Rhett L. Waller, Josh Walker, Trygve Nelson, Carly Selleck, Laura Bendzick, Philippa Kennedy, Jeffrey S. Miller, Martin Felices. Enhancing NK cell function in the ‘cold’ tumor microenvironment of prostate cancer with a novel tri-specific killer engager [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1240.

Networked Cluster Formation via Trigonal Lipid Modules for Augmented Ex Vivo NK Cell Priming.

Current cytokine-based natural killer (NK) cell priming techniques have exhibited limitations such as the deactivation of biological signaling molecules and subsequent insufficient maturation of the cell population during mass cultivation processes. In this study, we developed an amphiphilic trigonal 1,2-distearoyl-sn-glycero-3-phosphorylethanolamine (DSPE) lipid-polyethylene glycol (PEG) material to assemble NK cell clusters via multiple hydrophobic lipid insertions into cellular membranes. Our lipid conjugate-mediated ex vivo NK cell priming sufficiently augmented the structural modulation of clusters, facilitated diffusional signal exchanges, and finally activated NK cell population with the clusters. Without any inhibition in diffusional signal exchanges and intrinsic proliferative efficacy of NK cells, effectively prime NK cell clusters produced increased interferon-gamma, especially in the early culture periods. In conclusion, the present study demonstrates that our novel lipid conjugates could serve as a promising alternative for future NK cell mass production.

In Vivo Generation of Memory-like NK Cells for the Treatment of AML and Myelodysplastic Syndrome; Early Clinical Applications of INKmuneTM

Natural killer (NK) cells can have potent anti-tumour responses and recently highlighted potential for memory-like functions, but the best approach to generate memory-like NK (mlNK) cells remains unclear. Priming of NK cells with the pharmaceutical-grade, replication incompetent tumour cell product, INKmuneTM generates tumour-induced mlNK (TIML-NK) cells with enhanced cytokine production and cytotoxicity against multiple NK-resistant tumour target cell lines in vitro, analogous to previously reported cytokine-primed memory-like NK cells. In addition, proteomic profiling of TIML-NK cells revealed differential abundance of proteins involved in promoting mitochondrial survival and function as well as critical nutrient receptors, which may provide a unique benefit of NK cell activation whilst sparing mitochondrial damage typically associated with cytokine-mediated activation. INKmune priming increased NK glycolysis and oxidative phosphorylation whilst enhancing mitochondrial respiratory capacity and maintaining glycolytic reserve. Four patients have been treated with a cycle of three, weekly infusions of 1x108 INKmuneTM cells. All patients received all three doses without incident and without side effects. Patient 1 was a 78yr old man with A 3yr history of refractory MLD MDS who was transfusion and platelet dependent and required G-CSF. Within 7 days of first infusion, over 50% of his peripheral blood NK cells activated and this increased with each dose. By day +29, 72% of his NK cells were activated and this remained above 68% at day +119 when monitoring ended. His ECOG status fell from 2 pre-treatment to 0 at D+119 and his RAD-1 ctDNA from 45 to 38. Analysis of systemic cytokines showed increases in MIP-1a/b, TNF-a and sIL2R which paralleled the changes in percentages of CD69+ NK cells. IL6 levels peaked after the second infusion but levels remained low and there was no evidence of CRS. At one year follow-up he remained well, platelet and G-CSF independent, with reduced transfusion requirements and had resumed playing sport with friends. Subjects 2, 3 and 4, were all multiply relapsed and refractory AML patients; two of whom had relapsed after HLA-mismatched HSCT. All showed rapid activation of peripheral NK cells post infusion with sustained activity throughout follow-up. In all cases these activated NK cells killed NK-resistant tumour cells in vitro without additional activation. Subject 2 was a neutropenic 21yr old female with 70% a mixed chimera and refractory AML (M2) post VUD HSCT at time of treatment. Within 1 month of initiation of INKmune treatment she was discharged with PMN >500 and her mixed chimera resolved to full donor chimera. At Day 140 post INKmune her bone marrow NK cells remained highly activated (>60% CD69+, K562 and Raji lysis of >70%). Her AML recurred 4 months post INKmune infusion and she died from relapsed disease 8 months after INKmune treatment. Subject 3 was a 21yr old male with refractory AML (M6) after two failed HSCT (haplo m/m and VUD). He showed the same rapid generation of activated NK cells in vivo but to a lesser degree (maximum 35% CD69+, sustained increased lysis of K562 but no lysis of Raji cells) and no evidence of clinical improvement. Subject 4, a multiply relapsed AML patient has just been treated and has shown peripheral NK proliferation and maturation at D+8. The subject experienced no adverse effects post 1st and 2nd doses and further follow-up will be presented. INKmuneTM infusions proved safe and led to rapid and sustained activation of peripheral NK cells with concomitant increases in relevant systemic cytokines. The TIML-NK have a distinct phenotypic, proteomic and metabolomic signature which mirrors the activity in vitro and in vivo. This is the first time memory-like NK cells have been generated in vivo without the need for cytokine induction or support and our findings inform the development of more optimal NK cell-based immunotherapeutic strategies for cancer. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Nanomaterials for Natural Killer Cell-Based Immunoimaging and Immunotherapies in Cancer.

Natural killer (NK) cells are an important component of the tumor immunosurveillance; activated NK cells can recognize and directly lyse tumor cells eliciting a potent antitumor immune response. Due to their intrinsic ability to unleash cytotoxicity against tumor cells, NK cell-based adoptive cell therapies have gained rapid clinical significance, and many clinical trials are ongoing. However, priming and activating NK cells, infiltration of activated NK cells in the immunosuppressive tumor microenvironment, and tracking the infiltrated NK cells in the tumors remain a critical challenge. To address these challenges, NK cells have been successfully interfaced with nanomaterials where the morphology, composition, and surface characteristics of nanoparticles (NPs) were leveraged to enable longitudinal tracking of NK cells in tumors or deliver therapeutics to prime NK cells. Distinct from other published reviews, in this tutorial review, we summarize the recent findings in the past decade where NPs were used to label NK cells for immunoimaging or deliver treatment to activate NK cells and induce long-term immunity against tumors. We discuss the NP properties that are key to surmounting the current challenges in NK cells and the different strategies employed to advance NK cells-based diagnostics and therapeutics. We conclude the review with an outlook on future directions in NP-NK cell hybrid interfaces, and overall clinical impact and patient response to such interfaces that need to be addressed to enable their clinical translation.

Chemical priming of natural killer cells with branched polyethylenimine for cancer immunotherapy

BackgroundDue to their powerful immune surveillance activity and ability to kill and clear cancer cells, natural killer (NK) cells are an emerging anticancer immunotherapeutic agent. Therefore, there is much interest...

Phosphoantigen-Stimulated γδ T Cells Suppress Natural Killer-Cell Responses to Missing-Self.

γδ T cells stimulated by phosphoantigens (pAg) are potent effectors that secrete Th1 cytokines and kill tumor cells. Consequently, they are considered candidates for use in cancer immunotherapy. However, they have proven only moderately effective in several clinical trials. We studied the consequences of pAg-stimulated γδ T-cell interactions with natural killer (NK) cells and CD8+ T cells, major innate and adaptive effectors, respectively. We found that pAg-stimulated γδ T cells suppressed NK-cell responses to "missing-self" but had no effect on antigen-specific CD8+ T-cell responses. Extensive analysis of the secreted cytokines showed that pAg-stimulated γδ T cells had a proinflammatory profile. CMV-pp65-specific CD8+ T cells primed with pAg-stimulated γδ T cells showed little effect on responses to pp65-loaded target cells. By contrast, NK cells primed similarly with γδ T cells had impaired capacity to degranulate and produce IFNγ in response to HLA class I-deficient targets. This effect depended on BTN3A1 and required direct contact between NK cells and γδ T cells. γδ T-cell priming of NK cells also led to a downregulation of NKG2D and NKp44 on NK cells. Every NK-cell subset was affected by γδ T cell-mediated immunosuppression, but the strongest effect was on KIR+NKG2A- NK cells. We therefore report a previously unknown function for γδ T cells, as brakes of NK-cell responses to "missing-self." This provides a new perspective for optimizing the use of γδ T cells in cancer immunotherapy and for assessing their role in immune responses to pAg-producing pathogens. See related Spotlight by Kabelitz, p. 543.

Enhanced anticancer efficacy of primed natural killer cells via coacervate-mediated exogenous interleukin-15 delivery.

Effective exogenous delivery of interleukin (IL)-15 to natural killer (NK) cells with subsequent anticancer efficacy could be a promising immune cell-based cancer immunotherapy. For the protection of encapsulated cargo IL-15 while maintaining its bioactivity under physiological conditions, we utilized a coacervate (Coa) consisting of a cationic methoxy polyethylene glycol-poly(ethylene arginyl aspartate diglyceride) (mPEG-PEAD) polymer, anionic counterpart heparin, and cargo IL-15. mPEGylation into the backbone cation effectively preserved the colloidal stability of Coa in harsh environments and enhanced the protection of cargo IL-15 than normal Coa without mPEGylation. Proliferation and anticancer efficacy of primed NK cells through co-culture with multiple cancer cell lines were enhanced in the mPEG-Coa group due to the maintained bioactivity of cargo IL-15 during the ex vivo expansion of NK cells. These facilitated functions of NK cells were also supported by the increased expression of mRNAs related to anticancer effects of NK cells, including cytotoxic granules, death ligands, anti-apoptotic proteins, and activation receptors. In summary, our Coa-mediated exogenous IL-15 delivery could be an effective ex vivo priming technique for NK cells with sustained immune activation that can effectively facilitate its usage for cancer immunotherapy.

Gastric cancer mesenchymal stem cells inhibit natural killer cell function by up-regulating FBP1.

IntroductionThe dysfunction of natural killer (NK) cells has been widely reported in malignancies, including in solid tumours. Gastric cancer mesenchymal stem cells (GCMSCs) are one of the vital elements of stromal cells in the tumour environment (TME) which possess immunosuppressive activity. This study aimed to determine whether GCMSCs are involved in the inhibition of NK cell immune function and explore its underlying mechanism.Material and methodsCD107a and perforin expression of GCMSCs conditioned medium (GCMSCs-CM)-primed NK cells were detected by flow cytometry. To determine NK cell cytotoxicity, the CytoTox96 Non-Radioactive Cytotoxicity Assay kit was used. Glucose uptake and lactate production assay were performed to evaluate the metabolism state of NK cells treated with GCMSCs-CM. The expression of FBP1 in NK cells was analysed by immunoblotting.ResultsGCMSCs inhibited the degranulation capacity, perforin production and cytotoxicity of NK cells. GCMSCs-CM restrained NK cell glucose uptake and lactate production, thus weakening their glycolytic metabolism. FBP1 expression of NK cells was upregulated in the presence of GCMSCs-CM. Using FBP1 inhibitor could reverse the dysfunctional state of NK cells.ConclusionsThis study indicated that GCMSCs could exert immunosuppressive effects on NK cells by up-regulating FBP1 expression, opening up new avenues for NK cell-based GC immunotherapy.

Immunophenotypic and functional characterisation of Mesodermal Killer (MK) cells: a novel cell type and potential cellular therapy for cancer

Background & Aim We have identified a novel cell type, named mesodermal killer (MK) cells, derived from adult bone marrow under proprietary culture conditions. This study characterized the MK cells by immunophenotype, cytokine profile, immunomodulatory function and cytotoxicity. Methods, Results & Conclusion Methods: We investigated MK cells before and after activation with TNFα or IFNγ for immunophenotype using high throughput-FACS, for secretome and for inflammatory response in the murine air pouch assay. We evaluated MK cell cytotoxicity using the chromium release assay and immunomodulatory function by co-culture with natural killer (NK) cells in transwells. We used immunocytochemistry, flow cytometry and RT-PCR to detect expression of granzyme/perforin genes and proteins and investigated death pathways by siRNA silencing of FasL and TRAIL. Results MK cells displayed a novel immunophenotype (and different from BM-MSCs) including expression of CD16, CD96, CD112, CD137L, FasL, TRAIL and CD277 and some NK cell markers. In contrast to NK cells, MK cells lacked CD56 expression. TNFα and IFNγ activation increased FasL expression. MK cells secreted GROa, IL-12, IL-2Ra, IL-8, TRAIL and IL-6, and GROa and IL-8 secretion was increased by TNFα. MK cell cytotoxicity was observed against leukemia (K562), multiple myeloma (RPMI-8226) and breast cancer (MCF7) cell lines. MK cell conditioned media had no effect on MCF7 and antibody dependent cell mediated cytotoxicity (ADCC) experiments also proved negative. MK cells expressed granzymes and perforin and these were upregulated following exposure to RPMI-8226 (in contrast to BM-MSCs). Pre-treatment of MK cells with EGTA (a non-specific inhibitor of granule exocytosis) demonstrated >50% decrease in cytotoxicity against MCF7 suggesting a partial role of granule exocytosis in MK cell cytotoxicity. Inhibition of TRAIL expression by siRNA led to a significant reduction in MK cell cytotoxicity suggesting TRAIL may also be involved. In contrast to BM-MSCs, MK cells enhanced the cytotoxicity of primary NK cells against K562, RPMI-8226 and U226 (plasma cell myeloma) and IFNγ-activated MK cells enhanced migration of NK cells and monocytes into the murine air pouch suggesting a pro-inflammatory response. Conclusion MK cells exhibit cytotoxicity against a variety of cancer cell lines, are capable of priming NK cells in vitro and are pro-inflammatory in vivo. These results suggest the clinical translation of the MK cells is warranted and animal efficacy studies will begin shortly.

Priming NK cells for tumor destruction

Immunotherapy Some tumors can evade CD8+ T cells, which are used in several cancer immunotherapies, but natural killer (NK) cells provide another option to target such tumors for immune elimination. Nicolai et al. used several mouse models to investigate how a cyclic dinucleotide (CDN) agonist for an innate immune pathway called STING potentiates the antitumor activity of NK cells. CDN administration induced type I interferons that directly promoted NK cell activation and simultaneously enabled an indirect pathway of activation driven by induction of interleukin- 15 signaling in dendritic cells. Amplification of NK-based tumor immunity may offer a valuable adjunct to CD8+ T cell immunotherapy. Sci. Immunol. 5 , eaaz2738 (2020).

Abstract P5-04-11: Targeting hormone receptor positive breast cancer for immune destruction by natural killer cells

Abstract Hormone receptor positive (HR+) breast cancers that express estrogen receptor (ER) can recur 5-20 years after initial diagnosis and treatment, often as bone metastases. More than 30% of patients with early-stage HR+ breast cancer treated with endocrine or hormonal therapy will relapse and all patients with metastatic breast cancer expressing ER eventually acquire resistance to endocrine therapy. The metastases are caused by slowly dividing well-differentiated cancer cells. These cells are not susceptible to chemotherapy, which only acts on dividing cells, and they are also resistant to endocrine therapy. Recent analyses indeed show that chemotherapy added to endocrine therapy for patients with lower risk early stage breast cancer did not increase overall survival. Thus, patients with metastatic HR+ breast cancer need new therapeutic options. Unfortunately HR+ breast cancers have not responded to current T-cell based immunotherapies. Therefore the objective of this study is to target HR+ breast cancers using a natural killer (NK) cell based approach. The type I IGF receptor (IGF1R) is expressed on hormone receptor positive (HR+) breast cancers and its expression is tightly related to ER function. We hypothesized that a drug that would target NK cells to IGF1R expressing HR+ cells will be effective in eliminating them. We generated a novel NK cell based immunotherapy called Tri specific Killer Engagers (TriKE) and these are much smaller molecules than conventional trispecific antibodies. The IGF1R TriKE consists of humanized single domain antibody (VHH) sequence of a camelid nanobody against CD16 to engage and activate NK cells, humanized VHH sequence of a nanobody against IGF1R to target HR+ breast cancer cells, and an IL-15 molecule between the two single domain antibodies to drive NK cell priming, expansion and survival. The IGF1R TriKE specifically bound to breast cancer cells expressing IGF1R and did not bind mouse embryonal fibroblasts from IGF1R knock out mice (R- cells). It bound R-/IGF1R cells (R- cells engineered to express human IGF1R) indicating specific binding to IGF1R on cells. The IGF1R TriKE did not cause downregulation of cell surface IGF1R levels in multiple HR+ breast cancer cells. The IGF1R TriKE specifically induced NK cell degranulation, measured by surface CD107a expression, against IGF1R expressing HR+ target breast cancer cells (MCF-7, T-47D and ZR-75-1) in a functional assay compared to treatment with anti-IGF1R nanobody or IL-15 alone. It did not induce degranulation of NK cells against R- cells but enhanced degranulation against R-/IGF1R cells suggesting that the functionality was specific for IGF1R expressing targets. Furthermore, the IGF1R TriKE also enhanced cell killing of ZR-75-1 breast cancer cells compared to IGF1R nanobody as measured by number of live breast cancer cells at the end of a 48 hour cytotoxicity assay. We are currently testing the IGF1R TriKE’s functionality in causing degranulation of NK cells from blood of breast cancer patients. These data indicate that this NK cell based immunotherapeutic strategy could be an effective treatment for HR+ metastatic breast cancer and make them responsive to immunotherapy. Our approach generates targeted off-the-shelf immunotherapy that does not require a personalized approach for each patient that is required with CAR T-cell based approaches that is expensive and time consuming. Citation Format: Emily Chiu, Ivy R Johnson, Jeffrey S Miller, Martin Felices, Deepali Sachdev. Targeting hormone receptor positive breast cancer for immune destruction by natural killer cells [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 P5-04-11.

NK Cell Priming From Endogenous Homeostatic Signals Is Modulated by CIS

Natural killer (NK) cell activation is controlled by a balance of activating and inhibitory signals and cytokines such as IL-15. We previously identified cytokine-inducible SH2-containing protein (CIS) as a negative regulator of IL-15 signaling in NK cells under inflammatory conditions. While the functional effect of Cish-deficiency in NK cells was obvious by their increased anti-tumor immunity and hyper-proliferative response to IL-15, it remained unclear how CIS regulates NK cell biology in steady-state. Here, we investigated the role of CIS in the homeostatic maintenance of NK cells and found CIS-ablation promoted terminal differentiation of NK cells and increased turnover, suggesting that under steady-state conditions, CIS plays a role in maintaining IL-15 driven regulation of NK cells in vivo. However, hyper-responsiveness to IL-15 did not manifest in NK cell accumulation, even when the essential NK cell apoptosis mediator, Bcl2l11 (BIM) was deleted in addition to Cish. Instead, loss of CIS conferred a lower activation threshold, evidenced by augmented functionality on a per cell basis both in vitro and in vivo without prior priming. We conclude that Cish regulates IL-15 signaling in NK cells in vivo, and through the rewiring of several activation pathways leads to a reduction in activation threshold, decreasing the requirement for priming and improving NK cell anti-tumor function. Furthermore, this study highlights the tight regulation of NK cell homeostasis by several pathways which prevent NK cell accumulation when IL-15 signaling and intrinsic apoptosis are dysregulated.

Constitutive Activation of Natural Killer Cells in Primary Biliary Cholangitis.

Natural killer (NK) cells are innate immune cells that interface with the adaptive immune system to generate a pro-inflammatory immune environment. Primary Biliary Cholangitis (PBC) is a hepatic autoimmune disorder with extrahepatic associations including systemic sclerosis, Sjogren's syndrome and thyroiditis. Immunogenetic studies have identified polymorphisms of the IL-12/STAT4 pathway as being associated with PBC. As this pathway is important for NK cell function we investigated NK cells in PBC. Circulating NK cells from individuals with PBC were constitutively activated, with higher levels of CD49a and the liver-homing marker, CXCR6, compared to participants with non-autoimmune chronic liver disease and healthy controls. Stimulation with minimal amounts of IL-12 (0.005 ng/ml) led to significant upregulation of CXCR6 (p < 0.005), and enhanced IFNγ production (p < 0.02) on NK cells from PBC patients compared to individuals with non-autoimmune chronic liver disease, indicating dysregulation of the IL-12/STAT4 axis. In RNAseq studies, resting NK cells from PBC patients had a constitutively activated transcriptional profile and upregulation of genes associated with IL-12/STAT4 signaling and metabolic reprogramming. Consistent with these findings, resting NK cells from PBC patients expressed higher levels of pSTAT4 compared to control groups (p < 0.001 vs. healthy controls and p < 0.05 vs. liver disease controls). In conclusion NK cells in PBC are sensitive to minute quantities of IL-12 and have a “primed” phenotype. We therefore propose that peripheral priming of NK cells to express tissue-homing markers may contribute to the pathophysiology of PBC.

Combretastatin A4 Nanoparticles Combined with Hypoxia-Sensitive Imiquimod: A New Paradigm for the Modulation of Host Immunological Responses during Cancer Treatment.

Vascular disrupting agents (VDAs) have great potential in cancer treatment. However, in addition to their direct tumoral vascular collapse effect, VDAs activate host immunological responses, which can remarkably impair their anticancer efficacy. Here, a VDA nanomedicine, poly(l-glutamic acid)-graft-methoxy poly(ethylene glycol)/combretastatin A4 (CA4-NPs), is found to induce the intratumor infiltration of immature plasmacytoid dendritic cells (pDCs), thereby curtailing anticancer immunity. To overcome this problem, hypoxia-sensitive imiquimod (hs-IMQ) is developed, which is selectively activated into imiquimod (IMQ) in treated tumors following the catalysis of CA4-NPs-induced nitroreductase (NTR). The combination of hs-IMQ and CA4-NPs causes a 6.3-fold enhancement of active IMQ concentration in tumors, as compared to hs-IMQ treatment alone. The in situ-generated IMQ alters the tumor microenvironment from a state of immunosuppression to immune activation. Hs-IMQ achieves this effect through the conversion of immature pDCs into their active form, leading to the robust infiltration and priming of natural killer cells and cytotoxic T-lymphocytes in treated tumors. Thus, the CA4-NPs and hs-IMQ combination treatment synergistically inhibits tumor growth and metastasis in 4T1 tumor-bearing mice. This work offers new approaches to harness intratumor pDCs to reverse the immune suppression resulting from VDA treatment. These findings additionally provide a mechanistic rationale for the use of VDAs in combination with TLR agonists to trigger in situ immune activation and enhance anticancer efficacy.

NK cells are activated and primed for skin-homing during acute dengue virus infection in humans

Despite animal models showing that natural killer (NK) cells are important players in the early defense against many viral infections, the NK cell response is poorly understood in humans. Here we analyze the phenotype, temporal dynamics, regulation and trafficking of NK cells in a patient cohort with acute dengue virus infection. NK cells are robustly activated and proliferate during the first week after symptom debut. Increased IL-18 levels in plasma and in induced skin blisters of DENV-infected patients, as well as concomitant signaling downstream of the IL-18R, suggests an IL-18-dependent mechanism in driving the proliferative NK cell response. Responding NK cells have a less mature phenotype and a distinct chemokine-receptor imprint indicative of skin-homing. A corresponding NK cell subset can be localized to skin early during acute infection. These data provide evidence of an IL-18-driven NK cell proliferation and priming for skin-homing during an acute viral infection in humans.

A Brief IL-15 Pulse Results in JAK3-Dependent Phosphorylation of ITAM-Associated Signaling Molecules and a Long-Lasting Priming Imprint in Mouse NK Cells

Interleukin-15 (IL-15) is responsible for natural killer (NK) cell priming but the dynamics, kinetics and molecular mechanisms of NK cell priming are not well characterised. We here show that as little as 5 minutes of IL-15 treatment of NK cells followed by removal of excess cytokine, left a long-lasting but reversible priming imprint characterised by enhanced calcium flux, degranulation and INF-gamma production after activation of the ITAM-encoded receptor NK1.1. Enhanced function was linked to ROS production and to an increased steady-state phosphorylation of signalling molecules downstream of ITAM-containing activating receptors, including LCK and SLP-76. A crosstalk between the IL-15 receptor and activating receptors in NK cells was suggested by a dampening effect of IL-15-dependent priming by JAK3 inhibition. Increased STAT5 phosphorylation and calcium flux responses remained for at least three hours after IL-15 removal but then disappeared. Our study extends the understanding of NK cell priming and is relevant to fundamental and applied questions in NK cell biology.

Phenotype and Function of Activated Natural Killer Cells From Patients With Prostate Cancer: Patient-Dependent Responses to Priming and IL-2 Activation.

Background: Although immunotherapy has emerged as the “next generation” of cancer treatments, it has not yet been shown to be successful in the treatment of patients with prostate cancer, for whom therapeutic options remain limited to radiotherapy and androgen (hormone) deprivation therapy. Previous studies have shown that priming natural killer (NK) cells isolated from healthy individuals via co-incubation with CTV-1 cells derived from an acute lymphoblastic leukemia (ALL) enhances their cytotoxicity against human DU145 (metastatic) prostate cancer cells, but it remains unknown to what extent NK cells from patients with prostate cancer can be triggered to kill. Herein, we explore the phenotype of peripheral blood NK cells in patients with prostate cancer and compare the capacity of CTV-1 cell-mediated priming and IL-2 stimulation to trigger NK cell-mediated killing of the human PC3 (metastatic) prostate cancer cell line.Methods: The phenotype of resting, primed (co-incubation with CTV-1 cells for 17 h) and IL-2 activated (100 IU/ml IL-2 for 17 h) NK cells isolated from frozen-thawed peripheral blood mononuclear cell (PBMC) preparations from patients with benign disease (n = 6) and prostate cancer (n = 18) and their cytotoxicity against PC3 and K562 cells was determined by flow cytometry. Relationship(s) between NK cell phenotypic features and cytotoxic potential were interrogated using Spearman Rank correlation matrices.Results and Conclusions: NK cell priming and IL-2 activation of patient-derived NK cells resulted in similar levels of cytotoxicity, but distinct NK cell phenotypes. Importantly, the capacity of priming and IL-2 stimulation to trigger cytotoxicity was patient-dependent and mutually exclusive, in that NK cells from ~50% of patients preferentially responded to priming whereas NK cells from the remaining patients preferentially responded to cytokine stimulation. In addition to providing more insight into the biology of primed and cytokine-stimulated NK cells, this study supports the use of autologous NK cell-based immunotherapies for the treatment of prostate cancer. However, our findings also indicate that patients will need to be stratified according to their potential responsiveness to individual therapeutic approaches.

Inhibitory Receptor Crosslinking Quantitatively Dampens Calcium Flux Induced by Activating Receptor Triggering in NK Cells

Natural killer (NK) cell function is regulated by a balance between activating and inhibitory receptors, but the details of this receptor interplay are not extensively understood. We developed a flow cytometry-based assay system in which Ca2+ flux downstream of antibody-mediated activating receptor triggering was studied in the presence or absence of inhibitory receptor co-crosslinking. We show that the inhibitory influence on activating receptor-induced Ca2+ flux is quantitatively regulated, both on murine and human NK cells. Furthermore, both activating and inhibitory receptors operate in an additive way, suggesting that a fine-tuned balance between activating and inhibitory receptors regulate proximal NK cell signaling. We also demonstrate that murine NK cell expression of H2Dd lowered the capacity of Ly49A to deliver inhibitory signals after antibody crosslinking, suggesting that the cis interaction between H2Dd and Ly49A reduce the signaling capacity of Ly49A in this setting. Finally, we show that priming of NK cells by IL-15 rapidly augments Ca2+ flux after activating receptor signaling without attenuating the potential of inhibitory receptors to reduce Ca2+ flux. Our data shed new light on NK cell inhibition and raises new questions for further studies.

CD16-IL15-CLEC12A Trispecific Killer Engager (TriKE) Drives NK Cell Expansion, Activation, and Antigen Specific Killing of Cancer Stem Cells in Acute Myeloid Leukemia

Our group developed a 161533 trispecific killer engager (TriKE) molecule to target acute myeloid leukemia (AML) cells using Natural Killer (NK) cells. This molecule contains an anti-CD16 camelid nanobody to activate NK cells, an anti-CD33 single chain variable fragment (scFv) to engage cancer targets, and an IL-15 molecule that drives NK cell priming, expansion and survival. Using an earlier version of this molecule, we have shown that the CD33 TriKE is effective at activating NK cells against AML targets in vitro and in vivo. This preclinical data has lead to the establishment of a clinical trial in refractory AML patients at the University of Minnesota, set to open Q3 2018. While these previous studies have validated the use of TriKEs as an effective strategy of harnessing NK cells in cancer immunotherapy, CD33 has limitations as a target antigen.The high mortality and poor five-year survival rates (26%) for AML patients can be attributed to chemotherapy resistance and disease relapse. A majority of chemotherapy resistant leukemia stem cells (LSCs), that are hypothesized to facilitate relapse, do not express CD33. In addition, all hematopoietic stem cells and normal myeloid cells express CD33, thus targeting this antigen can lead to severe defects in hematopoiesis and on-target/off-tumor toxicity. To address these limitations, we developed a TriKE that targets CLEC12A or C-type lectin-like molecule 1 (CLL-1). CLEC12A is highly expressed on AML cells and over 70% of CD33 negative cells express CLEC12A. It has been attributed as a stem cell marker in AML, being selectively overexpressed in LSCs. CLEC12A is expressed by CD34+/CD38- LSCs but not normal CD34+/CD38- hematopoietic stem cells in regenerating bone marrow, thus minimizing off-target effects.The CLEC12A TriKE was developed in a mammalian cell system to ensure that appropriate post-translational modifications are present. We confirmed that the TriKE binds specifically to HL-60 and THP-1 target cells that express CLEC12A compared to Raji cells that do not express CLEC12A. Treatment of peripheral blood mononuclear cells (PBMCs) with the CLEC12A TriKE drives a significant increase in NK cell specific proliferation over 7 days as measured by CellTrace dilution compared to treatment with a CLEC12A scFv or IL-15 alone (69.7 ± 6.7% vs 11.9 ± 2.5% vs 38.4 ± 7.3%) (Figure 1A). To measure NK cell killing, we conducted an IncuCyte Zoom assay. Here, HL-60 target cells were labeled with a caspase 3/7 reagent where a color change indicates target cell death. The CLEC12A TriKE was able to induce more target cell killing than CLEC12A scFv or IL-15 as measured by number of live target cells at the end of the 48 hour assay (53.9 ± 1.9% vs 103.3 ± 3.4% vs 71.1 ± 1.4%). The CLEC12A TriKE induces an increase in NK cell degranulation, measured by CD107a expression against HL-60 AML tumor targets in a 4 hour functional assay compared to treatment with CLEC12A scFv or IL-15 alone (62.3 ± 1.1% vs 19.4 ± 3.8% vs 27.5 ± 4.9%). In this assay, there is also an increase in cytokine production, measured by IFNg and TNFa respectively (16.7 ± 4.2% vs 2.3 ± 1.5% vs 4.7 ± 1.9% and 18.0 ± 5.1% vs 2.5 ± 1.7% vs 4.6 ± 2.5%) (Figure 1B). We observe a similar enhanced functional response with THP-1 AML tumor targets. In these functional assays, treatment with the CLEC12A TriKE produced less background activation compared to the CD33 TriKE, indicating less off-target effects on PBMCs. To confirm the clinical relevance of this molecule, we tested the efficacy of the CLEC12A TriKE against primary AML targets. AML blasts were identified as SSC low, CD45 intermediate and CD34 high cells. Out of the 9 AML samples tested, 7 expressed high levels of CD33 (70.4 ± 6.3%) and CLEC12A (78.1 ± 5.2%). In functional assays with these samples, the CLEC12A TriKE was able to induce greater CD107a and IFNg expression, and enhanced killing of tumor targets as measured by a live/dead stain compared to CLEC12A scFv or IL-15 (Figure 1C). In these assays, the efficacy of the CLEC12A TriKE was comparable to the CD33 TriKE. Our data demonstrates that the CLEC12A TriKE drives NK cell specific proliferation, degranulation, cytokine secretion, and killing of tumor targets in vitro. Apart from AML, CLEC12A is expressed on cancer cells and LSCs in patients with myelodysplastic syndromes (MDS). These findings highlight the clinical potential of the CLEC12A TriKE individually or in combination with the CD33 TriKE for the treatment of MDS and AML. [Display omitted] DisclosuresVallera:GT Biopharma: Consultancy, Research Funding. Felices:GT Biopharma: Research Funding.

Tumor cGAMP Awakens the Natural Killers

Type I interferon (IFN) production within the tumor microenvironment is important in shaping the immune response to the tumor. In this issue of Immunity, Marcus etal. (2018) reveal that tumor cells produce 2'3'-cGAMP, which activates the STING pathway in non-tumor cells and leads to type I IFN production and the priming of natural killer cells for tumor rejection.