Natural Killer Cell Antitumor Activity Research Articles

Therapeutic Potential of Natural Killer Cells in Gastric Cancer.

Gastric cancer (GC) is one of the most common cancers, with a high incidence of cancer death. Despite various therapeutic approaches, the cures and prognosis of advanced GC remain poor. Natural killer (NK) cells, which are known as important lymphocytes in innate immunity, play vital roles in suppressing GC initiation, progression, and metastases. A wide range of clinical settings shows that increasing the number of NK cells or improving NK cell antitumor activity is promising in GC patients. NK cell adoptive therapy (especially expanded NK cells) is a safe and well-tolerated method, which can enhance NK cell cytotoxicity against GC. Meanwhile, cytokines, immunomodulatory drugs, immune checkpoint blockades, antibodies, vaccines, and gene therapy have been found to directly or indirectly activate NK cells to improve their killing activity toward GC. In this review, we summarize recent advancements in the relationship between NK cells and GC and point out all the innovative strategies that can enhance NK cells' function to inhibit the growth of GC.

Assessing Canonical and Adaptive Natural Killer Cell Function in Suppression Assays In Vitro.

The in vitro culture system outlined in this chapter allows for standardized protocols to examine canonical and adaptive natural killer (NK) cell responses while interacting with immune suppressor cells such as regulatory T cells (Treg) and myeloid-derived suppressor cells (MDSC). These interactions pathologically occur during tumorigenesis and tumor progression. Strategies to study the effects of ex vivo purified immune suppressor cells from cancer patients on the function of NK cell antitumor activity will help to understand suppressive mechanisms to improve immunotherapy. Immune checkpoint inhibitors have recently demonstrated tremendous clinical responses in patients with diverse types of cancers. However, their effect on NK cell function is not very well studied. Here, we have adapted a coculture system that previously has been utilized to study regulatory T cells. This approach can further be utilized to study the effects of immune checkpoint inhibitors in vitro and ex vivo. We focus on the differences between canonical NK cells and the newly identified subset of NK cells termed "adaptive NK cells." These cells are induced by cytomegalovirus (CMV) in CMV-seropositive individuals.

A Flow Cytometric NK Cell-Mediated Cytotoxicity Assay to Evaluate Anticancer Immune Responses In Vitro.

The immune system is able to detect and eliminate nascent and developing tumors. Thus, T lymphocytes of the adaptive immunity recognize cancer cells by detecting tumor-associated antigens, whereas certain innate immune cells scan for molecules that are mainly overexpressed on malignant and infected cells. The best example of the latter is natural killer (NK) cells. The activity of these immune cells is strictly governed by a balance between positive and negative signals provided by stress-regulated molecules that bind NK cell membrane receptors, such as the activatory receptor NKG2D. Given the key role of NK cells in eradicating cancer cells, the development of assays to study the cellular and molecular determinants of NK cell antitumor activity are of great relevance. In this chapter, we describe a flow cytometric in vitro assay to evaluate the cytotoxic activity of NK cells against a given target cell, including tumor cells. Moreover, this method is highly versatile, as it can be complemented with the use of antibodies blocking cell surface proteins (receptors or ligands) or a variety of chemical modulators, which allows the elucidation of molecules and signaling pathways that regulate NK cell anticancer function.

Abstract 4743: Inhibition of the Hif1a-mediated checkpoint refuels NK activation in cancer

Abstract Solid tumors remain a major challenge for immunotherapy of cancer due to their immunosuppressive microenvironment. Hypoxia is a common feature of solid tumors and might affect immune cell activation within the tumor tissue. Upon hypoxia the transcription factor Hypoxia inducible factor 1a (HIF-1α) that affects many cellular processes including cell proliferation, survival and effector function becomes induced. Its impact on innate immune cells such as Natural Killer (NK) cells, however, is still incompletely understood. Natural Killer (NK) cells are considered as highly potent effector cells against tumors. Within the tumor tissue, however, NK effector function is often impaired. Our microarray analyses of tumor-infiltrated NK cells revealed upregulated levels of Hif1a. To determine the role of the HIF-1α in NK cell function, we generated the mouse strain Hif1aΔ/ΔNcr1-iCreTg in which HIF-1α is deficient in the NKp46+ lineage. Hif1a deficient NK cells showed greatly enhanced anti-tumor activity compared to wild-type NK cells, leading to a pronounced reduction of tumor growth. Tumor-infiltrated Hif1a deficient NK cells expressed lower levels of checkpoint molecules and show enhanced responses to tumor cells indicating a less exhausted phenotype. Hif1a deficient NK cells preferentially utilize oxidative phosphorylation (OXPHOS) than glycolysis to fuel their energy resulting in enhanced IFN-γ production in the hypoxic environment. In addition, using HIF-1α inhibitors we were able to greatly enhance human NK cell anti-tumor activity. Accordingly, our data reveal HIF-1α as a negative metabolic checkpoint for NK cells. Moreover, inhibition of HIF-1α holds high promise to refuel NK cell activation in cancer with a high potential for improving current strategies of NK cell-based cancer immunotherapy. Citation Format: Jing Ni, Lea Bühler, Ana Stojanovic, Annette Arnold, Veronika Sexl, Adelheid Cerwenka. Inhibition of the Hif1a-mediated checkpoint refuels NK activation in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4743.

Killing without poking holes

Cancer Immunology Given the success of T cell–centric cancer immunotherapies, there is considerable interest in understanding exactly how tumors sometimes evade this form of treatment. Kearney et al. carried out a series of genome-wide CRISPR screens to identify mechanisms of tumor immune evasion from cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. Interferon-γ signaling and antigen presentation were critical for CTL-mediated killing of cancer cells, and TNF (tumor necrosis factor) signaling was a key effector mechanism for both CTL and NK cell antitumor activity. The same immune evasion mechanisms arose upon screening with perforin-deficient CTLs, suggesting that tumors evade the immune system by dampening the effects of cytokines, rather than reducing direct killing via perforin. Sci. Immunol. 3 , eaar3451 (2018).

CD16A Activation of NK Cells Promotes NK Cell Proliferation and Memory-Like Cytotoxicity against Cancer Cells.

CD16A is a potent cytotoxicity receptor on human natural killer (NK) cells, which can be exploited by therapeutic bispecific antibodies. So far, the effects of CD16A-mediated activation on NK cell effector functions beyond classical antibody-dependent cytotoxicity have remained poorly elucidated. Here, we investigated NK cell responses after exposure to therapeutic antibodies such as the tetravalent bispecific antibody AFM13 (CD30/CD16A), designed for the treatment of Hodgkin lymphoma and other CD30+ lymphomas. Our results reveal that CD16A engagement enhanced subsequent IL2- and IL15-driven NK cell proliferation and expansion. This effect involved the upregulation of CD25 (IL2Rα) and CD132 (γc) on NK cells, resulting in increased sensitivity to low-dose IL2 or to IL15. CD16A engagement initially induced NK cell cytotoxicity. The lower NK cell reactivity observed 1 day after CD16A engagement could be recovered by reculture in IL2 or IL15. After reculture in IL2 or IL15, these CD16A-experienced NK cells exerted more vigorous IFNγ production upon restimulation with tumor cells or cytokines. Importantly, after reculture, CD16A-experienced NK cells also exerted increased cytotoxicity toward different tumor targets, mainly through the activating NK cell receptor NKG2D. Our findings uncover a role for CD16A engagement in priming NK cell responses to restimulation by cytokines and tumor cells, indicative of a memory-like functionality. Our study suggests that combination of AFM13 with IL2 or IL15 may boost NK cell antitumor activity in patients by expanding tumor-reactive NK cells and enhancing NK cell reactivity, even upon repeated tumor encounters. Cancer Immunol Res; 6(5); 517-27. ©2018 AACR.

NK cell therapy for hematologic malignancies.

Natural killer (NK) cells are part of the innate immune system and represent the first line of defense against infections and tumors. In contrast to T cells, NK cells do not require prior antigen sensitization to induce cytotoxicity and do not cause graft-versus-host disease. These, along with other advantages, make NK cells an attractive candidate for adoptive cellular therapy. Herein, we describe the mechanisms of NK cell cytotoxicity, which is governed by an intricate balance between various activating and inhibitory receptors, including the killer cell immunoglobulin-like receptors (KIRs). We illustrate the advantages of NK alloreactivity as demonstrated in various types of hematopoietic stem cell transplants (HSCT), such as haploidentical, human leukocyte antigen-matched related or unrelated donor and umbilical cord blood transplant. We elaborate on different models used to predict NK cell alloreactivity in these studies, which are either based on the absence of the ligands for inhibitory KIRs, presence of activating NK cell receptors and KIR genes content in donors, or a combination of these. We will review clinical studies demonstrating anti-tumor efficacy of NK cells used either as a stand-alone immunotherapy or as an adjunct to HSCT and novel genetic engineering strategies to improve the anti-tumor activity of NK cells.

Tumor immunotherapy: New aspects of natural killer cells.

A group of impressive immunotherapies for cancer treatment, including immune checkpoint-blocking antibodies, gene therapy and immune cell adoptive cellular immunotherapy, have been established, providing new weapons to fight cancer. Natural killer (NK) cells are a component of the first line of defense against tumors and virus infections. Studies have shown dysfunctional NK cells in patients with cancer. Thus, restoring NK cell antitumor functionality could be a promising therapeutic strategy. NK cells that are activated and expanded ex vivo can supplement malfunctional NK cells in tumor patients. Therapeutic antibodies, chimeric antigen receptor (CAR), or bispecific proteins can all retarget NK cells precisely to tumor cells. Therapeutic antibody blockade of the immune checkpoints of NK cells has been suggested to overcome the immunosuppressive signals delivered to NK cells. Oncolytic virotherapy provokes antitumor activity of NK cells by triggering antiviral immune responses. Herein, we review the current immunotherapeutic approaches employed to restore NK cell antitumor functionality for the treatment of cancer.

Abstract 3696: Tumor-intrinsic PD-L1 alters tumor chemokines, NK cell trafficking and function, and renders distant PD-L1 null tumors responsive to αPD-L1

Abstract Programmed death ligand (PD-L) 1 is expressed on many tumors and inhibits anti-tumor T cells through programmed death (PD)-1. Tumor PD-L1 predicts αPD-L1 treatment effects, but mechanism(s) for PD-L1- tumor response to αPD-L1 are unclear. Our studies suggest tumor-intrinsic PD-L1 signals and spatially varied PD-L1 expression may contribute to response deviation. We used PD-L1+ B16 melanoma (ctrl) and made PD-L1KO by CRISPR. αPD-L1 slowed ctrl but not PD-L1KO B16 growth in mice as expected, but PD-L1KO also responded to αPD-L1 if ctrl B16 was on the trans flank. αPD-L1 elicited similar CD3+ T cell infiltration into ctrl vs. PD-L1KO tumors, but without detectable B16-specific T cell increase. CD11b+ cell infiltration was similar in ctrl and PD-L1KO. Strikingly, NKp46+ and NK1.1+ natural killer (NK) cells infiltrated PD-L1KO > ctrl. NK cells increased significantly (~2-fold) after αPD-L1 in PD-L1KO vs. ctrl, along with NK effector functions (e.g., IFN-γ). Tumor PD-L1 altered tumor chemokines (e.g., CXCL12, CCL2) that could explain trafficking. CD4+ and CD8+ T cells chemotaxed to PD-L1KO slightly > ctrl (with slightly > PD-1+ T cells) in transwells, but NK cells migrated ~2-fold more to PD-L1KO vs. ctrl (with increased activation (e.g., PD-1, CD69), also seen in vivo). We observed that tumor β2 microglobulin (B2M) expression was suppressed by tumor PD-L1. Tumor PD-L1 alteration of immune infiltrates by altering chemokines is a novel mechanism for PD-L1 TIL control. These data also indicate that tumor PD-L1 blunts NK cell infiltration in B16 and reduces B2M, which could boost NK cell anti-tumor activity. These changes correlate with rescued αPD-L1 response of PD-L1KO B16. As αPD-L1 did not increase B16-specific T cells in PD-L1low, we hypothesize that rescue of αPD-L1 response is in part NK cell-mediated. Preliminary in vivo NK cell depletion + αPD-L1 experiments implicate a role for NK cells in PD-L1KO tumor response, but more work is needed to identify mechanisms. Effects extend beyond B16 as ctrl (PD-L1+) MB49 bladder cancer cells in trans elicited αPD-L1 response of PD-L1KO MB49 tumors. Mouse breast and ovarian cancers, and human ovarian and bladder cells exhibit similar PD-L1-intrinsic effects. Finally, αPD-L1 directly altered tumor cell chemokine production in vitro, suggesting additional, novel αPD-L1 treatment mechanisms requiring more study. Our models are useful to understand αPD-L1 (and likely αPD-1) responses based on tumor PD-L1 expression and will help define strategies to improve responses in PD-L1low tumors, and possibly poorly responsive PD-L1+ tumors. Citation Format: Curtis A. Clark, Harshita B. Gupta, Alvaro Padron, Deyi Zhang, Vincent Hurez, Mary Jo Turk, Rong Li, Tyler Curiel. Tumor-intrinsic PD-L1 alters tumor chemokines, NK cell trafficking and function, and renders distant PD-L1 null tumors responsive to αPD-L1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3696. doi:10.1158/1538-7445.AM2017-3696

A Listeria-derived polypeptide promotes in vivo activation of NK cells for antitumor therapy.

Immunotherapies have shown promise in treatment of cancer, but more potent and targeted therapies are needed. Natural killer (NK) cells are lymphocytes with innate ability to recognize and lyse tumor cells. When activated, they also produce type II interferon (IFNγ) to orchestrate the activity of other immune cells. Strategies to elicit NK cell activation in vivo have potential usefulness in anti-tumor immunotherapies. Here, we report on a strategy to stimulate NK cell activation and anti-tumor activity in mice with established B16.F10 murine melanomas. We and others previously observed that NK cells are rapidly activated during infection by pathogens such as the bacterium Listeria monocytogenes (Lm). A secreted Lm virulence protein, p60, and a fragment of p60 termed L1S were previously shown to stimulate innate immune responses and promote NK cell activation. We purified recombinant L1S and characterized its activity in cell culture studies. Recombinant L1S protein was also observed to promote accumulation and robust NK cell activation in the lungs when given via intratracheal instillation to control and tumor-bearing mice. Importantly, therapeutic administration of a single L1S dose was found to significantly reduce the number and area of "metastatic" tumor nodules on the lungs of mice with established B16.F10 murine melanomas. Depletion studies showed that these antitumor effects were dependent on NK cells and IFNγ. These data provide proof of concept that administration of a single immune-modulating microbial polypeptide can be used to therapeutically boost NK cell in vivo activation and promote anti-tumor responses.

Shaping of Natural Killer Cell Antitumor Activity by Ex Vivo Cultivation

Natural killer (NK) cells are a promising tool for the use in adoptive immunotherapy, since they efficiently recognize and kill tumor cells. In this context, ex vivo cultivation is an attractive option to increase NK cells in numbers and to improve their antitumor potential prior to clinical applications. Consequently, various strategies to generate NK cells for adoptive immunotherapy have been developed. Here, we give an overview of different NK cell cultivation approaches and their impact on shaping the NK cell antitumor activity. So far, the cytokines interleukin (IL)-2, IL-12, IL-15, IL-18, and IL-21 are used to culture and expand NK cells. The selection of the respective cytokine combination is an important factor that directly affects NK cell maturation, proliferation, survival, distribution of NK cell subpopulations, activation, and function in terms of cytokine production and cytotoxic potential. Importantly, cytokines can upregulate the expression of certain activating receptors on NK cells, thereby increasing their responsiveness against tumor cells that express the corresponding ligands. Apart from using cytokines, cocultivation with autologous accessory non-NK cells or addition of growth-inactivated feeder cells are approaches for NK cell cultivation with pronounced effects on NK cell activation and expansion. Furthermore, ex vivo cultivation was reported to prime NK cells for the killing of tumor cells that were previously resistant to NK cell attack. In general, NK cells become frequently dysfunctional in cancer patients, for instance, by downregulation of NK cell activating receptors, disabling them in their antitumor response. In such scenario, ex vivo cultivation can be helpful to arm NK cells with enhanced antitumor properties to overcome immunosuppression. In this review, we summarize the current knowledge on NK cell modulation by different ex vivo cultivation strategies focused on increasing NK cytotoxicity for clinical application in malignant diseases. Moreover, we critically discuss the technical and regulatory aspects and challenges underlying NK cell based therapeutic approaches in the clinics.

Novel chemoimmunotherapeutic strategy for hepatocellular carcinoma based on a genome-wide association study.

Pharmacotherapeutic options are limited for hepatocellular carcinoma (HCC). Recently, we identified the anti-tumor ligand MHC class I polypeptide-related sequence A (MICA) gene as a susceptibility gene for hepatitis C virus-induced HCC in a genome-wide association study (GWAS). To prove the concept of HCC immunotherapy based on the results of a GWAS, in the present study, we searched for drugs that could restore MICA expression. A screen of the FDA-approved drug library identified the anti-cancer agent vorinostat as the strongest hit, suggesting histone deacetylase inhibitors (HDACis) as potent candidates. Indeed, the HDACi-induced expression of MICA specific to HCC cells enhanced natural killer (NK) cell-mediated cytotoxicity in co-culture, which was further reinforced by treatment with an inhibitor of MICA sheddase. Similarly augmented anti-tumor activity of NK cells via NK group 2D was observed in vivo. Metabolomics analysis revealed HDACi-mediated alterations in energy supply and stresses for MICA induction and HCC inhibition, providing a mechanism for the chemoimmunotherapeutic actions. These data are indicative of promising strategies for selective HCC innate immunotherapy.

Engineering Human Pluripotent Stem Cell-Derived Natural Killer Cells to Prevent CD16a Shedding for Enhanced Anti-Tumor Killing

Natural killer (NK) cells are lymphocytes able to recognize and lyse transformed cells without HLA-restriction and without prior sensitization, mediated through a repertoire of cell surface receptors. Solid tumors are typically more refractory to NK cell-mediated killing compared to hematologic malignancies. To improve NK cell-mediated killing of solid tumors, we have engineered NK cells to express a more stable form of CD16a. The IgG Fc receptor CD16a represents one of the most potent activators of NK cell anti-tumor activity. This receptor binds antibodies attached to target cells to initiate antibody-dependent cell-mediated cytotoxicity (ADCC). Importantly, CD16a undergoes a rapid down-regulation in expression by ADAM17 (A Disintegrin And Metalloprotease-17) mediated proteolytic cleavage upon cell activation. This loss of CD16a expression leads to reduced ADCC activity and decreased anti-tumor killing. Previous studies by our group identified cleavage sites in CD16a that are targeted by ADAM17 activity. Substitution of the serine at position 197 for a proline (S197P) in the middle of the cleavage region effectively blocks ADAM17-mediated CD16a cleavage. To evaluate the ability of this cleavage-resistant CD16a(S197P) to improve ADCC, we have used a system previously developed by our group to efficiently produce NK cells from both human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). These hESC/iPSC-derived NK cells have a phenotype and function similar to NK cells isolated from peripheral blood (PB-NK cells). However, unlike PB-NK cells, it is relatively easy to genetically-modify hESC/iPSC-derived NK cells by engineering at the stem cell level. Here, we use two strategies to generate iPSC-derived NK cells with stabilized CD16a expression. First, we used the Sleeping Beautytransposon system to stably express the cleavage-resistant CD16a(S197P) in iPSCs. Second, we have used the CRISPR/Cas9 system to delete the ADAM17 gene. This strategy allows us to compare specifically engineering stabilized CD16a expression to the broader effect of ADAM17-deletion which cleaves multiple cell-surface proteins.NK cells derived from the CD16a(S197P) iPSCs are resistant to CD16a shedding upon activation with IL-12 and IL-18 as 97.5% of iPSC-NK cells are CD16+ before stimulation and 95.2% of cells are CD16+ after stimulation. In contrast, PB-NK cells demonstrate a decrease from 85% CD16+ cells before stimulation to 67% CD16+ cells after stimulation. Next, we demonstrate improved ADCC and NK cell degranulation (measured by CD107a expression) in this engineered iPSC-derived NK cell compared to non-engineered cells upon addition of the anti-Her2 antibody (ab) Herceptin to ovarian cancer cells with differing levels of Her2 expression. Specifically, CD16a(S197P) iPSC-NK cells stimulated with the Her2-high SKOV3 ovarian carcinoma cells were 26.7% CD107a+ cells with addition of anti-Her2 and 10.8% CD107a+ cells without ab. In contrast, unmodified iPSC-NK cells were 9.6% vs. 6.9% CD107a+ with and without anti-Her2; and PB-NK cells were 12.4% vs. 9.4% CD107a+ with and without anti-Her2. However, when co-cultured with the Her2-low MA-148 ovarian carcinoma none of the three NK cell populations demonstrated increased CD107a+ cells with anti-Her2 treatment compared to no ab controls. Similar results were obtained when testing the ADCC of these cells using the Cr-51 release cytotoxicity assay. Killing of SKOV3 cells by CD16a(S197P) iPSC-NK cells was markedly increased upon addition of anti-Her2 ab from 32.2% cytoxicity with ab vs. 11% cytotoxicity without ab. In contrast, unmodified iPSC-NK cells had less killing of the SKOV3 cells (8.1% vs. 3.7% with or without anti-Her2 ab) and PB-NK cell killing of SKOV3 cells was 20.5% vs. 9.2% with or without anti-Her2 ab. Currently, we are comparing anti-tumor activity of the CD16a(S197P) iPSC-NK cells to the ADAM17-deleted iPSC-NK cells, both as resting NK cells and IL12+IL18-stimulated NK cells. Overall, this strategy will allow us to improve the ability of readily available anti-tumor antibodies to improve NK cell-mediated killing of solid tumors and use engineered iPSC-derived NK cells as an "off-the-shelf" approach for allogeneic cell-based immunotherapies. DisclosuresKaufman:Fate Therapeutics: Consultancy.

Cetuximab intensifies the ADCC activity of adoptive NK cells in a nude mouse colorectal cancer xenograft model.

Natural killer (NK) cells, discovered ~40 years ago, are believed to be the most effective cytotoxic lymphocytes to counteract cancer; however, adoptive NK cell therapy in vivo has encountered certain limitations, including a lack of specificity. The drug cetuximab can mediate antibody dependent cell mediated cytotoxicity (ADCC) activity through NK cells in vivo, and has been approved for the first-line treatment of epidermal growth factor receptor (EGFR)-positive metastatic colorectal cancer (CRC). However, the ADCC activity of adoptive NK cells, induced by cetuximab in a nude mouse CRC xenograft model, has not been previously reported. The aim of the present study was to explore the ADCC activity of cetuximab combined with adoptive NK cells in CRC xenograft models with various EGFR expressions. The nude mouse xenograft models were established by subcutaneously injecting LOVO or SW620 cells. The mice were then randomly divided into 6 groups: Phosphate-buffered saline, cetuximab, human immunoglobulin G (hIgG), NK cells, hIgG plus NK cells and cetuximab plus NK cells. The ADCC antitumor activity was evaluated in these CRC models. The results indicated that the cetuximab plus NK cells group showed the greatest tumor inhibition effect compared with the NK cells group in LOVO xenograft tumor models with positive EGFR expression. However, the combination of cetuximab and NK cells did not show a stronger tumor inhibitory effect against the SW620 xenograft tumor models compared with the efficiency of NK cells. In conclusion, cetuximab could intensify the ADCC antitumor activity of adoptive NK cells towards CRC with an increased EGFR expression. The combination of cetuximab and NK cells may be a potential immunotherapy for metastatic CRC patients with positive EGFR expression.

Natural killer cells require monocytic Gr-1+/CD11b+ myeloid cells to eradicate orthotopically engrafted glioma cells

ABSTRACTMalignant gliomas are resistant to natural killer (NK) cell immune surveillance. However, the mechanisms used by these cancers to suppress antitumor NK cell activity remain poorly understood. We have recently reported on a novel mechanism of innate immune evasion characterized by the overexpression of the carbohydrate-binding protein galectin-1 by both mouse and rat malignant glioma. Here, we investigate the cytokine profile of galectin-1-deficient GL26 cells and describe the process by which these tumors are targeted by the early innate immune system in RAG1−/− and C57BL/6J mice. Our data reveal that galectin-1 knockdown in GL26 cells heightens their inflammatory status leading to the rapid recruitment of Gr-1+/CD11b+ myeloid cells and NK1.1+ NK cells into the brain tumor microenvironment, culminating in tumor clearance. We show that immunodepletion of Gr-1+ myeloid cells in RAG1−/− mice permits the growth of galectin-1-deficient glioma despite the presence of NK cells, thus demonstrating an essential role for myeloid cells in the clearance of galectin-1-deficient glioma. Further characterization of tumor-infiltrating Gr-1+/CD11b+ cells reveals that these cells also express CCR2 and Ly-6C, markers consistent with inflammatory monocytes. Our results demonstrate that Gr-1+/CD11b+ myeloid cells, often referred to as myeloid-derived suppressor cells (MDSCs), are required for antitumor NK cell activity against galectin-1-deficient GL26 glioma. We conclude that glioma-derived galectin-1 represents an important factor in dictating the phenotypic behavior of monocytic Gr-1+/CD11b+ myeloid cells. Galectin-1 suppression may be a valuable treatment approach for clinical glioma by promoting their innate immune-mediated recognition and clearance through the concerted effort of innate myeloid and lymphoid cell lineages.

High dose CD11c-driven IL15 is sufficient to drive NK cell maturation and anti-tumor activity in a trans-presentation independent manner.

The common gamma (γc)-chain cytokine interleukin 15 (IL15) is a multifunctional immune-modulator which impacts the generation, maturation and activity of many cell types of the innate, as well as the adaptive immune system, including natural killer (NK) and CD8+ T cells. Using a new series of transgenic mice, we analyzed the in vivo potential of IL15 as an immune-regulator when available at different concentrations or delivery modes, i.e. soluble monomer or complexed to its specific receptor α (Rα)-chain. We have identified distinct effects on selected IL15-responsive populations. While CD8+ T cells required complexed forms of IL15/IL15Rα for full functionality, mature NK populations were rescued in an IL15/IL15Rα-deficient environment by high levels of CD11c-restricted IL15. These IL15-conditions were sufficient to limit tumor formation in a lung metastasis model indicating that the NK cell populations were fully functional. These data underline the potential of “free” IL15 in the absence of Rα-complex as a powerful and specific immuno-modulator, which may be beneficial where selective immune-activation is desired.

Abstract B45: Mitochondrial Ca2+ transport fine-tunes functional cross-talk between antitumor CD8+T lymphocytes and natural killer cells

Abstract Tumor antigen-specific T cell and natural killer (NK) cell collaboration is indispensable for the elimination of tumor cells, including antigen-deficient tumor escape variants before metastasis. While mechanistic details are available for the innate instruction of T lymphocyte responses, little is known for the adaptive control of NK cell activity. We observed in a mouse model of mastocytoma expressing a self tumor antigen P1A that effector CD8+T cells provided a necessary “help” to dormant NK cells in eliciting their antitumor effector function. Bioluminescence imaging of mastocytoma tumors following adoptive transfer of P1A-specific T cells in RAG-/- and RAG-/-gamma chain-/- mice showed that NK cell anti-tumor activity requires cytolytic T cells, whereas T cells can function independent of NK cells. The goal of present study was to dissect the molecular mechanisms underlying functional cross-talk between activated CD8+T lymphocytes and naïve NK cells. In 2D and 3D co-culture systems, we observed that activated CD8+T cells form multiple intercellular contacts with naïve NK lymphocytes. Moreover, co-culture of activated T cells with naïve NK cells results in the selective down-regulation of CD25 molecule in T cells while elevating CD25 and CD69 expression on naïve NK cells. Further, CD8+T and NK cells cross-regulate their mitochondrial homeostasis including calcium transport. This effect is dependent on both cytokines and intercellular contacts, and partially involves natural killer group 2 member D (NKG2D) receptor activation. Data also indicate that activated CD8+T cells may exchange mitochondria and activation molecules such as CD25 and CD69 with naïve NK cells. Alterations in phosporylation status of multiple signaling proteins during CD8+T-NK interaction suggest a cellular remodeling whereby NK cells shift activated CD8+T cells towards T central-memory (TCM) phenotype and activated CD8+T lymphocytes alter the naïve state of NK cells towards effector/regulatory phenotype. Inhibition of mitochondrial Ca2+ uptake (mCU) or Na+/Ca2+ exchanger (mNCE) with Ru360 and CGP37157, respectively, mimicked observed alterations in CD8+ T and NK cell phenotypes upon their interaction. These results highlight a novel role of mitochondrial Ca2+ homeostasis in the re-modeling of memory differentiation and activation signaling of interacting CD8+T cells and NK cells, respectively. Improved mechanistic understanding of CD8+T-NK interplay will help refine cellular immunotherapy approach against cancer. Citation Format: Roman V. Uzhachenko, J Shawn Goodwin, Lino Costa, Alexander Terekhov, Menaka C. Thounaojam, William H. Hofmeister, Anil Shanker. Mitochondrial Ca2+ transport fine-tunes functional cross-talk between antitumor CD8+T lymphocytes and natural killer cells. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B45.

In Vivo Activation of Human NK Cells by Treatment with an Interleukin-15 Superagonist Potently Inhibits Acute In Vivo HIV-1 Infection in Humanized Mice.

Natural killer (NK) cells with anti-HIV-1 activity may inhibit HIV-1 replication and dissemination during acute HIV-1 infection. We hypothesized that the capacity of NK cells to suppress acute in vivo HIV-1 infection would be augmented by activating them via treatment with an interleukin-15 (IL-15) superagonist, IL-15 bound to soluble IL-15Rα, an approach that potentiates human NK cell-mediated killing of tumor cells. In vitro stimulation of human NK cells with a recombinant IL-15 superagonist significantly induced their expression of the cytotoxic effector molecules granzyme B and perforin; their degranulation upon exposure to K562 cells, as indicated by cell surface expression of CD107a; and their capacity to lyse K562 cells and HIV-1-infected T cells. The impact of IL-15 superagonist-induced activation of human NK cells on acute in vivo HIV-1 infection was investigated by using hu-spl-PBMC-NSG mice, NOD-SCID-IL2rγ(-/-) (NSG) mice intrasplenically injected with human peripheral blood mononuclear cells (PBMCs) which develop productive in vivo infection after intrasplenic inoculation with HIV-1. IL-15 superagonist treatment potently inhibited acute HIV-1 infection in hu-spl-PBMC-NSG mice even when delayed until 3 days after intrasplenic HIV-1 inoculation. Removal of NK cells from human PBMCs prior to intrasplenic injection into NSG mice completely abrogated IL-15 superagonist-mediated suppression of in vivo HIV-1 infection. Thus, the in vivo activation of NK cells, integral mediators of the innate immune response, by treatment with an IL-15 superagonist increases their anti-HIV activity and enables them to potently suppress acute in vivo HIV-1 infection. These results indicate that in vivo activation of NK cells may represent a new immunotherapeutic approach to suppress acute HIV-1 infection. Epidemiological studies have indicated that NK cells contribute to the control of HIV-1 infection, and in vitro studies have demonstrated that NK cells can selectively kill HIV-1-infected cells. We demonstrated that in vivo activation of NK cells by treatment with an IL-15 superagonist that potently stimulates the antitumor activity of NK cells markedly inhibited acute HIV-1 infection in humanized mice, even when activation of NK cells by IL-15 superagonist treatment is delayed until 3 days after HIV-1 inoculation. NK cell depletion from PBMCs prior to their intrasplenic injection abrogated the suppression of in vivo HIV-1 infection observed in humanized mice treated with the IL-15 superagonist, demonstrating that activated human NK cells were mediating IL-15 superagonist-induced inhibition of acute HIV-1 infection. Thus, in vivo immunostimulation of NK cells, a promising therapeutic approach for cancer therapy, may represent a new treatment modality for HIV-1-infected individuals, particularly in the earliest stages of infection.

Distinct Mechanisms of Tumor Resistance to NK Killing: Of Mice and Men

Tumors cells can release natural killer (NK) cell ligands for activating receptor NKG2D that are thought to inhibit NK cell function. In a recent issue of Science, Deng et al. (2015) show that, unexpectedly, a soluble NKG2D ligand can enhance anti-tumor NK cell activity.

Cracking the glioma-NK inhibitory code: toward successful innate immunotherapy

Natural killer (NK) cells eradicate galectin-deficient malignant gliomas without the necessity for T cell cooperation. This phenomenon was discovered as a consequence of reducing glioma-derived galectin-1. We propose that stimulation of endogenous antitumor NK cell activity may be achieved by reducing potent tumor-derived NK cell inhibitors, such as galectin-1, and that such agents be tested in the clinic to treatbrain tumors.