Targeting natural killer cells in cancer immunotherapy.

Alteration in the expression of cell-surface proteins is a common consequence of malignant transformation. Natural killer (NK) cells use an array of germline-encoded activating and inhibitory receptors that scan for altered protein-expression patterns, but tumor evasion of detection by the immune system is now recognized as one of the hallmarks of cancer. NK cells display rapid and potent immunity to metastasis or hematological cancers, and major efforts are now being undertaken to fully exploit NK cell anti-tumor properties in the clinic. Diverse approaches encompass the development of large-scale NK cell-expansion protocols for adoptive transfer, the establishment of a microenvironment favorable to NK cell activity, the redirection of NK cell activity against tumor cells and the release of inhibitory signals that limit NK cell function. In this Review we detail recent advances in NK cell-based immunotherapies and discuss the advantages and limitations of these strategies.

Regulatory T cells limit unconventional memory to preserve the capacity to mount protective CD8 memory responses to pathogens.

Immunological memory exists so that following infection an expanded population of pathogen-specific lymphocytes can rapidly and efficiently control infection in the case of reexposure. However, in the case of CD8+ T lymphocytes, a population of unconventional CD44+CD122+ virtual memory T cells (TVM) has been described that possesses many, though not all, features of "true memory" T cells, without the requirement of first encountering cognate antigen. Here, we demonstrate a role for regulatory T cell-mediated restraint of TVM at least in part through limiting IL-15 trans-presentation by CD11b+ dendritic cells. Further, we show that keeping TVM in check ensures development of functional, antigen-specific "true" memory phenotype CD8+ T cells that can assist in pathogen control upon reexposure.

Gene deregulation and chronic activation in natural killer cells deficient in the transcription factor ETS1.

Multiple transcription factors guide the development of mature functional natural killer (NK) cells, yet little is known about their function. We used global gene expression and genome-wide binding analyses combined with developmental and functional studies to unveil three roles for the ETS1 transcription factor in NK cells. ETS1 functions at the earliest stages of NK cell development to promote expression of critical transcriptional regulators including T-BET and ID2, NK cell receptors (NKRs) including NKp46, Ly49H, and Ly49D, and signaling molecules essential for NKR function. As a consequence, Ets1(-/-) NK cells fail to degranulate after stimulation through activating NKRs. Nonetheless, these cells are hyperresponsive to cytokines and have characteristics of chronic stimulation including increased expression of inhibitory NKRs and multiple activation-associated genes. Therefore, ETS1 regulates a broad gene expression program in NK cells that promotes target cell recognition while limiting cytokine-driven activation.

Interleukin-15 in Outcomes of Pregnancy.

Interleukin-15 (IL-15) is a pleiotropic cytokine that classically acts to support the development, maintenance, and function of killer lymphocytes. IL-15 is abundant in the uterus prior to and during pregnancy, but it is subject to tight spatial and temporal regulation. Both mouse models and human studies suggest that homeostasis of IL-15 is essential for healthy pregnancy. Dysregulation of IL-15 is associated with adverse outcomes of pregnancy. Herein, we review producers of IL-15 and responders to IL-15, including non-traditional responders in the maternal uterus and fetal placenta. We also review regulation of IL-15 at the maternal-fetal interface and propose mechanisms of action of IL-15 to facilitate additional study of this critical cytokine in the context of pregnancy.

Tissue maintenance of CMV-specific inflationary memory T cells by IL-15.

Cytomegalovirus (CMV) infection induces an atypical CD8 T cell response, termed inflationary, that is characterised by accumulation and maintenance of high numbers of effector memory like cells in circulation and peripheral tissues-a feature being successfully harnessed for vaccine purposes. Although stability of this population depends on recurrent antigen encounter, the requirements for prolonged survival in peripheral tissues remain unknown. Here, we reveal that murine CMV-specific inflationary CD8 T cells are maintained in an antigen-independent manner and have a half-life of 12 weeks in the lung tissue. This half-life is drastically longer than the one of phenotypically comparable inflationary effector cells. IL-15 alone, and none of other common γ-cytokines, was crucial for survival of inflationary cells in peripheral organs. IL-15, mainly produced by non-hematopoietic cells in lung tissue and being trans-presented, promoted inflationary T cell survival by increasing expression of Bcl-2. These results indicate that inflationary CD8 T cells are not just simply effector-like cells, rather they share properties of both effector and memory CD8 T cells and they appear to be long-lived cells compared to the effector cells from acute virus infections.

Prostate cancer cells enhance interleukin-15-mediated expansion of NK cells.

OBJECTIVES: To identify cytokines that can activate and expand NK cells in the presence of prostate cancer cells in order to determine whether these agents may be useful in future intra-tumoural administration in pre-clinical and clinical prostate cancer trials. MATERIALS AND METHODS: Lymphocytes isolated from normal donor blood were set up in co-cultures with either cancer or non-cancerous prostate cell lines, together with each of the cytokines interleukin (IL)-2, IL-12, IL-15, interferon (IFN)-γ or IL-21 for a period of 7 days. Then, expansion of NK cells, NKT cells and CD8 T cells was measured by flow cytometry and compared with the expansion of the same cells in the absence of prostate cells. The cytotoxic activity of NK cells, as measured by perforin and tumour cell killing, was also assessed. NK cell receptors and their corresponding ligands on prostate tumour cells were analysed to determine whether any of these were modulated by co-culture. The role of the tumour-secreted heat shock proteins HSP90 and HSP70 in the expansion of NK cells in the co-cultures was also investigated because of their effects on NK and CD8 T-cell activation. RESULTS: We showed that, among a panel of cytokines known to cause NK cell activation and expansion, only IL-15 could actively induce expansion of NK, NKT and CD8 T cells in the presence of prostate cancer cell lines. Furthermore, the expansion of NK cells was far greater (up to 50% greater) in the presence of the cancer cells (LNCaP, PC3) than when lymphocytes were incubated alone. In contrast, non-cancerous cell lines (PNT2 and WPMY-1) did not exert any expansion of NK cells. The cytolytic activity of the NK cells, as measured by perforin, CD107a and killing of tumour cells, was also greatest in co-cultures with IL-15. Examination of NK cell receptors shows that NKG2D is upregulated to a greater degree in the presence of prostate cancer cells, compared with the upregulation with IL-15 in lymphocytes alone. However, blocking of NKG2D does not inhibit the enhanced expansion of NK cells in the presence of tumour cells. CONCLUSIONS: Among a panel of NK cell-activating cytokines, IL-15 was the only cytokine that could stimulate expansion of NK cells in the presence of prostate cancer cells; therefore IL-15 may be a good candidate for novel future intra-tumoural therapy of the disease.

IL-15 Enables Septic Shock by Maintaining NK Cell Integrity and Function.

Interleukin 15 is essential for the development and differentiation of NK and memory CD8+ (mCD8+) T cells. Our laboratory previously showed that NK and CD8+ T lymphocytes facilitate the pathobiology of septic shock. However, factors that regulate NK and CD8+ T lymphocyte functions during sepsis are not well characterized. We hypothesized that IL-15 promotes the pathogenesis of sepsis by maintaining NK and mCD8+ T cell integrity. To test our hypothesis, the pathogenesis of sepsis was assessed in IL-15-deficient (IL-15 knockout, KO) mice. IL-15 KO mice showed improved survival, attenuated hypothermia, and less proinflammatory cytokine production during septic shock caused by cecal ligation and puncture or endotoxin-induced shock. Treatment with IL-15 superagonist (IL-15 SA, IL-15/IL-15Rα complex) regenerated NK and mCD8+ T cells and re-established mortality of IL-15 KO mice during septic shock. Preventing NK cell regeneration attenuated the restoration of mortality caused by IL-15 SA. If given immediately prior to septic challenge, IL-15-neutralizing IgG M96 failed to protect against septic shock. However, M96 caused NK cell depletion if given 4 d prior to septic challenge and conferred protection. IL-15 SA treatment amplified endotoxin shock, which was prevented by NK cell or IFN-γ depletion. IL-15 SA treatment also exacerbated septic shock caused by cecal ligation and puncture when given after the onset of sepsis. In conclusion, endogenous IL-15 does not directly augment the pathogenesis of sepsis but enables the development of septic shock by maintaining NK cell numbers and integrity. Exogenous IL-15 exacerbates the severity of sepsis by activating NK cells and facilitating IFN-γ production.

IL-15 Complexes Induce Migration of Resting Memory CD8 T Cells into Mucosal Tissues.

IL-15 is an essential cytokine known to promote T cell survival and activate the effector function of memory phenotype CD8 T cells. Blocking IL-15 signals also significantly impacts tissue-specific effector and memory CD8 T cell formation. In this study, we demonstrate that IL-15 influences the generation of memory CD8 T cells by first promoting their accumulation into mucosal tissues and second by sustaining expression of Bcl-6 and T-bet. We show that the mechanism for this recruitment is largely dependent on mammalian target of rapamycin and its subsequent inactivation of FoxO1. Last, we show that IL-15 complexes delivered locally to mucosal tissues without reinfection is an effective strategy to enhance establishment of tissue resident memory CD8 T cells within mucosal tissues. This study provides mechanistic insight into how IL-15 controls the generation of memory CD8 T cells and influences their trafficking and ability to take up residence within peripheral tissues.

Short Term Hypoxia Synergizes with Interleukin 15 Priming in Driving Glycolytic Gene Transcription and Supports Human Natural Killer Cell Activities.

Natural killer (NK) cells induce apoptosis in infected and transformed cells and are important producers of immunoregulatory cytokines. Therefore, they operate under low oxygen conditions (hypoxia) in inflammatory and tumor environments. In vitro studies of NK cells are, however, commonly performed in ambient air (normoxia). We used global gene expression profiling to evaluate changes in transcriptional pathways in primary human NK cells following short term culture under hypoxia compared with normoxia and in response to interleukin 15 (IL-15) priming using a 2 × 2 factorial design. The largest contrasts observed were priming dependences for associations between hypoxia and the hypoxia-inducible factor (Hif) 1 signaling and glycolysis pathways. RT-PCR confirmed positive synergistic hypoxia/IL-15 interactions for genes of key regulatory and metabolic enzymes. IL-15 primes NK cells for effector functions, which were recently demonstrated to depend on glycolytic switching. We did not, however, observe important increases in glycolytic flux through hypoxia and priming alone. Chemical Hif-1α inhibition suggested equal importance of this transcription factor for glycolysis and energy production under normoxia and hypoxia. Hypoxia promoted secretion of CC chemokines Ccl3/4/5 and macrophage migration inhibitory factor. Unexpectedly, hypoxia also stimulated migration of NK cells through the extracellular matrix and shifted amounts of susceptible leukemia target cells toward late apoptosis in a cell killing assay. We conclude that short term hypoxia supports these activities by positively interacting with NK cell priming at the level of glycolytic gene transcription. Hypoxic conditioning of NK cells may thus benefit their use in cell-based immunotherapy of cancer.

IL-15 and RANKL Play a Synergistically Important Role in Osteoclastogenesis.

Interleukin-15 (IL-15), a cytokine secreted by several cell types, has important physiological roles in the activity, proliferation, and viability of immune cells. It has both chemoattractant and proinflammatory properties, and may promote bone destruction. A previous study has shown that IL-15 alone exerts no effect on osteoclastogenesis. Therefore, the current study addressed the synergistic effect of IL-15 on osteoclast formation using RAW264.7 (RAW) cells by co-stimulation with receptor activator of nuclear factor (NF)-κB ligand (RANKL) that has a major role in osteoclastogenesis involving the pathogenesis of rheumatoid arthritis and periodontal disease. Co-stimulation of RAW cells by IL-15 and RANKL significantly increased the gene expression of osteoclast differentiation and osteoclastogenesis markers compared with stimulation by RANKL or IL-15 independently as evaluated by tartrate-resistant acid phosphate-positive cell numbers, the fusion index, a pit formation assay with Alizarin red staining (calcification estimation), and quantitative polymerase chain reaction. Phosphorylation of extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase, p38 mitogen-activated protein kinase, and NF-κB was significantly increased by RANKL and IL-15 (P < 0.05) compared with RANKL alone. In addition, these differentiation activities induced by RANKL and IL-15 were comparatively suppressed by inhibition of ERK, suggesting that this synergistic effect on osteoclastogenesis is mainly mediated by ERK. Taken together, our results demonstrate that IL-15 and RANKL induce osteoclastogenesis synergistically, and IL-15 might play a novel and major role in destructive inflammatory bone diseases. J. Cell. Biochem. 118: 739-747, 2017. © 2016 Wiley Periodicals, Inc.

IL-15 stimulates NKG2D while promoting IgM expression of B-1a cells.

Interleukin (IL)-15, a key manipulator of T-cell function also modulates B-1a cell activity by augmenting activation markers, turning them towards type 1 polarization and immunoglobulin (Ig) expression which is significant in the context of gut immunity. Here we show, for the first time, IL-15 mediated up-regulation of the activation receptor NKG2D and its adaptor DAP10 in B-1a cells indicating their essential coupling with IL-15 receptor signaling pathway. Our results demonstrate IL-15 treatment increases phosphorylation of STAT5 and p38 leading to translocation of NF-κB onto the nucleus, an attribute that delineates activation of B-1a cells and its role in inflammation. In parallel, increase of anti-apoptotic Bcl-xL suggests its role in long term survival of B-1a cells in culture by IL-15. The cytokine induced overexpression of the plasma cell differentiation transcription factor BLIMP-1 while reducing PAX-5a that could be responsible for the spontaneous Ig secretion by B-1a cells. Up-regulation of IgM transcripts in presence of IL-15 validates mucosal response of the cells through natural Abs to counter pathogens.

Insulin-dependent cytoplasmic distribution of Rab4a in mouse adipocytes is inhibited by interleukin-6, -8, and -15.

The purpose of the study was to examine the effect of interleukins, IL-6, IL-8, and IL-15, on insulin-mediated redistribution of Rab4a, an early endosome marker, in mouse 3T3-L1 adipocytes. The interleukins did not affect cell viability; however, cell number was slightly but significantly higher in cultures exposed to IL-8 and IL-15. IL-8 and IL-15 decreased lipid storage in adipocytes, whereas IL-6 had no effect. Rab4A showed cytoplasmic localization, and in control unstimulated adipocytes it was found primarily nearby nucleus, that was supported by cellular fluorescence distribution profile, and by calculated indices, that is, high percentage of near-nuclear area fluorescence and a low mean peripheral cytoplasmic fluorescence/mean near-nuclear fluorescence ratio. Insulin stimulation (100 nmol/l, 30 min) altered the cytoplasmic localization of Rab4a in control adipocytes, which was manifested by its redistribution towards plasma membrane. This effect of insulin was prevented in adipocytes exposed to IL-6, IL-8, or IL-15. We concluded that insulin-dependent Rab4a redistribution, probably reflecting stimulation of vesicle-mediated transport, is inhibited in adipocytes subjected to differentiation in the presence of IL-6, IL-8, or IL-15. Such alterations may be involved in the mechanisms contributing to development of insulin resistance associated with inflammation; however, further studies in this field are required.

The biology of interleukin-2 and interleukin-15: implications for cancer therapy and vaccine design.

Interleukin-2 and interleukin-15 have pivotal roles in the control of the life and death of lymphocytes. Although their heterotrimeric receptors have two receptor subunits in common, these two cytokines have contrasting roles in adaptive immune responses. The unique role of interleukin-2 is in the elimination of self-reactive T cells to prevent autoimmunity. By contrast, interleukin-15 is dedicated to the prolonged maintenance of memory T-cell responses to invading pathogens. As discussed in this Review, the biology of these cytokines will affect the development of novel therapies for malignancy and autoimmune diseases, as well as the design of vaccines against infectious diseases.

Regulation of Retinoic Acid Receptor Beta by Interleukin-15 in the Lung during Cigarette Smoking and Influenza Virus Infection.

Virus-induced exacerbations often lead to further impairment of lung function in chronic obstructive pulmonary disease. IL-15 is critical in antiviral immune responses. Retinoic acid (RA) signaling plays an important role in tissue maintenance and repair, particularly in the lung. We studied RA signaling and its relation to IL-15 in the lung during cigarette smoke (CS) exposure and influenza virus infection. In vivo studies show that RA signaling is diminished by long-term CS exposure or influenza virus infection alone, which is further attenuated during infection after CS exposure. RA receptor ß (RARß) is specifically decreased in the lung of IL-15 transgenic (overexpression; IL-15Tg) mice, and a greater reduction in RARß is found in these mice compared with wild-type (WT) mice after infection. RARß is increased in IL-15 knockout (IL-15KO) mice compared with WT mice after infection, and the additive effect of CS and virus on RARß down-regulation is diminished in IL-15KO mice. IL-15 receptor α (IL-15Rα) is increased and RARß is significantly decreased in lung interstitial macrophages from IL-15Tg mice compared with WT mice. In vitro studies show that IL-15 down-regulates RARß in macrophages via IL-15Rα signaling during influenza virus infection. These studies suggest that RA signaling is significantly diminished in the lung by CS exposure and influenza virus infection. IL-15 specifically down-regulates RARß expression, and RARß may play a protective role in lung injury caused by CS exposure and viral infections.

IgG1 anti-epidermal growth factor receptor antibodies induce CD8-dependent antitumor activity.

Anti-EGFR monoclonal antibodies (mAb) like Cetuximab are commonly used for treatment of EGFR+ solid tumors mainly by exerting their therapeutic effect through inhibition of signal transduction. Additionally, IgG1 is a potent mediator of antibody-dependent cytotoxicity (ADCC). In case of the IgG1, Cetuximab induction of ADCC in vivo is controversially discussed. In our study, we investigated the efficiency of Cetuximab-mediated ADCC in a humanized mouse tumor model in vivo and analyzed the contribution of immunologic processes toward antitumor activity. Therefore, we used immunodeficient NOD/Scid mice transgenic for human MHC class I molecule HLA-A2 and adoptively transferred human HLA-A2+ PBMC after engraftment of human epidermoid cell carcinoma A431. Here, we show that high doses of anti-EGFR mAb induced strong tumor regression independent of the immune system. However, tumor regression by low doses of anti-EGFR mAb treatment was ADCC dependent and mediated by tumor infiltrating CD8+ T effector cells. This novel mechanism of ADCC conducted by CD8+ T effector cells was restricted to IgG1 anti-EGFR mAb, dependent of binding to CD16 on T cells and could be inhibited after EGFR blockade on tumor cells. Furthermore, CD8+ T effector cell-mediated ADCC was enhanced in the presence of IL-15 and strongly improved after glycosylation of anti-EGFR mAb indicating the potential of glycoengineered therapeutic mAb as efficient biologicals in cancer therapy.

Interleukin-15 enhances the expansion and function of natural killer T cells from adult peripheral and umbilical cord blood.

Invariant natural killer T cells (iNKT cells) are innate-like non-conventional T cells restricted by the CD1d molecule that are unique in their ability to play a pivotal role in immune regulation. Deficient iNKT function has been reported in patients receiving umbilical cord blood (UCB) transplantation. We sought to determine the effect of interleukin (IL)-15 on α-galactosylceramide (α-GalCer)-expanded iNKT cell function from UCB and adult peripheral blood (APB) mononuclear cells (MNCs). Fresh APB and UCB MNCs were cultured with IL-15 (50 ng/ml) in the presence or absence of α-GalCer (100 ng/ml) for 10 days. Cells were harvested for examination of cell yield, apoptosis, cytokine production and cytotoxic function of Vα24(+)/Vß11(+) iNKT cells. We observed that α-GalCer-expanded APB and UCB iNKT cells and such expansion was further enhanced with IL-15. The percentage of CD3(+)CD56(+) NKT-like cells in both APB and UCB MNCs was increased with IL-15 but not with α-GalCer. Apoptosis of UCB iNKT cells was ameliorated by IL-15. Although APB and UCB iNKT cells secreted lower IFN-γ, it could be enhanced with IL-15. The expression of perforin in APB iNKT cells can also be enhanced with IL-15. UCB Vα24(+)Vß11(+) iNKT cells further augmented K562 cytotoxicity mediated by IL-15. Taken together, these results demonstrated the relative functional deficiencies of α-GalCer induced UCB iNKT cells, which can be ameliorated by IL-15. Our findings suggest a therapeutic benefit of IL-15 immunotherapy during the post-UCB transplant period when iNKT function remains poor.

IL-7- and IL-15-mediated TCR sensitization enables T cell responses to self-antigens.

Regulation of the ERK pathway is intimately involved in determining whether TCR stimulation is productive or induces anergy. T cells from patients with rheumatoid arthritis (RA) have increased ERK responsiveness, which may be relevant for disease pathogenesis. Inflammatory cytokines such as TNF-α did not reproduce the TCR hypersensitivity typical for RA in T cells from healthy individuals. In contrast, priming with the homeostatic cytokines (HCs) IL-7 and IL-15 amplified ERK phosphorylation to TCR stimulation 2- to 3-fold. The underlying mechanism involved a priming of the SOS-dependent amplification loop of RAS activation. The sensitization of the TCR signaling pathway has downstream consequences, such as increased proliferation and preferential Th1 differentiation. Importantly, priming with IL-7 or IL-15 enabled T cell responses to autoantigens associated with RA. Production of HCs is induced in lymphopenic conditions, which have been shown to predispose for autoimmunity and which appear to be present in the preclinical stages of RA. We propose that HCs, possibly induced by lymphopenia, decrease the signaling threshold for TCR activation and are thereby partly responsible for autoimmunity in RA.

What does it take to make a natural killer?

We know how B and T cells develop, what they 'see' and the receptors they 'see with'. By contrast, and despite an unprecedented increase in the number of receptors and ligands known to regulate the activity of natural killer (NK) cells, we still have many questions regarding how these cells develop. Nevertheless, we are beginning to understand the transcriptional programmes of NK-cell maturation and the role of the effector functions of NK cells in the regulation of immune responses. An improved knowledge of NK-cell development in mice and humans might be useful to harness the power of these natural killers in the clinic to fight autoimmune diseases, infection and cancer.

Cytokine control of memory T-cell development and survival.

Evidence has accumulated that cytokines have a fundamental role in the differentiation of memory T cells. Here, we follow the CD8+ T cell from initial activation to memory-cell generation, indicating the checkpoints at which cytokines determine the fate of the T cell. Members of the common cytokine-receptor gamma-chain (gammac)-cytokine family--in particular, interleukin-7 (IL-7) and IL-15--act at each stage of the immune response to promote proliferation and survival. In this manner, a stable and protective, long-lived memory CD8+ T-cell pool can be propagated and maintained.

Dysregulation of interferon regulatory factors impairs the expression of immunostimulatory molecules in hepatitis C virus genotype 1-infected hepatocytes.

BACKGROUND: IL-7 and IL-15 are produced by hepatocytes and are critical for the expansion and function of CD8 T cells. IL-15 needs to be presented by IL-15Rα for efficient stimulation of CD8 T cells. METHODS: We analysed the hepatic levels of IL-7, IL-15, IL-15Rα and interferon regulatory factors (IRF) in patients with chronic hepatitis C (CHC) (78% genotype 1) and the role of IRF1 and IRF2 on IL-7 and IL-15Rα expression in Huh7 cells with or without hepatitis C virus (HCV) replicon. RESULTS: Hepatic expression of both IL-7 and IL-15Rα, but not of IL-15, was reduced in CHC. These patients exhibited decreased hepatic IRF2 messenger RNA levels and diminished IRF2 staining in hepatocyte nuclei. We found that IRF2 controls basal expression of both IL-7 and IL-15Rα in Huh7 cells. IRF2, but not IRF1, is downregulated in cells with HCV genotype 1b replicon and this was accompanied by decreased expression of IL-7 and IL-15Rα, a defect reversed by overexpressing IRF2. Treating Huh7 cells with IFNα plus oncostatin M increased IL-7 and IL-15Rα mRNA more intensely than either cytokine alone. This effect was mediated by strong upregulation of IRF1 triggered by the combined treatment. Induction of IRF1, IL-7 and IL-15Rα by IFNα plus oncostatin M was dampened in replicon cells but the combination was more effective than either cytokine alone. CONCLUSIONS: HCV genotype 1 infection downregulates IRF2 in hepatocytes attenuating hepatocellular expression of IL-7 and IL-15Rα. Our data reveal a new mechanism by which HCV abrogates specific T-cell responses and point to a novel therapeutic approach to stimulate anti-HCV immunity.