Sophie M. Poznanski

M2-polarized and tumor-associated macrophages alter NK cell phenotype and function in a contact-dependent manner

The crosstalk between NK cells and M1 macrophages has a vital role in the protection against infections and tumor development. However, macrophages in the tumor resemble an M2 phenotype, and, at present, their effect on NK cells is less clear. This study investigated whether tumor‐associated macrophages (TAMs) have a role in altering NK cell function and phenotype using in vitro cocultures of murine NK cells with peritoneal or bone marrow‐derived, M2‐polarized macrophages or TAMs isolated from spontaneous mouse breast tumors. We report here that both peritoneal and bone marrow‐derived M2 macrophages, as well as TAMs, substantially inhibit NK cell activation and concordant cytotoxicity against tumor cells. The mechanism for this inhibition was found to require contact between the respective cell types. Both M2 macrophages and TAMs are producers of the immunosuppressive cytokine TGF‐β. The inhibition of TGF‐β restored the cytotoxicity of NK cells in contact with M2 macrophages, implicating TGF‐β in the mechanism for NK cell inhibition. In addition to affecting NK cell function, TAMs also induced a CD27lowCD11bhigh‐exhausted NK cell phenotype, which corresponds with the reduced activation and cytotoxicity observed. This study reveals a novel implication of TAMs in the tumor‐associated inhibition of NK cell function by demonstrating their capacity to directly alter NK cell cytotoxicity and phenotype in a contact‐dependent mechanism involving TGF‐β. These findings identify the interaction between NK cells and TAMs as a prospective therapeutic target to enhance NK cell effector function for effective NK cell cancer therapies.

IL-18/IL-15/IL-12 synergy induces elevated and prolonged IFN-γ production by ex vivo expanded NK cells which is not due to enhanced STAT4 activation

HIGHLIGHTSExpanded NK cells respond to IL18/IL12 with robust IFN&ggr; and TNF&agr; production.NK cells maintain elevated IFN&ggr; production but not TNF&agr; after removal of IL18/IL12.IL18/IL12 induces unique IFN&ggr;+ population distinct from unstimulated IFN&ggr;+ NK cells.IL18/IL12 induction and maintenance of IFN&ggr; is not due to enhanced STAT4 activity.Elevated and prolonged IFN&ggr; production is mediated by up‐regulation of IFN&ggr; mRNA. ABSTRACT The synergistic effect of IL‐18/IL‐15/IL‐12 stimulation potently activates NK cells, inducing high levels of IFN‐&ggr; production. As a result of this potent stimulatory effect, NK cell pre‐activation with IL‐18/IL‐15/IL‐12 is being developed as a cancer immunotherapy. Ex vivo expansion of NK cells enables the efficient generation of large numbers of NK cells for wide‐scale and repeated therapeutic use, and is thus an important source of NK cells for clinical application. However, the effects of IL‐18/IL‐15/IL‐12 stimulation on ex vivo expanded NK cells have not yet been assessed. Thus, the present study assessed the effects of IL‐18/IL‐15/IL‐12 stimulation on NK cells expanded ex vivo using K562‐based artificial antigen presenting cells expressing membrane‐bound IL‐21. We report that ex vivo expanded NK cells stimulated with IL‐18/IL‐15/IL‐12 produce high levels of IFN‐&ggr; and TNF&agr;, have potent cytotoxicity, and maintain prolonged IFN‐&ggr; production following removal of stimulation. IL‐18/IL‐15/IL‐12 stimulation induces a phenotypically unique IFN‐&ggr;‐producing population with reduced CD16 expression and greater CD25 expression as compared to stimulated IFN‐&ggr;‐ NK cells and unstimulated NK cells. We elucidate that the mechanism of synergy for induction and maintenance of IFN‐&ggr; production is not due to a further enhancement of STAT4 activation compared to stimulation with IL‐12 alone. Furthermore, we demonstrate that the synergistic increase in IFN‐&ggr; is not solely under translational regulation, as elevated levels of IFN‐&ggr; mRNA contribute to the synergistic increase in IFN‐&ggr;. Overall, this study characterizes the response of ex vivo expanded NK cells to IL‐18/IL‐15/IL‐12 stimulation and supports the use of ex vivo expanded NK cells as a feasible and efficient source of IL‐18/IL‐15/IL‐12 pre‐activated NK cells for adoptive transfer in cancer immunotherapies.

Ex vivo-expanded NK cells from blood and ascites of ovarian cancer patients are cytotoxic against autologous primary ovarian cancer cells

Ovarian cancer (OC) is the leading cause of gynecological cancer-related death in North America. Most ovarian cancer patients (OCPs) experience disease recurrence after first-line surgery and chemotherapy; thus, there is a need for novel second-line treatments to improve the prognosis of OC. Although peripheral blood-derived NK cells are known for their ability to spontaneously lyse tumour cells without prior sensitization, ascites-derived NK cells (ascites-NK cells) isolated from OCPs exhibit inhibitory phenotypes, impaired cytotoxicity and may play a pro-tumourigenic role in cancer progression. Therefore, it is of interest to improve the cytotoxic effector function of impaired OCP ascites-NK cells at the tumour environment. We investigated the efficacy of using an artificial APC-based ex vivo expansion technique to generate cytotoxic, expanded NK cells from previously impaired OCP ascites-NK cells, for use in an autologous model of NK cell immunotherapy. We are the first to obtain a log-scale expansion of OCP ascites-NK cells that upregulate the surface expression of activating receptors NKG2D, NKp30, NKp44, produce robust amounts of anti-tumour cytokines in the presence of OC cells and mediate direct tumour cytotoxicity against ascites-derived, primary OC cells obtained from autologous patients. Our findings demonstrate that it is possible to generate cytotoxic OCP ascites-NK cells from previously impaired OCP ascites-NK cells, which presents a promising immunotherapeutic target for the second-line treatment of OC. Future work should focus on evaluating the in vivo efficacy of autologous NK cell immunotherapy through the intraperitoneal delivery of NK cell expansion factors to a preclinical xenograft mouse model of human OC.

Type I interferon signalling is not required for the induction of endotoxin tolerance

HighlightsType I interferon signalling is not involved in the induction of LPS tolerance.Peritoneal macrophages from BL6 and IFNAR1−/− mice are equally tolerized towards LPS.IFNAR1−/− mice have increased survival post LPS challenge than BL6 mice. Abstract Endotoxin, or LPS tolerance, is an immunomodulatory mechanism that results in a significantly diminished response to secondary LPS exposure, which may serve to protect the host against endotoxic shock. Type I interferons (IFNs) are cytokines released upon LPS binding to TLR4 and have been shown to have immunomodulatory properties. Due to this regulatory function of type I IFN, we aimed to investigate the role of type I IFN signalling in LPS tolerance. Our data suggests that type I IFN does not play a role in LPS tolerance in vitro, as both wild type and IFNAR1−/− peritoneal macrophages showed reduced cytokine production after secondary LPS exposure. Furthermore, both wild type and IFNAR1−/− mice were protected from a lethal dose of LPS after receiving three small doses to induce tolerance. However, IFNAR−/− mice seemed to recover faster than wild type mice, suggesting type I IFN signalling plays a detrimental role in LPS‐induced sepsis. Although type I IFN may have a regulatory function in microbial infections, it does not seem to play a role in endotoxin tolerance. Type I IFN involvement in bacterial infection remains complex and further studies are needed to define the regulatory function of type I IFN signalling.

Immunometabolism of T cells and NK cells: metabolic control of effector and regulatory function

Metabolic flux can dictate cell fate, including immune cell effector and regulatory function. The metabolic regulation of cell function is well characterized with respect to effector, memory, and regulatory T cells. This knowledge may allow for manipulation of T cell metabolic pathways that set the stage for more effective T cell therapy. Natural Killer (NK) and T-lymphocytes have complementary roles in the defense against pathogens. However, studies of NK cell metabolism are only beginning to emerge and there is comparatively little knowledge on the metabolic regulation of NK-cell activation and effector function. Given their common lymphoid lineage, effector functions and cellular memory potential our current knowledge on T cell metabolism could inform investigation of metabolic reprogramming in NK cells. In this review, we compare the current knowledge of metabolic regulation in T cell and NK cell development, activation, effector and memory function. Commonalties in glucose transport, hypoxia-inducible factors and mTOR highlight metabolic control points in both cells types. Contrasting the glycolytic and oxidative nodes of metabolic regulation in T cells versus NK cells may provide insight into the contribution of specific immune responses to disease and promote the development of immunotherapeutic approaches targeting both innate and adaptive immune responses.

Ex Vivo Expanded Human NK Cells Survive and Proliferate in Humanized Mice with Autologous Human Immune Cells

Adoptive immune cell therapy is emerging as a promising immunotherapy for cancer. Particularly, the adoptive transfer of NK cells has garnered attention due to their natural cytotoxicity against tumor cells and safety upon adoptive transfer to patients. Although strategies exist to efficiently generate large quantities of expanded NK cells ex vivo, it remains unknown whether these expanded NK cells can persist and/or proliferate in vivo in the absence of exogenous human cytokines. Here, we have examined the adoptive transfer of ex vivo expanded human cord blood-derived NK cells into humanized mice reconstituted with autologous human cord blood immune cells. We report that ex vivo expanded NK cells are able to survive and possibly proliferate in vivo in humanized mice without exogenous cytokine administration, but not in control mice that lack human immune cells. These findings demonstrate that the presence of autologous human immune cells supports the in vivo survival of ex vivo expanded human NK cells. These results support the application of ex vivo expanded NK cells in cancer immunotherapy and provide a translational humanized mouse model to test the lifespan, safety, and functionality of adoptively transferred cells in the presence of autologous human immune cells prior to clinical use.

Combined Stimulation with Interleukin-18 and Interleukin-12 Potently Induces Interleukin-8 Production by Natural Killer Cells

The combination of interleukin (IL)-18 and IL-12 (IL-18+IL-12) potently stimulates natural killer (NK) cells, triggering an innate immune response to infections and cancers. Strategies exploiting the effects of IL-18+IL-12 have shown promise for cancer immunotherapy. However, studies have primarily characterized the NK cell response to IL-18+IL-12 in terms of interferon (IFN)-γ production, with little focus on other cytokines produced. IL-8 plays a critical role in activating and recruiting immune cells, but it also has tumor-promoting functions. IL-8 is classically produced by regulatory NK cells; however, cytotoxic NK cells do not typically produce IL-8. In this study, we uncover that stimulation with IL-18+IL-12 induces high levels of IL-8 production by ex vivo expanded and freshly isolated NK cells and NK cells in peripheral blood mononuclear cells. We further report that tumor necrosis factor (TNF)-α, produced by NK cells following IL-18+IL-12 stimulation, regulates IL-8 production. The IL-8 produced is in turn required for maximal IFN-γ and TNF-α production. These findings may have important implications for the immune response to infections and cancer immunotherapies. This study broadens our understanding of NK cell function and IL-18+IL-12 synergy by uncovering an unprecedented ability of IL-18+IL-12-activated peripheral blood NK cells to produce elevated levels of IL-8 and identifying the requirement for intermediates induced by IL-18+IL-12 for maximal cytokine production following stimulation.

Shining light on the significance of NK cell CD56 brightness

CD56 is a fundamental marker in the determination of human natural killer (NK) cell subsets. The degree of CD56 expression is ubiquitously used to define human NK cell maturation, functional, and tissue-specific subsets, yet a unifying implication for the degree of CD56 expression in NK cells remains elusive. Peripheral blood (PB)-NK cells are dichotomized into CD56, which highly express CD56, and CD56, which have lower CD56 expression. Classically, CD56 NK cells are considered to be a less mature, immunoregulatory subset with a greater propensity for cytokine production, whereas CD56 are more mature with greater cytotoxic abilities. While PB-NK cells are primarily CD56 with only a minor CD56 population, NK cells in secondary lymphoid and other tissues are predominantly CD56. It has been shown that the interaction of CD56 with human fibroblast cells can promote the differentiation of CD56 to CD56 NK cells. However, considering the importance and the extent of use of this marker in NK cell biology, relatively little is known about how downregulation of CD56 contributes to NK cell maturation, what role the degree of CD56 expression plays in contexts separate from NK cell maturation, and whether CD56 has an active functional role in mature NK cells. Moreover, while CD56 NK cells were classically considered immunoregulatory, evidence is emerging to suggest that CD56 NK cells also comprise the most highly cytotoxic of NK cell subsets, producing contradictory implications for CD56 brightness. Given this, we ask: is there a unifying implication for the degree of CD56 brightness across NK cells? Herein, we explore the wide-ranging functions of CD56 NK cells in order to determine a unified meaning for the degree of CD56 brightness and shed new light on the implications of CD56 brightness in NK cell biology. In different settings, CD56 NK cells can have vastly divergent functions. In their most classic definition, they are immunoregulatory and play important regulatory roles in numerous contexts. A seminal study by Hanna et al. revealed that decidual NK cells (dNK), which are superbright for CD56 expression, play a critical role in tissue remodeling during pregnancy. They demonstrated that noncytotoxic CD56 dNK cells promote trophoblast invasion through secretion of IL-8 and IP-10, and support angiogenesis through secretion of VEGF and PLGF. In contrast to the classic anti-tumor and anti-viral functions induced by NK cell activation, receptor-mediated activation of dNK cells enhanced the secretion of these tissuemodeling factors, revealing a distinct activation-induced functional profile. Due to these tissue-remodeling capabilities, dNK cells in fact improved tumor growth and vascularization of trophoblast choriocarcinomas. Indeed, regulatory mechanisms have important implications that also extend to NK cells in the context of cancer. In contrast to anti-tumor splenic NK cells, our group has demonstrated that tumor-associated murine NK cells have a less mature phenotype and exhibit reduced expression of activation receptors, perforin, and granzyme B. Similarly, in humans, tumor-associated NK cells are enriched in the less mature CD56 subset and in many ways resemble dNK cells. The presence of CD56 cells within a tumor correlates with poor prognosis and reduced time to disease recurrence following therapy. In addition, Crome et al. demonstrated that CD56 TILs have impaired anti-tumor cytotoxicity and negatively regulate T cell expansion, TNFα, and IFN-γ production. The regulation of T cells by CD56 NK cells has also been reported to play an important role in the attenuation of autoimmune disease. For example, Bielekova et al. demonstrated that daclizumab therapy, a mAb against the IL-2Rα chain, exerted its therapeutic effects in patients with multiple sclerosis by inducing the expansion of CD56 NK cells. The expansion of CD56 cells is correlated with reduced brain inflammation and reduced T cell populations. While regulatory CD56 NK cells are considered to have poor cytotoxicity against tumor targets, these CD56 NK cells inhibited the survival of activated autologous T cells through direct cytotoxicity. Thus, in these studies, CD56 cells have a strong propensity for regulatory function in response to activation signals. In contrast to their classic regulatory roles, recent evidence has demonstrated that in certain contexts, CD56 NK cells have the capacity for anti-viral and anti-tumor cytotoxicity, which plays an important role in driving responses to therapies. For instance, in patients with chronic hepatitis B infection, Pegylated InterferonAlpha (PegIFNα) therapy induced preferential expansion of CD56 NK cells compared to CD56 cells. PegIFNα treatment enhanced the expression of activation receptors, degranulation, and IFN-γ production in CD56 NK cells. Importantly, this increase in degranulation and IFN-γ was only observed in patients who responded to therapy and was not observed in nonresponders. While CD56 NK cells are typically considered to have poor cytotoxicity against tumor cells, a recent study demonstrated that IL-15-priming enhanced degranulation and cytotoxicity of CD56 PB-NK cells against tumor cells to levels that surpassed even those of IL-15-primed CD56 NK cells. In fact, these CD56 NK cells had a more comprehensive antitumor functional profile as they combined a superior ability to secrete high levels of IFN-γ with enhanced cytotoxic function. Similarly, in the clinical setting, IL-15 infusion in cancer patients was shown to predominantly impact CD56 NK cells. IL-15 infusion induced greater expansion of CD56 NK cells and these expanded CD56 cells upregulated anti-tumor cytokine secretion and displayed strong anti-tumor cytotoxicity. Ex vivo expansion of NK cells has also been shown to induce the growth