The tumor microenvironment drives NK cell metabolic dysfunction leading to impaired antitumor activity.

NK cells represent key players capable of driving antitumor immune responses. However, the potent immunosuppressive activity of the tumor microenvironment (TME) may impair their effector function. Here, we strengthen the importance of metabolic interactions between NK cells and TME and propose metabolic dysfunction as one of the major mechanisms behind NK failure in cancer treatment. In particular, we described that TME has a direct negative impact on NK cell function by disrupting their mitochondrial integrity and function in pediatric and adult patients with primary and metastatic cancer. Our results will help to design new strategies aimed at increasing the NK cell antitumor efficacy by their metabolic reprogramming. In this regard, we reveal an unprecedented role of IL15 in the metabolic reprogramming of NK cells enhancing their antitumor functions. IL15 prevents the inhibitory effect of soluble factors present in TME and restores both the metabolic characteristics and the effector function of NK cells inhibited by exposure to malignant pleural fluid. Thus, we propose here that IL15 may be exploited as a new strategy to metabolically reprogram NK cells with the aim of increasing the efficacy of NK-based immunotherapy in a wide range of currently refractory adult and pediatric solid tumors.

ILC3s: Rhythmic Keepers of Gut Integrity at Mealtime.

Cyclically, during the day, increased permeability of the intestinal epithelial barrier, allowing nutrient uptake, must be compensated for, to achieve increased protection against potentially harmful components. Seillet et al. demonstrate that, upon food intake, enteric neuron-derived VIP promotes anticipatory mucosal immunity by inducing ILC3s to produce protective IL-22.

Human RORγt(+)CD34(+) cells are lineage-specified progenitors of group 3 RORγt(+) innate lymphoid cells.

Group 3 innate lymphoid cells (ILC3s) are defined by the expression of the transcription factor RORγt, which is selectively required for their development. The lineage-specified progenitors of ILC3s and their site of development after birth remain undefined. Here we identified a population of human CD34(+) hematopoietic progenitor cells (HPCs) that express RORγt and share a distinct transcriptional signature with ILC3s. RORγt(+)CD34(+) HPCs were located in tonsils and intestinal lamina propria (LP) and selectively differentiated toward ILC3s. In contrast, RORγt(-)CD34(+) HPCs could differentiate to become either ILC3s or natural killer (NK) cells, with differentiation toward ILC3 lineage determined by stem cell factor (SCF) and aryl hydrocarbon receptor (AhR) signaling. Thus, we demonstrate that in humans RORγt(+)CD34(+) cells are lineage-specified progenitors of IL-22(+) ILC3s and propose that tonsils and intestinal LP, which are enriched both in committed precursors and mature ILC3s, might represent preferential sites of ILC3 lineage differentiation.

Human NK cells, their receptors and function.

NK cells are cytotoxic components of innate lymphoid cells (ILC) that provide a first line of defense against viral infections and contribute to control tumor growth and metastasis. Their function is finely regulated by an array of HLA-specific and non-HLA-specific inhibitory and activating receptors which allow to discriminate between healthy and altered cells. Human NK cells gained a major attention in recent years because of the important progresses in understanding their biology and of some promising data in tumor therapy. In this review, we will outline well-established issues of human NK cells and discuss some of the open questions, debates, and recent advances regarding their origin, differentiation, and tissue distribution. Newly defined NK cell specializations, including the impact of inhibitory checkpoints on their function, their crosstalk with other cell types, and the remarkable adaptive features acquired in response to certain virus infections will also be discussed.

Immune Checkpoint Inhibitors: Anti-NKG2A Antibodies on Board.

Antibodies directed towards checkpoint inhibitors have unveiled extraordinary potential in cancer therapy. An article by the Vivier group (Cell 2018;175:1731-1743) shows that blocking the HLA-E-specific NKG2A inhibitory receptor, expressed by NK and inducible in T cells, results in benefits against poor prognosis tumors. Moreover, NKG2A and PD-1/PD-L1 mAb combinations unleash tumor-specific T cell proliferation and memory.

Glucocorticoids and the cytokines IL-12, IL-15, and IL-18 present in the tumor microenvironment induce PD-1 expression on human natural killer cells.

BACKGROUND: Programmed cell death protein 1 (PD-1)-immune checkpoint blockade has provided significant clinical efficacy across various types of cancer by unleashing both T and natural killer (NK) cell-mediated antitumor responses. However, resistance to immunotherapy occurs for many patients, rendering the identification of the mechanisms that control PD-1 expression extremely important to increase the response to the therapy. OBJECTIVE: We sought to identify the stimuli and the molecular mechanisms that induce the de novo PD-1 expression on human NK cells in the tumor setting. METHODS: NK cells freshly isolated from peripheral blood of healthy donors were stimulated with different combinations of molecules, and PD-1 expression was studied at the mRNA and protein levels. Moreover, ex vivo analysis of tumor microenvironment and NK cell phenotype was performed. RESULTS: Glucocorticoids are indispensable for PD-1 induction on human NK cells, in cooperation with a combination of cytokines that are abundant at the tumor site. Mechanistically, glucocorticoids together with IL-12, IL-15, and IL-18 not only upregulate PDCD1 transcription, but also activate a previously unrecognized transcriptional program leading to enhanced mRNA translation and resulting in an increased PD-1 amount in NK cells. CONCLUSIONS: These results provide evidence of a novel immune suppressive mechanism of glucocorticoids involving the transcriptional and translational control of an important immune checkpoint.

Presence of innate lymphoid cells in pleural effusions of primary and metastatic tumors: Functional analysis and expression of PD-1 receptor.

The tumor microenvironment (TM) contains a wide variety of cell types and soluble factors capable of suppressing immune responses. While the presence of NK cells in pleural effusions (PE) has been documented, no information exists on the presence of other innate lymphoid cell (ILC) subsets and on the expression of programmed cell death-1 (PD-1) in NK and ILC. The presence of ILC was assessed in PE of 54 patients (n = 33 with mesothelioma, n = 15 with adenocarcinoma and n = 6 with inflammatory pleural diseases) by cell staining with suitable antibody combinations and cytofluorimetric analysis. The cytokine production of ILC isolated from both PE and autologous peripheral blood was analyzed upon cell stimulation and intracytoplasmic staining. We show that, in addition to NK cells, also ILC1, ILC2 and ILC3 are present in malignant PE and that the prevalent subset is ILC3. PE-ILC subsets produced their typical sets of cytokines upon activation. In addition, we analyzed the PD-1 expression on NK/ILC by multiparametric flow-cytometric analysis, while the expression of PD-1 ligand (PD-L1) was evaluated by immunohistochemical analysis. Both NK cells and ILC3 expressed functional PD-1, moreover, both tumor samples and malignant PE-derived tumor cell lines were PD-L1+ suggesting that the interaction between PD-1+ ILC and PD-L1+ tumor cells may hamper antitumor immune responses mediated by NK and ILC.

A conserved energetic footprint underpins recognition of human leukocyte antigen-E by two distinct αß T cell receptors.

αß T cell receptors (TCRs) interact with peptides bound to the polymorphic major histocompatibility complex class Ia (MHC-Ia) and class II (MHC-II) molecules as well as the essentially monomorphic MHC class Ib (MHC-Ib) molecules. Although there is a large amount of information on how TCRs engage with MHC-Ia and MHC-II, our understanding of TCR/MHC-Ib interactions is very limited. Infection with cytomegalovirus (CMV) can elicit a CD8+ T cell response restricted by the human MHC-Ib molecule human leukocyte antigen (HLA)-E and specific for an epitope from UL40 (VMAPRTLIL), which is characterized by biased TRBV14 gene usage. Here we describe an HLA-E-restricted CD8+ T cell able to recognize an allotypic variant of the UL40 peptide with a modification at position 8 (P8) of the peptide (VMAPRTLVL) that uses the TRBV9 gene segment. We report the structures of a TRBV9+ TCR in complex with the HLA-E molecule presenting the two peptides. Our data revealed that the TRBV9+ TCR adopts a different docking mode and molecular footprint atop HLA-E when compared with the TRBV14+ TCR-HLA-E ternary complex. Additionally, despite their differing V gene segment usage and different docking mechanisms, mutational analyses showed that the TCRs shared a conserved energetic footprint on the HLA-E molecule, focused around the peptide-binding groove. Hence, we provide new insights into how monomorphic MHC molecules interact with T cells.

Development of human natural killer cells and other innate lymphoid cells.

Innate lymphoid cells (ILC) have recently gained much attention in immunology. They represent a novel developmentally related family. Three distinct subsets have been identified on the basis of phenotypic and functional criteria and termed ILC1, ILC2, and ILC3. The available data suggest that ILC play an important role in innate defenses against different pathogens, in lymphoid organogenesis, and in tissue remodeling. All these aspects are relevant in hematopoietic stem cell transplantation (HSCT), particularly in the haplo-HSCT setting, in which donor NK cells are known to play a major therapeutic role, while the involvement of other ILC is still undefined. In this context, it has been postulated that all ILC share a common precursor expressing the ID2 transcription factor. While the differentiation of human NK cells (belonging to ILC1) is now well characterized both in vitro and in vivo, limited information is available on the development of human ILC2 and ILC3 and of their relationships with NK cells. In this review, we will summarize the present knowledge on the developmental relationship among different ILC, with particular focus on early stages of NK cell differentiation, and their features shared with ILC2 and ILC3.

The generation of human innate lymphoid cells is influenced by the source of hematopoietic stem cells and by the use of G-CSF.

NK cells play a central role in the haploidentical HSC transplantation (HSCT) to cure high-risk leukemias. Other innate lymphoid cells (ILCs) have been proposed to exert a protective role in graft-versus-host disease and could also contribute to anti-microbial defence and to lymphoid tissue remodeling. Thus, we investigated the ILC differentiation potential of HSCs isolated from BM, mobilized peripheral blood (PB), and umbilical cord blood (UCB). BM CD34(+) cells are enriched in lymphoid-committed precursors, while PB CD34(+) cells preferentially contain myeloid precursors. In vitro differentiation experiments revealed that the highest and the lowest CD56(+) CD161(+) ILC recovery was detected in UCB and PB HSC cultures, respectively. Among CD56(+) CD161(+) ILCs, the ratio between NK cells and ILC3s was similar for all HSC analyzed. ILC recovery in PB CD34(+) cultures was lower for G-CSF-mobilized HSCs (good mobilizers) than for G-CSF+plerixafor-mobilized HSC (poor mobilizers). Moreover, G-CSF inhibited in vitro ILC recovery and the degree of inhibition was proportional to the time of exposure to the cytokine. Thus, although all common sources of HSC for transplant differentiate towards ILCs, substantial differences exist among different sources and G-CSF may influence ILC recovery. These data offer new clues for a better understanding of the immune reconstitution after HSCT.

IL-1ß inhibits ILC3 while favoring NK-cell maturation of umbilical cord blood CD34(+) precursors.

NK cells are innate lymphocytes characterized by the expression of nuclear factor interleukin 3 regulated (NFIL3 or E4BP4), eomesodermin (EOMES) transcription factors (TFs), and by the ability to exert cytolytic activity and release IFN-γ. In the haploidentical-hematopoietic stem cell transplantation (haplo-HSCT) setting, CD34(+) donor derived NK cells play a major role in the control of leukemic relapses. Therefore, it is important to better define cytokines that influence NK-cell differentiation from CD34(+) precursors. We analyzed the effects of IL-1ß on NK-cell differentiation from umbilical cord blood (UCB) CD34(+) cells. While IL-1ß inhibited CD161(+) CD56(+) cell proliferation, an increased expression of LFA-1, CD94/NKG2A, KIRs, and perforin on CD56(+) cells was detected. In addition, within the CD161(+) CD56(+) IL-1RI(+) LFA-1(-) cell fraction (representing group 3 innate lymphoid cells, ILC3-like cells), a significant increase of EOMES, NKp46, and CD94/NKG2A receptors, cytolytic granules, and IFN-γ was detected. This increase was paralleled by a decrease of related orphan receptors (RORγt) TF, NKp44 expression, and IL-22 production. These data suggest that IL-1ß inhibits ILC3- while favoring NK-cell maturation. Since in haplo-HSCT conditioning regimen, infections or residual leukemia cells may induce IL-1ß production, this may influence the NK/ILC3 development from donor-derived CD34(+) precursors.

Human natural killer cells: news in the therapy of solid tumors and high-risk leukemias.

It is well established that natural killer (NK) cells play an important role in the immunity against cancer, while the involvement of other recently identified, NK-related innate lymphoid cells is still poorly defined. In the haploidentical hematopoietic stem cell transplantation for the therapy of high-risk leukemias, NK cells have been shown to exert a key role in killing leukemic blasts residual after conditioning. While the clinical results in the cure of leukemias are excellent, the exploitation of NK cells in the therapy of solid tumors is still limited and unsatisfactory. In solid tumors, NK cell function may be inhibited via different mechanisms, occurring primarily at the tumor site. The cellular interactions in the tumor microenvironment involve tumor cells, stromal cells and resident or recruited leukocytes and may favor tumor evasion from the host's defenses. In this context, a number of cytokines, growth factors and enzymes synthesized by tumor cells, stromal cells, suppressive/regulatory myeloid and lymphoid cells may substantially impair the function of different tumor-reactive effector cells, including NK cells. The identification and characterization of such mechanisms may offer clues for the development of new immunotherapeutic strategies to restore effective anti-tumor responses. In order to harness NK cell-based immunotherapies, several approaches have been proposed, including reinforcement of NK cell cytotoxicity by means of specific cytokines, antibodies or drugs. These new tools may improve NK cell function and/or increase tumor susceptibility to NK-mediated killing. Hence, the integration of NK-based immunotherapies with conventional anti-tumor therapies may increase chances of successful cancer treatment.

CD56(bright)perforin(low) noncytotoxic human NK cells are abundant in both healthy and neoplastic solid tissues and recirculate to secondary lymphoid organs via afferent lymph.

As limited information is available regarding the distribution and trafficking of NK cells among solid organs, we have analyzed a wide array of tissues derived from different human compartments. NK cells were widely distributed in most solid tissues, although their amount varied significantly depending on the tissue/organ analyzed. Interestingly, the distribution appeared to be subset specific, as some tissues were preferentially populated by CD56(bright)perforin(low) NK cells, with others by the CD56(dim)perforin(high) cytotoxic counterpart. Nevertheless, most tissues were highly enriched in CD56(bright)perforin(low) cells, and the distribution of NK subsets appeared in accordance with tissue gene expression of chemotactic factors, for which receptors are differently represented in the two subsets. Remarkably, chemokine expression pattern of tissues was modified after neoplastic transformation. As a result, although the total amount of NK cells infiltrating the tissues did not significantly change upon malignant transformation, the relative proportion of NK subsets infiltrating the tissues was different, with a trend toward a tumor-infiltrating NK population enriched in noncytotoxic cells. Besides solid tissues, CD56(bright)perforin(low) NK cells were also detected in seroma fluids, which represents an accrual of human afferent lymph, indicating that they may leave peripheral solid tissues and recirculate to secondary lymphoid organs via lymphatic vessels. Our results provide a comprehensive mapping of NK cells in human tissues, demonstrating that discrete NK subsets populate and recirculate through most human tissues and that organ-specific chemokine expression patterns might affect their distribution. In this context, chemokine switch upon neoplastic transformation might represent a novel mechanism of tumor immune escape.

Membrane transfer from tumor cells overcomes deficient phagocytic ability of plasmacytoid dendritic cells for the acquisition and presentation of tumor antigens.

The potential contribution of plasmacytoid dendritic cells (pDCs) in the presentation of tumor cell Ags remains unclear, and some controversies exist with regard to the ability of pDCs to phagocytose cell-derived particulate Ags and cross-present them to MHC class I-restricted T lymphocytes. In this study, we show that human pDCs, although inefficient in the internalization of cell membrane fragments by phagocytosis, can efficiently acquire membrane patches and associated molecules from cancer cells of different histotypes. The transfer of membrane patches to pDCs occurred in a very short time and required cell-to-cell contact. Membrane transfer also included intact HLA complexes, and the acquired Ags could be efficiently recognized on pDCs by tumor-specific CD8(+) T cells. Remarkably, pDCs isolated from human colon cancer tissues displayed a strong surface expression of epithelial cell adhesion molecule, indicating that the exchange of exogenous Ags between pDCs and tumor cells also can occur in vivo. These data demonstrate that pDCs are well suited to acquire membrane patches from contiguous tumor cells by a cell-to-cell contact-dependent mechanism that closely resembles "trogocytosis." This phenomenon may allow pDCs to proficiently present tumor cell-derived Ags, despite limited properties of endophagocytosis.

Effect of tumor cells and tumor microenvironment on NK-cell function.

The ability of tumors to manage an immune-mediated attack has been recently included in the "next generation" of cancer hallmarks. In solid tumors, the microenvironment that is generated during the first steps of tumor development has a pivotal role in immune regulation. An intricate net of cross-interactions occurring between tumor components, stromal cells, and resident or recruited immune cells skews the possible acute inflammatory response toward an aberrant ineffective chronic inflammatory status that favors the evasion from the host's defenses. Natural killer (NK) cells have powerful cytotoxic activity, but their activity may be eluded by the tumor microenvironment. Immunosubversion, immunoediting or immunoselection of poorly immunogenic tumor cells and interference with tumor infiltration play a major role in evading NK-cell responses to tumors. Tumor cells, tumor-associated fibroblasts and tumor-induced aberrant immune cells (i.e. tolerogenic or suppressive macrophages, dendritic cells (DCs) and T cells) can interfere with NK-cell activation pathways or the complex receptor array that regulate NK-cell activation and antitumor activity. Thus, the definition of tumor microenvironment-related immunosuppressive factors, along with the identification of new classes of tissue-residing NK-like innate lymphoid cells, represent key issues to design effective NK-cell-based therapies of solid tumors.

Characterization of human afferent lymph dendritic cells from seroma fluids.

Dendritic cells (DCs) migrate from peripheral tissues to secondary lymphoid organs (SLOs) through the afferent lymph. Owing to limitations in investigating human lymph, DCs flowing in afferent lymph have not been properly characterized in humans until now. In this study, DCs present in seroma, an accrual of human afferent lymph occurring after lymph node surgical dissection, were isolated and analyzed in detail. Two main DC subsets were identified in seroma that corresponded to the migratory DC subsets present in lymph nodes, that is, CD14(+) and CD1a(+). The latter also included CD1a(bright) Langerhans cells. The two DC subsets appeared to share the same monocytic precursor and to be developmentally related; both of them spontaneously released high levels of TGF-ß and displayed similar T cell-activating and -polarizing properties. In contrast, they differed in the expression of surface molecules, including TLRs; in their phagocytic activity; and in the expression of proteins involved in Ag processing and presentation. It is worth noting that although both subsets were detected in seroma in the postsurgical inflammatory phase, only CD1a(+) DCs migrated via afferent lymph under steady-state conditions. In conclusion, the high numbers of DCs contained in seroma fluids allowed a proper characterization of human DCs migrating via afferent lymph, revealing a continuous stream of DCs from peripheral regions toward SLOs under normal conditions. Moreover, we showed that, in inflammatory conditions, distinct subsets of DCs can migrate to SLOs via afferent lymph.

Polymorphism in human cytomegalovirus UL40 impacts on recognition of human leukocyte antigen-E (HLA-E) by natural killer cells.

Natural killer (NK) cell recognition of the nonclassical human leukocyte antigen (HLA) molecule HLA-E is dependent on the presentation of a nonamer peptide derived from the leader sequence of other HLA molecules to CD94-NKG2 receptors. However, human cytomegalovirus can manipulate this central innate interaction through the provision of a "mimic" of the HLA-encoded peptide derived from the immunomodulatory glycoprotein UL40. Here, we analyzed UL40 sequences isolated from 32 hematopoietic stem cell transplantation recipients experiencing cytomegalovirus reactivation. The UL40 protein showed a "polymorphic hot spot" within the region that encodes the HLA leader sequence mimic. Although all sequences that were identical to those encoded within HLA-I genes permitted the interaction between HLA-E and CD94-NKG2 receptors, other UL40 polymorphisms reduced the affinity of the interaction between HLA-E and CD94-NKG2 receptors. Furthermore, functional studies using NK cell clones expressing either the inhibitory receptor CD94-NKG2A or the activating receptor CD94-NKG2C identified UL40-encoded peptides that were capable of inhibiting target cell lysis via interaction with CD94-NKG2A, yet had little capacity to activate NK cells through CD94-NKG2C. The data suggest that UL40 polymorphisms may aid evasion of NK cell immunosurveillance by modulating the affinity of the interaction with CD94-NKG2 receptors.

NK cells from malignant pleural effusions are not anergic but produce cytokines and display strong antitumor activity on short-term IL-2 activation.

NK cells are a major component of innate immunity and exert a potent antitumor effect both in vitro and in vivo. However, NK cells infiltrating solid tumors have been shown to display severely impaired functional capabilities. In this study, we analyzed NK cells present in pleural effusions (PEs) of patients with primary or metastatic tumors of different origin, including mesothelioma and lung, breast, colon, gastric, bladder, and uterus carcinoma. In all instances, freshly isolated PE-NK cells displayed a CD56(bright) phenotype and expressed normal levels of both activating receptors and HLA class I-specific inhibitory receptors. In addition, they rapidly released large amounts of IFN-γ and TNF-α after stimulation. Upon culture in IL-2, they acquired a potent cytolytic activity against both allogeneic and autologous tumor cells. Tumor cell lysis was primarily mediated by NKG2D and NKp30 and partially by NKp46 and DNAM-1, in agreement with the expression of the corresponding ligands on tumor cells. The finding that PE-NK cells are not functionally impaired and that they can efficiently kill tumor cells upon short-term IL-2 activation may offer important clues for the development of novel approaches in tumor immunotherapy.

Hypoxia downregulates the expression of activating receptors involved in NK-cell-mediated target cell killing without affecting ADCC.

In certain infection sites or tumor tissues, the disruption of homeostasis can give rise to a hypoxic microenvironment, which, in turn, can alter the function of different immune cell types and favor the progression of the disease. Natural killer (NK) cells are directly involved in the elimination of virus-infected or transformed cells, however it is unknown whether their function is affected by hypoxia or not. In this study, we show that NK cells adapt to a hypoxic environment by upregulating the hypoxia-inducible factor 1α. However, NK cells lose their ability to upregulate the surface expression of the major activating NK-cell receptors (NKp46, NKp30, NKp44, and NKG2D) in response to IL-2 (or other activating cytokines, including IL-15, IL-12, and IL-21). These altered phenotypic features correlate with reduced responses to triggering signals resulting in impaired capability of killing infected or tumor target cells. Remarkably, hypoxia does not significantly alter the surface density and the triggering function of the Fc-γ receptor CD16, thus allowing NK cells to maintain their capability of killing target cells via antibody-dependent cellular cytotoxicity. This finding offers an important clue for exploitation of NK cell in antibody-based immunotherapy of cancer.