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Backgroundwe evaluated the concordance between immunohistochemical p53 staining and TP53 mutations in a series of HGSOC. Moreover, we searched for prognostic differences between p53 overexpression and null expression groups.Methodspatients affected by HGSOC were included. For each case p53 immunohistochemical staining and molecular assay (Sanger sequencing) were performed. Kaplan-Meier survival analyses were undertaken to determine whether the type of TP53 mutation, or p53 staining pattern influenced overall survival (OS) and progression free survival (PFS).Results34 HGSOC were considered. All cases with a null immunohistochemical p53 expression (n=16) showed TP53 mutations (n=9 nonsense, n=4 in-frame deletion, n=2 splice, n=1 in-frame insertion). 16 out of 18 cases with p53 overexpression showed TP53 missense mutation. Follow up data were available for 33 out of 34 cases (median follow up time 15 month). We observed a significant reduction of OS in p53 null group [HR = 3.64, 95% CI 1.01-13.16].Conclusionimmunohistochemical assay is a reliable surrogate for TP53 mutations in most cases. Despite the small cohort and the limited median follow up, we can infer that HGSOC harboring p53 null mutations are a more aggressive subgroup.

IntroductionOral squamous cell carcinoma (OSCC) accounts for approximately 90% of oral malignancies and has a 5-year mortality rate close to 50%. A consistent part (70%) of all oral cancers is diagnosed at an advanced stage since available screening techniques are ineffective. Therefore, it would be urgent to improve them. The diagnostic gold standard is tissue biopsy with histological and immunohistochemical assessment. This method presents some limitations. Biopsy is invasive and the histopathological evaluation is semi-quantitative, and the absolute abundance of the target cannot be reliably determined. In addition, tissue is highly processed and may lead to loss of information of the natural state. The search for classical and new clinical biomarkers on fragments of tissue/cells collected with a cytobrush is a highly hopeful technique for early detection and diagnosis of OSCC, because of its non-invasive sampling and easy collection method.MethodsHere we analyzed cytobrush biopsies samples collected from the oral cavity of 15 patients with already diagnosed OSCC by applying an innovative high-sensitivity ELISA technique, in order to verify if this approach may provide useful information for detection, diagnosis, and prognosis of OSCC. To this end, we selected six biomarkers, already used in clinical practice for the diagnosis of OSCC (EGFR, Ki67, p53) or selected based on recent scientific and clinical data which indicate their presence or over-expression in cells undergoing transformation and their role as possible molecular targets in immunecheckpoints blockade therapies (PD-L1, HLA-E, B7-H6).ResultsThe selected tumor biomarkers were highly expressed in the tumor core, while were virtually negative in healthy tissue collected from the same patients. These differences were highly statistically significant and consistent with those obtained using the gold standard test clearly indicating that the proposed approach, i.e. analysis of biomarkers by a custom ELISA technique, is strongly reliable.DiscussionThese preliminary data suggest that this non-invasive rapid phenotyping technique could be useful as a screening tool for phenotyping oral lesions and support clinical practice by precise indications on the characteristics of the lesion, also with a view to the application of new anti-tumor treatments, such as immunotherapy, aimed at OSCC patients.

BackgroundNatural Killer cells (NKs) represent the innate counterpart of TCRαβ lymphocytes and are characterized by a high anti-tumor and an anti-viral cytotoxic activity. Recently, it has been demonstrated that NKs can express PD-1 as an additional inhibitory receptor. Specifically, PD-1 was identified on a subpopulation of terminally differentiated NKs from healthy adults with previous HCMV infection. So far it is unknown whether PD-1 appears during NK-cell development and whether this process is directly or indirectly related to HCMV infection.MethodsIn this study, we analyzed the expression and function of PD-1 on Cord Blood derived NKs (CB-NKs) on a large cohort of newborns through multiparametric cytofluorimetric analysis.ResultsWe identified PD-1 on CB-NKs in more than of half the newborns analyzed. PD-1 was present on CD56dim NKs, and particularly abundant on CD56neg NKs, but only rarely present on CD56bright NKs. Importantly, unlike in adult healthy donors, in CB-NKs PD-1 is co-expressed not only with KIR, but also with NKG2A. PD-1 expression was independent of HCMV mother seropositivity and occurs in the absence of HCMV infection/reactivation during pregnancy. Notably, PD-1 expressed on CB-NKs was functional and mediated negative signals when triggered.ConclusionTo our understanding, this study is the first to report PD-1 expression on CB derived NKs and its features in perinatal conditions. These data may prove important in selecting the most suitable CB derived NK cell population for the development of different immunotherapeutic treatments.

Chronic myeloid leukemia (CML) is a hematologic malignancy associated to an unregulated growth of myeloid cells in bone marrow (BM) and peripheral blood (PB), characterized by the BCR-ABL1 translocation. Given the known cytokine impairment in the leukemic niche of CML, we investigated the impact of this microenvironmental dysregulation on innate lymphoid cells (ILC), whose role in cancer has recently emerged. Three ILC subsets are identified based on transcriptional profiles and cytokine secretion. We observed that interleukin 18 (IL-18) and vascular endothelial growth factor A (VEGF-A) are increased in CML patients’ sera and that ILC2 are enriched in CML PB and BM. We found that IL-18 drives ILC2 proliferation and that CML ILC2 highly express CXCR4 and CXCR7 BM-homing receptors, potentially explaining their enrichment in PB and BM, respectively. Next, we showed that ILC2 are hyper-activated through a tumor-derived VEGF-Adependent mechanism, which leads to higher IL-13 secretion. In response to IL-13, leukemic cells increase their clonogenic capacity. Finally, we discovered that the pro-tumoral axis involving VEGF-A, IL-18 and ILC2 was disrupted upon tyrosine kinase inhibitor treatment, normalizing the levels of all these players in CML patients responding to therapy. Overall, our study uncovers the involvement of ILC2 in CML progression, mediated by VEGF-A and IL-18.

Host versus graft reaction is a major impediment to CAR-T cell immune therapy in allogeneic settings. Authors show here that CAR-T cells, engineered to be deficient in MHC I expression but to express the NK inhibitor HLA-E, are resistant to destruction by both T and NK cells of the host.

The characterization of frequency and phenotypes of natural killer (NK) cells and T cells in BAL and peripheral blood of patients with sarcoidosis was evaluated, to discriminate the differential status of these cells in these two compartments. The analysis revealed that CD56brightCD16neg resulted higher in BAL than PB of sarcoidosis and healthy subjects, while CD56dimCD16+ showed a different proportion between BAL and PB of both Sarcoidosis patients and HC. Moreover, in comparison with autologous PB, BAL was characterized by a higher expression of activated NK cell markers NKp44, CD69 and CD25. Significantly increased levels of PD-1+ NK cells in the BAL of patients were detected. Regarding the maturation of CD4 and CD8, an increase of Effector Memory T cells (TEM) was reported in BAL compared to PB. A better characterization of NK and T cells may lead to an improvement of the pathogenetic mechanisms in sarcoidosis.

Despite the results achieved with the evolution of conventional chemotherapy and the inclusion of targeted therapies in the treatment of acute myeloid leukemia (AML), survival is still not satisfying, in particular in the setting of relapsed/refractory (R/R) disease or elderly/unfit patients. Among the most innovative therapeutic options, cellular therapy has shown great results in different hematological malignancies such as acute lymphoblastic leukemia and lymphomas, with several products already approved for clinical use. However, despite the great interest in also expanding the application of these new treatments to R/R AML, no product has been approved yet for clinical application. Furthermore, cellular therapy could indeed represent a powerful tool and an appealing alternative to allogeneic hematopoietic stem cell transplantation for ineligible patients. In this review, we aim to provide an overview of the most recent clinical research exploring the effectiveness of cellular therapy in AML, moving from consolidated approaches such as post- transplant donor’s lymphocytes infusion, to modern adoptive immunotherapies such as alloreactive NK cell infusions, engineered T and NK cells (CAR-T, CAR-NK) and novel platforms of T and NK cells engaging (i.e., BiTEs, DARTs and ANKETTM).

Despite recent advances in ovarian cancer (OC) treatment, including the introduction of bevacizumab and PARP-inhibitors, OC remains a lethal disease. Other therapeutic options are being explored, such as immunotherapy (IT), which has been proved effective in many solid tumors. Findings about tumor-infiltrating cytotoxic and regulatory T cells, together with the expression of PD-1 on immune cells and of PD-L1 on tumor cells, gave the rationale for an attempt to the use of IT also in OC. We treated two patients with avelumab, an anti-PD-L1 monoclonal antibody, after the first line of chemotherapy: Patient A underwent 19 cycles of maintenance therapy with avelumab with a disease-free interval of 12 months, whereas patient B showed a slight progression of disease after only eight cycles. A higher PD-L1 expression in tumor cells of patient A was detected. She also underwent a genomic assessment that described the presence of a high Tumor Mutational Burden (TMB) and a status of Loss of Heterozygosity (LoH). This different response to the same treatment puts in evidence that some genomic and immune features might be investigated.

We report a case of inflammatory colitis after SARS-CoV-2 infection in a patient with no additional co-morbidity who died within three weeks of hospitalization. As it is becoming increasingly clear that SARS-CoV-2 infection can cause immunological alterations, we investigated the expression of the inhibitory checkpoint PD-1 and its ligand PD-L1 to explore the potential role of this axis in the break of self-tolerance. The presence of the SARS-CoV-2 virus in colon tissue was demonstrated by qRT-PCR and immunohistochemical localization of the nucleocapsid protein. Expression of lymphocyte markers, PD-1, and PD-L1 in colon tissue was investigated by IHC. SARS-CoV-2-immunoreactive cells were detected both in the ulcerated and non-ulcerated mucosal areas. Compared to healthy tissue, where PD-1 is weakly expressed and PD-L1 is absent, PD-1 and PD-L1 expression appears in the inflamed mucosal tissue, as expected, but was mainly confined to non-ulcerative areas. At the same time, these markers were virtually undetectable in areas of mucosal ulceration. Our data show an alteration of the PD-1/PD-L1 axis and suggest a link between SARS-CoV-2 infection and an aberrant autoinflammatory response due to concomitant breakdown of the PD-1/PD-L1 interaction leading to early death of the patient.

Abstract: An understanding of natural killer (NK) cell physiology in acute myeloid leukemia (AML) has led to the use of NK cell transfer in patients, demonstrating promising clinical results. However, AML is still characterized by a high relapse rate and poor overall survival. In addition to conventional NKs that can be considered the innate counterparts of CD8 T cells, another family of innate lymphocytes has been recently described with phenotypes and functions mirroring those of helper CD4 T cells. Here, in blood and tissues, we identified a CD56+ innate cell population harboring mixed transcriptional and phenotypic attributes of conventional helper innate lymphoid cells (ILCs) and lytic NK cells. These CD56+ ILC1-like cells possess strong cytotoxic capacities that are impaired in AML patients at diagnosis but are restored upon remission. Their cytotoxicity is KIR independent and relies on the expression of TRAIL, NKp30, NKp80, and NKG2A. However, the presence of leukemic blasts, HLA-E–positive cells, and/or transforming growth factor-β1 (TGF-β1) strongly affect their cytotoxic potential, at least partially by reducing the expression of cytotoxic-related molecules. Notably, CD56+ ILC1-like cells are also present in the NK cell preparations used in NK transfer–based clinical trials. Overall, we identified an NK cell–related CD56+ ILC population involved in tumor immunosurveillance in humans, and we propose that restoring their functions with anti-NKG2A antibodies and/or small molecules inhibiting TGF-β1 might represent a novel strategy for improving current immunotherapies.

Immune checkpoint inhibitors (CI) have demonstrated clinical activity in Hodgkin Lymphoma (HL) patients relapsing after autologous stem cell transplantation (ASCT), although only 20% complete response (CR) rate was observed. The efficacy of CI is strictly related to the host immune competence, which is impaired in heavily pre-treated HL patients. Here, we aimed to enhance the activity of early post-ASCT CI (nivolumab) administration with the infusion of autologous lymphocytes (ALI). Twelve patients with relapse/refractory (R/R) HL (median age 28.5 years; range 18-65), underwent lymphocyte apheresis after first line chemotherapy and then proceeded to salvage therapy. Subsequently, 9 patients with progressive disease at ASCT received early post-transplant CI supported with four ALI, whereas 3 responding patients received ALI alone, as a control cohort. No severe adverse events were recorded. HL-treated patients achieved negative PET scan CR and 8 are alive and disease-free after a median follow-up of 28 months. Four patients underwent subsequent allogeneic SCT. Phenotypic analysis of circulating cells showed a faster expansion of highly differentiated NK cells in ALI plus nivolumab-treated patients as compared to control patients. Our data show anti-tumor activity with good tolerability of ALI + CI for R/R HL and suggest that this setting may accelerate NK cell development/maturation and favor the expansion of the “adaptive” NK cell compartment in patients with HCMV seropositivity, in the absence of HCMV reactivation.

Summary: Human Vδ2 cells are innate-like γδ T effectors performing potent immune surveillance against tumors. The constitutive expression of NKG2A identifies a subset of Vδ2 T cells licensed with an intrinsic hyper-responsiveness against cancer. Indeed, the transcriptomic profiles of NKG2A+ and NKG2A− cells characterize two distinct “intralineages” of Vδ2 T lymphocytes that appear early during development, keep their phenotypes, and show self-renewal capabilities in adult life. The hyper-responsiveness of NKG2A+ Vδ2 T cells is counterbalanced by the inhibitory signaling delivered by human leukocyte antigen E (HLA-E) expressed on malignant cells as a tumor-escape mechanism. However, either masking or knocking out NKG2A restores the capacity of Vδ2 T cells to exert the highest effector functions even against HLA-E+ tumors. This is highly relevant in the clinic, as the different degrees of engagement of the NKG2A-HLA-E checkpoint in hepatocellular carcinoma, glioblastoma, and non-small cell lung cancer directly impact patients’ overall survival. These findings open avenues for developing combined cellular and immunologic anticancer therapies.

Haploidentical hematopoietic stem cell transplantation (h-HSCT) represents an efficient curative approach for patients affected by hematologic malignancies in which the reduced intensity conditioning induces a state of immunologic tolerance between donor and recipient. However, opportunistic viral infections greatly affect h-HSCT clinical outcomes. NK cells are the first lymphocytes that recover after transplant and provide a prompt defense against human cytomegalovirus (HCMV) infection/reactivation. By undertaking a longitudinal single-cell computational profiling of multiparametric flow cytometry, we show that HCMV accelerates NK cell immune reconstitution together with the expansion of CD158b1b2jpos/NKG2Aneg/NKG2Cpos/NKp30lo NK cells. The frequency of this subset correlates with HCMV viremia, further increases in recipients experiencing multiple episodes of viral reactivations, and persists for months after the infection. The transcriptional profile of FACS-sorted CD158b1b2jpos NK cells confirmed the ability of HCMV to deregulate NKG2C, NKG2A, and NKp30 gene expression, thus inducing the expansion of NK cells with adaptive traits. These NK cells are characterized by the downmodulation of several gene pathways associated with cell migration, the cell cycle, and effector-functions, as well as by a state of metabolic/cellular exhaustion. This profile reflects the functional impairments of adaptive NK cells to produce IFN-γ, a phenomenon also due to the viral-induced expression of lymphocyte-activation gene 3 (LAG-3) and programmed cell death protein 1 (PD-1) checkpoint inhibitors.

Human Natural Killer (NK) cells are all round players in immunity thanks to their powerful and immediate response against transformed cells and the ability to modulate the subsequent adaptive immune response. The potential of immunotherapies based on NK cell involvement has been initially revealed in the hematological setting but has inspired the design of different immune tools to also be applied against solid tumors, including colorectal cancer (CRC). Indeed, despite cancer prevention screening plans, surgery, and chemotherapy strategies, CRC is one of the most widespread cancers and with the highest mortality rate. Therefore, further efficient and complementary immune-based therapies are in urgent need. In this review, we gathered the most recent advances in NK cell-based immunotherapies aimed at fighting CRC, in particular, the use of monoclonal antibodies targeting tumor-associated antigens (TAAs), immune checkpoint blockade, and adoptive NK cell therapy, including NK cells modified with chimeric antigen receptor (CAR-NK).

Innate lymphoid cells (ILCs) represent the most recently identified subset of effector lymphocytes, with key roles in the orchestration of early immune responses. Despite their established involvement in the pathogenesis of many inflammatory disorders, the role of ILCs in cancer remains poorly defined. Here we assessed whether human ILCs can actively interact with the endothelium to promote tumor growth control, favoring immune cell adhesion. We show that, among all ILC subsets, ILCPs elicited the strongest upregulation of adhesion molecules in endothelial cells (ECs) in vitro, mainly in a contact-dependent manner through the tumor necrosis factor receptor- and RANK-dependent engagement of the NF-κB pathway. Moreover, the ILCP-mediated activation of the ECs resulted to be functional by fostering the adhesion of other innate and adaptive immune cells. Interestingly, pre-exposure of ILCPs to human tumor cell lines strongly impaired this capacity. Hence, the ILCP–EC interaction might represent an attractive target to regulate the immune cell trafficking to tumor sites and, therefore, the establishment of an anti-tumor immune response.

The incidence of certain forms of tumors has increased progressively in recent years and is expected to continue growing as life expectancy continues to increase. Tumor-infiltrating NK cells may contribute to develop an anti-tumor response. Optimized combinations of different cancer therapies, including NK cell-based approaches for targeting tumor cells, have the potential to open new avenues in cancer immunotherapy. Functional inhibitory receptors on NK cells are needed to prevent their attack on healthy cells. Nevertheless, disruption of inhibitory receptors function on NK cells increases the cytotoxic capacity of NK cells against cancer cells. MicroRNAs (miRNAs) are small non-coding RNA molecules that target mRNA and thus regulate the expression of genes involved in the development, maturation, and effector functions of NK cells. Therapeutic strategies that target the regulatory effects of miRNAs have the potential to improve the efficiency of cancer immunotherapy. Interestingly, emerging evidence points out that some miRNAs can, directly and indirectly, control the surface expression of immune checkpoints on NK cells or that of their ligands on tumor cells. This suggests a possible use of miRNAs in the context of anti-tumor therapy. This review provides the current overview of the connections between miRNAs and regulation of NK cell functions and discusses the potential of these miRNAs as innovative biomarkers/targets for cancer immunotherapy.

In the last years, natural killer (NK) cell-based immunotherapy has emerged as a promising therapeutic approach for solid tumors and hematological malignancies. NK cells are innate lymphocytes with an array of functional competences, including anti-cancer, anti-viral, and anti-graft-vs.-host disease potential. The intriguing idea of harnessing such potent innate immune system effectors for cancer treatment led to the development of clinical trials based on the adoptive therapy of NK cells or on the use of monoclonal antibodies targeting the main NK cell immune checkpoints. Indeed, checkpoint immunotherapy that targets inhibitory receptors of T cells, reversing their functional blocking, marked a breakthrough in anticancer therapy, opening new approaches for cancer immunotherapy and resulted in extensive research on immune checkpoints. However, the clinical efficacy of T cell-based immunotherapy presents a series of limitations, including the inability of T cells to recognize and kill HLA-Ineg tumor cells. For these reasons, new strategies for cancer immunotherapy are now focusing on NK cells. Blockade with NK cell checkpoint inhibitors that reverse their functional block may overcome the limitations of T cell-based immunotherapy, mainly against HLA-Ineg tumor targets. Here, we discuss recent anti-tumor approaches based on mAb-mediated blocking of immune checkpoints (either restricted to NK cells or shared with T cells), used either as a single agent or in combination with other compounds, that have demonstrated promising clinical responses in both solid tumors and hematological malignancies.

Neutrophils are potent effector cells and contain a battery of harmful substances and degrading enzymes. A silent neutrophil death, i.e., apoptosis, is therefore of importance to avoid damage to the surrounding tissue and to enable termination of the acute inflammatory process. There is a pile of evidence supporting the role for pro-inflammatory cytokines in extending the life-span of neutrophils, but relatively few studies have been devoted to mechanisms actively driving apoptosis induction in neutrophils. We have previously demonstrated that natural killer (NK) cells can promote apoptosis in healthy neutrophils. In this study, we set out to investigate how neutrophil sensitivity to NK cell-mediated cytotoxicity is regulated under inflammatory conditions. Using in vitro-activated neutrophils and a human skin chamber model that allowed collection of in vivo-transmigrated neutrophils, we performed a comprehensive characterization of neutrophil expression of ligands to NK cell receptors. These studies revealed a dramatic downregulation of HLA class I molecules in inflammatory neutrophils, which was associated with an enhanced susceptibility to NK cell cytotoxicity. Collectively, our data shed light on the complex regulation of interactions between NK cells and neutrophils during an inflammatory response and provide further support for a role of NK cells in the resolution phase of inflammation.

Glycine is an important neurotransmitter in vertebrates, performing both excitatory and inhibitory actions. Synaptic levels of glycine are tightly controlled by the action of two glycine transporters, GlyT1 and GlyT2, located on the surface of glial cells and neurons, respectively. Only limited information is available on glycinergic neurotransmission in invertebrates, and the evolution of glycinergic neurotransmission is poorly understood. Here, by combining phylogenetic and gene expression analyses, we characterized the glycine transporter complement of amphioxus, an important invertebrate model for studying the evolution of chordates. We show that amphioxus possess three glycine transporter genes. Two of these (GlyT2.1 and GlyT2.2) are closely related to GlyT2 of vertebrates, whereas the third (GlyT) is a member of an ancestral clade of deuterostome glycine transporters. GlyT2.2 expression is predominantly non-neural, whereas GlyT and GlyT2.1 are widely expressed in the amphioxus nervous system and are differentially expressed, respectively, in neurons and glia. Vertebrate glycinergic neurons express GlyT2 and glia GlyT1, suggesting that the evolution of the chordate glycinergic system was accompanied by a paralog-specific inversion of gene expression. Despite this genetic divergence between amphioxus and vertebrates, we found strong evidence for conservation in the role glycinergic neurotransmission plays during larval swimming, the implication being that the neural networks controlling the rhythmic movement of chordate bodies may be homologous.

Group 2 innate lymphoid cells (ILC2s) modulate inflammatory and allergic responses, but their function in cancer immunity is still unclear. Here the authors show that, in acute promyelocytic leukaemia, tumour-activated ILC2s secrete IL-13 to induce myeloid-derived suppressor cells and support tumour growth.

Peritoneal carcinomatosis (PC) is a rare disease defined as diffused implantation of neoplastic cells in the peritoneal cavity. This clinical picture occurs during the evolution of peritoneal tumors, and it is the main cause of morbidity and mortality of patients affected by these pathologies, though cytoreductive surgery with heated intra-peritoneal chemotherapy (CRS/HIPEC) is yielding promising results. In the present study, we evaluated whether the tumor microenvironment of low-grade and high-grade PC could affect the phenotypic and functional features and thus the anti-tumor potential of NK cells. We show that while in the peritoneal fluid (PF) of low-grade PC most CD56dim NK cells show a relatively immature phenotype (NKG2A+KIR–CD57–CD16dim), in the PF of high-grade PC NK cells are, in large majority, mature (CD56dimKIR+CD57+CD16bright). Furthermore, in low-grade PC, PF-NK cells are characterized by a sharp down-regulation of some activating receptors, primarily NKp30 and DNAM-1, while, in high-grade PC, PF-NK cells display a higher expression of the PD-1 inhibitory checkpoint. The compromised phenotype observed in low-grade PC patients corresponds to a functional impairment. On the other hand, in the high-grade PC patients PF-NK cells show much more important defects that only partially reflect the compromised phenotype detected. These data suggest that the PC microenvironment may contribute to tumor escape from immune surveillance by inducing different NK cell impaired features leading to altered anti-tumor activity. Notably, after CRS/HIPEC treatment, the altered NK cell phenotype of a patient with a low-grade disease and favorable prognosis was reverted to a normal one. Our present data offer a clue for the development of new immunotherapeutic strategies capable of restoring the NK-mediated anti-tumor responses in association with the CRS/HIPEC treatment to increase the effectiveness of the current therapy.

Natural killer (NK) cells were originally defined as effector lymphocytes of innate immunity characterized by the unique ability of killing tumor and virally infected cells without any prior priming and expansion of specific clones. The “missing-self” theory, proposed by Klas Karre, the seminal discovery of the first prototypic HLA class I-specific inhibitory receptors, and, later, of the Natural Cytotoxicity Receptors (NCRs) by Alessandro Moretta, provided the bases to understand the puzzling behavior of NK cells. Actually, those discoveries proved crucial also for many of the achievements that, along the years, have contributed to the modern view of these cells. Indeed, NK cells, besides killing susceptible targets, are now known to functionally interact with different immune cells, sense pathogens using TLR, adapt their responses to the local environment, and, even, mount a sort of immunological memory. In this review, we will specifically focus on the main activating NK receptors and on their crucial role in the ever-increasing number of functions assigned to NK cells and other innate lymphoid cells (ILCs).

The identification of inhibitory NK cell receptors specific for HLA-I molecules (KIRs and NKG2A) provided the molecular basis for clarifying the mechanism by which NK cells kill transformed cells while sparing normal cells. The direct interactions between inhibitory NK cell receptors and their HLA-I ligands enable NK cells to distinguish healthy from transformed cells, which frequently show an altered expression of HLA-I molecules. Indeed, NK cells can kill cancer cells that have lost, or under express, HLA-I molecules, but not cells maintaining their expression. In this last case, it is possible to use anti-KIR or anti-NKG2A monoclonal antibodies to block the inhibitory signals generated by these receptors and to restore the anti-tumor NK cell activity. These treatments fall within the context of the new immunotherapeutic strategies known as “immune checkpoint blockade.” These antibodies are currently used in clinical trials in the treatment of both hematological and solid tumors. However, a more complex scenario has recently emerged. For example, NK cells can also express additional immune checkpoints, including PD-1, that was originally described on T lymphocytes, and whose ligands (PD-Ls) are usually overexpressed on tumor cells. Thus, it appears that the activation of NK cells and their potentially harmful effector functions are under the control of different immune checkpoints and their simultaneous expression could provide additional levels of suppression to anti-tumor NK cell responses. This review is focused on PD-1 immune checkpoint in NK cells, its potential role in immunosuppression, and the therapeutic strategies to recover NK cell cytotoxicity and anti-tumor effect.

Under physiological conditions, PD-1/PD-L1 interactions regulate unwanted over-reactions of immune cells and contribute to maintain peripheral tolerance. However, in tumor microenvironment, this interaction may greatly compromise the immune-mediated anti-tumor activity. PD-1+ NK cells have been detected in high percentage in peripheral blood and ascitic fluid of ovarian carcinoma patients. To acquire information on PD-1 expression and physiology in human NK cells, we analyzed whether PD-1 mRNA and protein are present in resting, surface PD-1−, NK cells from healthy donors. Both different splicing isoforms of PD-1 mRNA and a cytoplasmic pool of PD-1 protein were detected. Similar results were obtained also from both in vitro-activated and tumor-associated NK cells. PD-1 mRNA and protein were higher in CD56dim than in CD56bright NK cells. Confocal microscopy analyses revealed that PD-1 protein is present in virtually all NK cells analyzed. The present findings are compatible with a rapid surface expression of PD-1 in NK cells in response to appropriate, still undefined, stimuli.

Natural killer cells are cytotoxic innate lymphoid cells that play an important role for early host defenses against infectious pathogens and surveillance against tumor. In humans, NK cells may be divided in various subsets on the basis of the relative CD56 expression and of the low-affinity FcγRIIIA CD16. In particular, the two main NK cell subsets are represented by the CD56bright/CD16−/dim and the CD56dim/CD16bright NK cells. Experimental evidences indicate that CD56bright and CD56dim NK cells represent different maturative stages of the NK cell developmental pathway. We identified multiple miRNAs differentially expressed in CD56bright/CD16− and CD56dim/CD16bright NK cells using both univariate and multivariate analyses. Among these, we found a few miRNAs with a consistent differential expression in the two NK cell subsets, and with an intermediate expression in the CD56bright/CD16dim NK cell subset, representing a transitional step of maturation of NK cells. These analyses allowed us to establish the existence of a miRNA signature able to efficiently discriminate the two main NK cell subsets regardless of their surface phenotype. In addition, by analyzing the putative targets of representative miRNAs we show that hsa-miR-146a-5p, may be involved in the regulation of killer Ig-like receptor (KIR) expression. These results contribute to a better understanding of the physiologic significance of miRNAs in the regulation of the development/function of human NK cells. Moreover, our results suggest that hsa-miR-146a-5p targeting, resulting in KIR down-regulation, may be exploited to generate/increment the effect of NK KIR-mismatching against HLA-class I+ tumor cells and thus improve the NK-mediated anti-tumor activity.

EC is the most common cancer in the female genital tract in developed countries, and with its increasing incidence due to risk factors, such as aging and obesity, tends to become a public health issue. Although EC is a hormone-dependent neoplasm, there are no recommendations for the determination of steroid hormone receptors in the tumor tissue and no hormone therapy has ever been assessed in the adjuvant setting. Furthermore, its immune environment has been slightly characterized, but recent evidences point out how EC microenvironment may increase self-tolerance by reducing the recruitment of cytotoxic immune cells to the tumor site and/or modifying their phenotype, making these cells no longer able to suppress tumor growth. Here we highlight insights for EC management from diagnosis to a desirable trend of personalized treatment.

Human NK cells can control tumor growth and metastatic spread thanks to their powerful cytolytic activity which relies on the expression of an array of activating receptors. Natural cytotoxicity receptors (NCRs) NKG2D and DNAM-1 are those non-HLA-specific activating NK receptors that are mainly involved in sensing tumor transformation by the recognition of different ligands, often stress-induced molecules, on the surface of cancer cells. Tumors display several mechanisms aimed at dampening/evading NK-mediated responses, a relevant fraction of which is based on the downregulation of the expression of activating receptors and/or their ligands. In this review, we summarize the role of the main non-HLA-specific activating NK receptors, NCRs, NKG2D and DNAM-1, in controlling tumor growth and metastatic spread in solid malignancies affecting the gastrointestinal tract with high incidence in the world population, i.e., pancreatic ductal adenocarcinoma (PDAC), colorectal cancer (CRC), and gastric cancer (GC), also describing the phenotypic and functional alterations induced on NK cells by their tumor microenvironment.

Immune checkpoints refer to a plethora of inhibitory pathways of the immune system that play a crucial role in maintaining self-tolerance and in tuning the duration and amplitude of physiological immune responses to minimize collateral tissue damages. The breakdown of this delicate balance leads to pathological conditions, including cancer. Indeed, tumor cells can develop multiple mechanisms to escape from immune system defense, including the activation of immune checkpoint pathways. The development of monoclonal antibodies, targeting inhibitory immune checkpoints, has provided an immense breakthrough in cancer therapy. Immune checkpoint inhibitors (ICI), initially developed to reverse functional exhaustion in T cells, recently emerged as important actors in natural killer (NK)-cell-based immunotherapy. Moreover, the discovery that also helper innate lymphoid cells (ILCs) express inhibitory immune checkpoints, suggests that these molecules might be targeted on ILCs, to modulate their functions in the tumor microenvironment. Recently, other strategies to achieve immune checkpoint blockade have been developed, including miRNA exploiting systems. Herein, we provide an overview of the current knowledge on inhibitory immune checkpoints on NK cells and ILCs and we discuss how to target these innate lymphocytes by ICI in both solid tumors and hematological malignancies.

For many years, the therapeutic advances in gynecological neoplasms have remained steady, however, in recent years, the application of the most modern “-omics” sciences has shed light on the pathogenesis and on neoplastic progression, with important implications in the introduction of targeted treatments that are more effective and less toxic [...]

Natural killer cells are the first lymphocyte population to reconstitute early after non-myeloablative and T cell-replete haploidentical hematopoietic stem cell transplantation with post-transplant infusion of cyclophosphamide. The study herein characterizes the transient and predominant expansion starting from the second week following haploidentical hematopoietic stem cell transplantation of a donor-derived unconventional subset of NKp46neg-low/CD56dim/CD16neg natural killer cells expressing remarkably high levels of CD94/NKG2A. Both transcription and phenotypic profiles indicated that unconventional NKp46neg-low/CD56dim/CD16neg cells are a distinct natural killer cell subpopulation with features of late stage differentiation, yet retaining proliferative capability and functional plasticity to generate conventional NKp46pos/CD56bright/CD16neg-low cells in response to interleukin-15 plus interleukin-18. While present at low frequency in healthy donors, unconventional NKp46neg-low/CD56dim/CD16neg cells are greatly expanded in the seven weeks following haploidentical hematopoietic stem cell transplantation, and express high levels of the activating receptors NKG2D and NKp30 as well as of the lytic granules Granzyme-B and Perforin. Nonetheless, NKp46neg-low/CD56dim/CD16neg cells displayed a markedly defective cytotoxicity that could be reversed by blocking the inhibitory receptor CD94/NKG2A. These data open new and important perspectives to better understand the ontogenesis/homeostasis of human natural killer cells and to develop a novel immune-therapeutic approach that targets the inhibitory NKG2A check-point, thus unleashing natural killer cell alloreactivity early after haploidentical hematopoietic stem cell transplantation.

Mutations of the recombinase-activating genes 1 and 2 (RAG1 and RAG2) in humans are associated with a broad range of phenotypes. For patients with severe clinical presentation, hematopoietic stem cell transplantation (HSCT) represents the only curative treatment; however, high rates of graft failure and incomplete immune reconstitution have been observed, especially after unconditioned haploidentical transplantation. Studies in mice have shown that Rag−/− natural killer (NK) cells have a mature phenotype, reduced fitness, and increased cytotoxicity. We aimed to analyze NK cell phenotype and function in patients with mutations in RAG and in non-homologous end joining (NHEJ) genes. Here, we provide evidence that NK cells from these patients have an immature phenotype, with significant expansion of CD56bright CD16−/int CD57− cells, yet increased degranulation and high perforin content. Correlation was observed between in vitro recombinase activity of the mutant proteins, NK cell abnormalities, and in vivo clinical phenotype. Addition of serotherapy in the conditioning regimen, with the aim of depleting the autologous NK cell compartment, may be important to facilitate engraftment and immune reconstitution in patients with RAG and NHEJ defects treated by HSCT.

Previous studies suggested that the cross talk between NK cells and other cell types is crucial for the regulation of both innate and adaptive immune responses. In the present study, we analyzed the phenotypic and functional outcome of the interaction between resting or cytokine-activated NK cells and eosinophils derived from non-atopic donors. Our results provide the first evidence that a natural cytotoxicity receptor (NCR)/NCR ligand-dependent cross talk between NK cells and eosinophils may be important to upregulate the activation state and the effector function of cytokine-primed NK cells. This interaction also promotes the NK-mediated editing process of dendritic cells that influence the process of Th1 polarization. In turn, this cross talk also resulted in eosinophil activation and acquisition of the characteristic features of antigen-presenting cells. At higher NK/eosinophil ratios, cytokine-primed NK cells were found to kill eosinophils via NKp46 and NKp30, thus suggesting a potential immunoregulatory role for NK cells in dampening inflammatory responses involving eosinophils.

In the present study, we report an extremely rare case of a 31-year-old woman with neuroblastoma arising in an ovarian cystic teratoma. We analyzed the expression of activating receptors on natural killer (NK) cells derived from the patient's peripheral blood and peritoneal fluid. In addition, we investigated the presence of specific ligands recognized by different NK cell receptors on tumor cells. We show that NK cells isolated from peritoneal fluid expressed certain triggering receptors including DNAM-1 (CD226) and CD16 with lower intensity as compared to peripheral blood NK cells. Remarkably, at variance with most cases of childhood neuroblastoma, the tumor cells from this patient expressed substantial amounts of HLA class-I molecules. These molecules are known to be protective against NK cell-mediated lysis. In addition, neuroblastoma cells expressed B7-H3 (CD276), another surface molecule that inhibits NK cell function. Finally, this tumor did not express the PVR (CD155) and nectin-2 (CD112) ligands for the DNAM-1 activating NK receptor, which plays a crucial role in NK/neuroblastoma interactions. Altogether, these findings indicate that the neuroblastoma cells of this patient express an NK-resistant surface phenotype, which is at least in part similar to that previously described in a fraction of childhood neuroblastoma.

There is increasing evidence that natural killer (NK) cells exhibit regulatory features. Among them, CD56bright natural killer cells have been suggested to play a major role in controlling T-cell responses and maintaining homeostasis. Dysfunction in NK cell-mediated regulatory features has been recently described in untreated multiple sclerosis (MS), suggesting a contribution to MS pathogenesis. Moreover, biological disease-modifying treatments effective in MS apparently enhance the frequencies and/or regulatory function of NK cells, further pointing toward an immuno-protective role of NK cells in MS. Here we summarize the current knowledge on the regulatory functions of NK cells, based on their interactions with other cells belonging to the innate compartment, as well as with adaptive effector cells. We review the more recent data reporting disruption of NK-cell/T-cell interactions in MS, and discuss how disease-modifying treatments for MS affect NK cells.

Innate and adaptive immunity has evolved complex molecular mechanisms regulating immune cell migration to facilitate the dynamic cellular interactions required for its function involving the chemokines and their receptors.One important chemokine receptor in the immune system is represented by CCR7. Together with its ligands CCL19 and CCL21, this chemokine receptor controls different arrays of migratory events, both in innate and adaptive immunity, including homing of CD56bright NK cells, T cells and DCs to lymphoid compartments where T cell priming occurs. Only recently, a key role for CCR7 in promoting CD56dim NK cell migration towards lymphoid tissues has been described. Remarkably, this event can influence the shaping and polarization of adaptive T cell responses.In this review, we describe recent progress in understanding the mechanisms and the site where CD56dim KIR+ NK cells can acquire the capability to migrate towards lymph nodes. The emerging significance of this event in clinical transplantation is also discussed.

The crosstalk between cancer cells and host cells is a crucial prerequisite for tumor growth and progression. The cells from both the innate and adaptive immune systems enter into a perverse relationship with tumor cells to create a tumor-promoting and immunosuppressive tumor microenvironment (TME). Epithelial ovarian cancer (EOC), the most lethal of all gynecological malignancies, is characterized by a unique TME that paves the way to the formation of metastasis and mediates therapy resistance through the deregulation of immune surveillance. A characteristic feature of the ovarian cancer TME is the ascites/peritoneal fluid, a malignancy-associated effusion occurring at more advanced stages, which enables the peritoneal dissemination of tumor cells and the formation of metastasis. The standard therapy for EOC involves a combination of debulking surgery and platinum-based chemotherapy. However, most patients experience disease recurrence. New therapeutic strategies are needed to improve the prognosis of patients with advanced EOC. Harnessing the body’s natural immune defenses against cancer in the form of immunotherapy is emerging as an innovative treatment strategy. NK cells have attracted attention as a promising cancer immunotherapeutic target due to their ability to kill malignant cells and avoid healthy cells. Here, we will discuss the recent advances in the clinical application of NK cell immunotherapy in EOC.

Primary infection with the human oncogenic Epstein-Barr virus (EBV) can result in infectious mononucleosis (IM), a self-limiting disease caused by massive lymphocyte expansion that predisposes for the development of distinct EBV-associated lymphomas. Why some individuals experience this symptomatic primary EBV infection, whereas the majority acquires the virus asymptomatically, remains unclear. Using a mouse model with reconstituted human immune system components, we show that depletion of human natural killer (NK) cells enhances IM symptoms and promotes EBV-associated tumorigenesis mainly because of a loss of immune control over lytic EBV infection. These data suggest that failure of innate immune control by human NK cells augments symptomatic lytic EBV infection, which drives lymphocyte expansion and predisposes for EBV-associated malignancies.

Human NK cells are distinguished into CD56brightCD16- cells and CD56dimCD16+ cells. These two subsets are conventionally associated with differential functional outcomes and are heterogeneous with respect to the expression of KIR and CD94/NKG2 heterodimers that represent the two major types of HLA class I-specific receptors. Recent studies indicated that immature CD56bright NK cells, homogeneously expressing the inhibitory CD94/NKG2A receptor, are precursors of CD56dim NK cells that in turn during their process of differentiation lose expression of CD94/NKG2A, and subsequentially acquire inhibitory KIRs and LIR-1. The terminally differentiated phenotype of CD56dim cells is marked by the expression of the CD57 molecule that is associated with poor responsiveness to cytokine stimulation, but retained cytolytic capacity.Remarkably, this CD56dimNKG2A-KIR+LIR-1+CD57+ NK cell subset when derived from individuals previously exposed to pathogens, such as HCMV, may contain memory-like NK cells. These cells are generally characterized by an up-regulation of the activating receptor CD94/NKG2C and a down-regulation of the inhibitory receptor Siglec-7. The memory-like NK cells are persistent over-time and display some hallmarks of adaptive immunity, i.e. clonal expansion, more effective anti-tumor and anti-viral immune responses, longevity as well as given epigenetic modifications. Interestingly, unknown co-factors associated to HCMV infection may induce the onset of a recently identified fully mature NK cell subset, characterized by marked downregulation of the activating receptors NKp30 and NKp46 and by the unexpected expression of the inhibitory PD-1 receptor. This phenotype correlates with an impaired anti-tumor NK cell activity that can be partially restored by antibody-mediated disruption of PD-1/PD-L interaction.

The KIR2DS4 receptor is the oldest KIR2DS expressed by human NK lymphocytes. The specificity of recognition of this receptor for various HLA class I alleles has been demonstrated; however it remains poorly understood whether these interactions may result in the activation of some specific functions in NK cells. Here, we examined the functional outcome of the KIR2DS4/HLA class I interaction by the use of an alternative functional system based on the ability of KIR2DS4 to regulate the mechanism of trogocytosis by NK cells. We demonstrate that KIR2DS4 can induce the uptake of CCR7 by KIR2DS4+ NKG2A+ NK cell clones after interacting with CCR7+ target cells expressing HLA-Cw4 and HLA-Cw6 alleles. However this interaction is not always sufficient to override the inhibition generated by NKG2A expressed on the same NK cells. The recognition of HLA-Cw4 was confirmed by experiments of cytotoxicity against HLA-C-transfected cells. We also show that, different from resting NK cells, the acquisition of CCR7 in response to IL-18 cannot occur in IL2-activated NK cells because of a marked downregulation in their IL-18Rα expression. As a consequence trogocytosis represents the major mechanism by which KIR2DS4+ activated NK cells acquire the expression of this chemokine receptor.

Understanding the cellular mechanisms that ensure an appropriate innate immune response against viral pathogens is an important challenge of biomedical research. In vitro studies have shown that natural killer (NK) cells purified from healthy donors can kill heterologous cell lines or autologous CD4+ T cell blasts exogenously infected with several strains of HIV-1. However, it is not known whether the deleterious effects of high HIV-1 viremia interferes with the NK cell-mediated cytolysis of autologous, endogenously HIV-1-infected CD4+ T cells. Here, we stimulate primary CD4+ T cells, purified ex vivo from HIV-1-infected viremic patients, with PHA and rIL2 (with or without rIL-7). This experimental procedure allows for the significant expansion and isolation of endogenously infected CD4+ T cell blasts detected by intracellular staining of p24 HIV-1 core antigen. We show that, subsequent to the selective down-modulation of MHC class-I (MHC-I) molecules, HIV-1-infected p24(pos) blasts become partially susceptible to lysis by rIL-2-activated NK cells, while uninfected p24(neg) blasts are spared from killing. This NK cell-mediated killing occurs mainly through the NKG2D activation pathway. However, the degree of NK cell cytolytic activity against autologous, endogenously HIV-1-infected CD4+ T cell blasts that down-modulate HLA-A and -B alleles and against heterologous MHC-I(neg) cell lines is particularly low. This phenomenon is associated with the defective surface expression and engagement of natural cytotoxicity receptors (NCRs) and with the high frequency of the anergic CD56(neg)/CD16(pos) subsets of highly dysfunctional NK cells from HIV-1-infected viremic patients. Collectively, our data demonstrate that the chronic viral replication of HIV-1 in infected individuals results in several phenotypic and functional aberrancies that interfere with the NK cell-mediated killing of autologous p24(pos) blasts derived from primary T cells.