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The Innate Lymphoid Cells (ILCs) are a family of innate immune cells composed by the Natural Killer (NK) cells and the helper ILCs (hILCs) (ILC1, ILC2, ILC3), both developing from a common ILC precursor (ILCP) derived from hematopoietic stem cells (HSCs). A correct ILC reconstitution is crucial, particularly in patients receiving HSC transplantation (HSCT), the only therapeutic option for many adult and pediatric high-risk hematological malignancies. Indeed, mainly thanks to their cytotoxic activity, NK cells have a strong Graft-versus-Leukemia (GvL) effect. On the other hand, hILCs, that are mainly tissue resident, are involved in tissue repair and homeostasis, Graft-versus-Host Disease (GvHD) prevention and immune response to infections. Unlike NK cell development, hILC-poiesis is still poorly characterized in humans. Here, we provide a protocol for the in vitro ILC differentiation from healthy donor peripheral blood-derived CD34 + HSCs. This could represent a useful model to dissect the molecular mechanisms by which the distinct ILC subsets are generated from ILCP leading to the development of novel strategies to improve the HSCT clinical outcome., (Copyright © 2025 Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Background: In early infected or severe coronavirus disease 2019 (COVID-19) patients, circulating NK cells are consistently reduced, despite being highly activated or exhausted. The aim of this paper was to establish whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein (SP) may directly trigger NK cells and through which receptor(s)., Methods: SP-stimulated human NK cells have been evaluated for the expression of activation markers, cytokine release, and cytotoxic activity, as well as for gene expression profiles and NF-kB phosphorylation, and they have been silenced with specific small interfering RNAs., Results: SPs from the Wuhan strain and other variants of concern (VOCs) directly bind and stimulate purified NK cells by increasing activation marker expression, cytokine release, and cytolytic activity, prevalently in the CD56 bright NK cell subset. VOC-SPs differ in their ability to activate NK cells, G614, and Delta-Plus strains providing the strongest activity in the majority of donors. While VOC-SPs do not trigger ACE2, which is not expressed on NK cells, or other activating receptors, they directly and variably bind to both Toll-like receptor 2 (TLR2) and TLR4. Moreover, SP-driven NK cell functions are inhibited upon masking such receptors or silencing the relative genes. Lastly, VOC-SPs upregulate CD56 dim NK cell functions in COVID-19 recovered, but not in non-infected, individuals., Conclusions: TLR2 and TLR4 are novel activating receptors for SP in NK cells, suggesting a new role of these cells in orchestrating the pathophysiology of SARS-CoV-2 infection. The pathogenic relevance of this finding is highlighted by the fact that free SP providing NK cell activation is frequently detected in a SARS-CoV-2 inflamed environment and in plasma of infected and long-COVID-19 subjects., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Landolina, Ricci, Veneziani, Alicata, Mariotti, Pelosi, Quatrini, Mortari, Carsetti, Vacca, Tumino, Azzarone, Moretta and Maggi.)

Natural Killer (NK) cells are important components of the immune system in the defense against tumor growth and metastasis. They release exosomes containing proteins and nucleic acids, including microRNAs (miRNAs). NK-derived exosomes play a role in the anti-tumor NK cell function since they are able to recognize and kill cancer cells. However, the involvement of exosomal miRNAs in the function of NK exosomes is poorly understood. In this study, we explored the miRNA content of NK exosomes by microarray as compared to their cellular counterparts. The expression of selected miRNAs and lytic potential of NK exosomes against childhood B acute lymphoblastic leukemia cells after co-cultures with pancreatic cancer cells were also evaluated. We identified a small subset of miRNAs, including miR-16-5p, miR-342-3p, miR-24-3p, miR-92a-3p and let-7b-5p that is highly expressed in NK exosomes. Moreover, we provide evidence that NK exosomes efficiently increase let-7b-5p expression in pancreatic cancer cells and induce inhibition of cell proliferation by targeting the cell cycle regulator CDK6. Let-7b-5p transfer by NK exosomes could represent a novel mechanism by which NK cells counteract tumor growth. However, both cytolytic activity and miRNA content of NK exosomes were reduced upon co-culture with pancreatic cancer cells. Alteration in the miRNA cargo of NK exosomes, together with their reduced cytotoxic activity, could represent another strategy exerted by cancer to evade the immune response. Our study provides new information on the molecular mechanisms used by NK exosomes to exert anti-tumor-activity and offers new clues to integrate cancer treatments with NK exosomes., Competing Interests: No potential conflict of interest was reported by the author(s)., (© 2023 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Innate lymphoid cells (ILCs) comprise cytotoxic NK cells and helper-ILCs, which are further divided in ILC1, ILC2, and ILC3. Helper-ILCs mirror the effector functions of helper T-cell subsets and contribute to host immune defense, tissue homeostasis and repair through cytokine secretion. Although they are mainly tissue-resident, helper ILCs are also found in the peripheral blood (PB). In the human setting, it may be needed to analyze circulating helper ILCs to compare pathological to physiological conditions. In this review, we provide simple guidelines and a list of markers useful to study human PB helper ILCs phenotype and function by flow cytometry., (© 2023 The Authors. Cytometry Part A published by Wiley Periodicals LLC on behalf of International Society for Advancement of Cytometry.)

Monoclonal antibodies that target specific ligand-receptor signaling pathways and act as immune checkpoint inhibitors have been designed to remove the brakes in T cells and restore strong and long-term antitumor-immunity. Of note, many of these inhibitory receptors are also expressed by Innate Lymphoid Cells (ILCs), suggesting that also blockade of inhibitory pathways in innate lymphocytes has a role in the response to the treatment with checkpoint inhibitors. ILCs comprise cytotoxic NK cells and "helper" subsets and are important cellular components in the tumor microenvironment. In addition to killing tumor cells, ILCs release inflammatory cytokines, thus contributing to shape adaptive cell activation in the context of immunotherapy. Therefore, ILCs play both a direct and indirect role in the response to checkpoint blockade. Understanding the impact of ILC-mediated response on the treatment outcome would contribute to enhance immunotherapy efficacy, as still numerous patients resist or relapse., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Different programmed cell death-ligand 1 (PD-L1) assays and scoring algorithms are being used in the evaluation of PD-L1 expression for the selection of patients for immunotherapy in specific settings of advanced urothelial carcinoma (UC). In this paper, we sought to investigate three approved assays (Ventana SP142 and SP263, and Dako 22C3) in UC with emphasis on implications for patient selection for atezolizumab/pembrolizumab as the first line of treatment. Tumors from 124 patients with invasive UC of the bladder were analyzed using tissue microarrays (TMA). Serial sections were stained with SP263 and SP142 on Ventana Benchmark Ultra and with 22C3 on Dako Autostainer Link 48. Stains were evaluated independently by two observers and scored using the combined positive score (CPS) and tumor infiltrating immune cells (IC) algorithms. Differences in proportions (DP), overall percent agreement (OPA), positive percent agreement (PPA), negative percent agreement (NPA), and Cohen κ were calculated for all comparable cases. Good overall concordance in analytic performance was observed for 22C3 and SP263 with both scoring algorithms; specifically, the highest OPA was observed between 22C3 and SP263 (89.6%) when using CPS. On the other hand, SP142 consistently showed lower positivity rates with high differences in proportions (DP) compared with 22C3 and SP263 with both CPS and IC, and with a low PPA, especially when using the CPS algorithm. In conclusion, 22C3 and SP263 assays show comparable analytical performance while SP142 shows divergent staining results, with important implications for the selection of patients for both pembrolizumab and atezolizumab., Competing Interests: FC was Chair of the Scientific and Medical Advisory Board of TRIBVN Healthcare, France, and received advisory board fees from TRIBVN Healthcare, France in the last five years. He is shareholder of Aiosyn BV, the Netherlands. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Munari, Querzoli, Brunelli, Marconi, Sommaggio, Cocchi, Martignoni, Netto, Caliò, Quatrini, Mariotti, Luchini, Girolami, Eccher, Segala, Ciompi, Zamboni, Moretta and Bogina.)

Background: Innate lymphoid cells (ILCs) comprise cytotoxic natural killer (NK) cells and helper ILCs (hILCs). Human hILC development is less characterized as compared with that of NK cells, although all ILCs are developmentally related. It has been reported that the immunosuppressive drugs glucocorticoids (GCs) regulate ILC function, but whether they control ILC differentiation from hematopoietic stem cells (HSCs) is unknown., Objectives: This study sought to analyze the effect of GCs on ILC development from HSCs., Methods: This study exploited an in vitro system to generate and expand from peripheral blood HSCs a multipotent CD56 + ILC precursor able to differentiate into NK cells, ILC1s, and ILC3s. We also analyzed ex vivo, at different time points, the peripheral blood of recipients of allogeneic HSC transplantation who were or were not treated with GCs and compared ILC subset reconstitution., Results: Invitro, GCs favor the generation of NK cells from myeloid precursors, while they strongly impair lymphoid development. In support of these data, recipients of HSC transplantation who had been treated with GCs display a lower number of circulating hILCs, including the ILC precursor (ILCP) previously identified as a systemic substrate for tissue ILC differentiation., Conclusions: GCs impair the development of the CD117 + ILCP from CD34 + HSCs, while they do not affect the further steps of ILCP differentiation toward NK cells and hILC subsets. This reflects an association of GC treatment with a marked reduction of circulating hILCs in the recipients of HSC transplantation., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Tumor microenvironment (TME) includes a wide variety of cell types and soluble factors capable of suppressing immune-responses. While the role of NK cells in TME has been analyzed, limited information is available on the presence and the effect of polymorphonuclear (PMN) myeloid-derived suppressor cells, (MDSC). Among the immunomodulatory cells present in TME, MDSC are potentially efficient in counteracting the anti-tumor activity of several effector cells. We show that PMN-MDSC are present in high numbers in the PB of patients with primary or metastatic lung tumor. Their frequency correlated with the overall survival of patients. In addition, it inversely correlated with low frequencies of NK cells both in the PB and in tumor lesions. Moreover, such NK cells displayed an impaired anti-tumor activity, even those isolated from PB. The compromised function of NK cells was consequent to their interaction with PMN-MDSC. Indeed, we show that the expression of major activating NK receptors, the NK cytolytic activity and the cytokine production were inhibited upon co-culture with PMN-MDSC through both cell-to-cell contact and soluble factors. In this context, we show that exosomes derived from PMN-MDSC are responsible of a significant immunosuppressive effect on NK cell-mediated anti-tumor activity. Our data may provide a novel useful tool to implement the tumor immunoscore. Indeed, the detection of PMN-MDSC in the PB may be of prognostic value, providing clues on the presence and extension of both adult and pediatric tumors and information on the efficacy not only of immune response but also of immunotherapy and, possibly, on the clinical outcome., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Tumino, Besi, Martini, Di Pace, Munari, Quatrini, Pelosi, Fiore, Fiscon, Paci, Scordamaglia, Covesnon, Bogina, Mingari, Moretta and Vacca.)

Cells of the innate immunity play an important role in tumor immunotherapy. Thus, NK cells can control tumor growth and metastatic spread. Thanks to their strong cytolytic activity against tumors, different approaches have been developed for exploiting/harnessing their function in patients with leukemia or solid tumors. Pioneering trials were based on the adoptive transfer of autologous NK cell-enriched cell populations that were expanded in vitro and co-infused with IL-2. Although relevant results were obtained in patients with advanced melanoma, the effect was mostly limited to certain metastatic localizations, particularly to the lung. In addition, the severe IL-2-related toxicity and the preferential IL-2-induced expansion of Treg limited this type of approach. This limitation may be overcome by the use of IL-15, particularly of modified IL-15 molecules to improve its half-life and optimize the biological effects. Other approaches to harness NK cell function include stimulation via TLR, the use of bi- and tri-specific NK cell engagers (BiKE and TriKE) linking activating NK receptors (e.g. CD16) to tumor-associated antigens and even incorporating an IL-15 moiety (TriKE). As recently shown, in tumor patients, NK cells may also express inhibitory checkpoints, primarily PD-1. Accordingly, the therapeutic use of checkpoint inhibitors may unleash NK cells against PD-L1 + tumors. This effect may be predominant and crucial in tumors that have lost HLA cl-I expression, thus resulting "invisible" to T lymphocytes. Additional approaches in which NK cells may represent an important tool for cancer therapy, are to exploit the unique properties of the "adaptive" NK cells. These CD57 + NKG2C + cells, despite their mature stage and a potent cytolytic activity, maintain a strong proliferating capacity. This property revealed to be crucial in hematopoietic stem cell transplantation (HSCT), particularly in the haplo-HSCT setting, to cure high-risk leukemias. T depleted haplo-HSCT (e.g. from one of the parents) allowed to save the life of thousands of patients lacking a HLA-compatible donor. In this setting, NK cells have been shown to play an essential role against leukemia cells and infections. Another major advance is represented by chimeric antigen receptor (CAR)-engineered NK cells. CAR-NK, different from CAR-T cells, may be obtained from allogeneic donors since they do not cause GvHD. Accordingly, they may represent "off-the-shelf" products to promptly treat tumor patients, with affordable costs. Different from NK cells, helper ILC (ILC1, ILC2 and ILC3), the innate counterpart of T helper cell subsets, remain rather ambiguous with respect to their anti-tumor activity. A possible exception is represented by a subset of ILC3: their frequency in peri-tumoral tissues in patients with NSCLC directly correlates with a better prognosis, possibly reflecting their ability to contribute to the organization of tertiary lymphoid structures, an important site of T cell-mediated anti-tumor responses. It is conceivable that innate immunity may significantly contribute to the major advances that immunotherapy has ensured and will continue to ensure to the cure of cancer., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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., (© 2021 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

Through the release of hormones, the neuro-endocrine system regulates the immune system function promoting adaptation of the organism to the external environment and to intrinsic physiological changes. Glucocorticoids (GCs) and sex hormones not only regulate immune responses, but also control the hematopoietic stem cell (HSC) differentiation and subsequent maturation of immune cell subsets. During the development of an organism, this regulation has long-term consequences. Indeed, the effects of GC exposure during the perinatal period become evident in the adulthood. Analogously, in the context of HSC transplantation (HSCT), the immune system development starts de novo from the donor HSCs. In this review, we summarize the effects of GCs and sex hormones on the regulation of HSC, as well as of adaptive and innate immune cells. Moreover, we discuss the short and long-term implications on hematopoiesis of sex steroid ablation and synthetic GC administration upon HSCT., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Quatrini, Ricci, Ciancaglini, Tumino and Moretta.)

The immune infiltrate within tumors has proved to be very powerful in the prognostic stratification of patients and much attention is also being paid towards its predictive value. In this work we therefore aimed at clarifying the significance and impact of PD-L1 and PD-1 expression on the prognostic value of CD8 + tumor infiltrating lymphocytes (TILs) in a cohort of consecutive patients with primary resected non-small cell lung cancer (NSCLC). Tissue microarrays (TMA) were built using one representative formalin fixed paraffin embedded block for every case, with 5 cores for each block. TMA sections were stained with PD-L1 (clone SP263), PD-1 (clone NAT105) and CD8 (clone SP57). Number of CD8 + cells per mm 2  were automatically counted; median, 25 th and 75 th percentiles of CD8 + cells were used as threshold for statistical clinical outcome analysis and evaluated in patients subgroups defined by expression of PD-L1 and PD-1 within tumors. We found an overall strong prognostic value of CD8 + cells in our cohort of 314 resected NSCLC, especially in PD-L1 negative tumors lacking PD-1 + TILs, and demonstrated that in PD-L1 positive tumors a higher density of CD8 + lymphocytes is necessary to improve the prognosis. Our data strengthen the concept of the importance of the assessment and quantification of the immune contexture in cancer and, similarly to what has been carried on in colorectal cancer, promote the efforts for the establishment of an Immunoscore for NSCLC for prognostic and possibly predictive purposes., Competing Interests: FC is a consultant and member of the scientific advisory board of TRIBVN Healthcare, France. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Munari, Marconi, Querzoli, Lunardi, Bertoglio, Ciompi, Tosadori, Eccher, Tumino, Quatrini, Vacca, Rossi, Cavazza, Martignoni, Brunelli, Netto, Moretta, Zamboni and Bogina.)

Host protection against cutaneous herpes simplex virus 1 (HSV-1) infection relies on the induction of a robust adaptive immune response. Here, we show that Nav 1.8 + sensory neurons, which are involved in pain perception, control the magnitude of CD8 T cell priming and expansion in HSV-1-infected mice. The ablation of Nav 1.8 -expressing sensory neurons is associated with extensive skin lesions characterized by enhanced inflammatory cytokine and chemokine production. Mechanistically, Nav 1.8 + sensory neurons are required for the downregulation of neutrophil infiltration in the skin after viral clearance to limit the severity of tissue damage and restore skin homeostasis, as well as for eliciting robust CD8 T cell priming in skin-draining lymph nodes by controlling dendritic cell responses. Collectively, our data reveal an important role for the sensory nervous system in regulating both innate and adaptive immune responses to viral infection, thereby opening up possibilities for new therapeutic strategies.

Immune evasion is a key strategy adopted by tumor cells to escape the immune system while promoting their survival and metastatic spreading. Indeed, several mechanisms have been developed by tumors to inhibit immune responses. PD-1 is a cell surface inhibitory receptor, which plays a major physiological role in the maintenance of peripheral tolerance. In pathological conditions, activation of the PD-1/PD-Ls signaling pathway may block immune cell activation, a mechanism exploited by tumor cells to evade the antitumor immune control. Targeting the PD-1/PD-L1 axis has represented a major breakthrough in cancer treatment. Indeed, the success of PD-1 blockade immunotherapies represents an unprecedented success in the treatment of different cancer types. To improve the therapeutic efficacy, a deeper understanding of the mechanisms regulating PD-1 expression and signaling in the tumor context is required. We provide an overview of the current knowledge of PD-1 expression on both tumor-infiltrating T and NK cells, summarizing the recent evidence on the stimuli regulating its expression. We also highlight perspectives and limitations of the role of PD-L1 expression as a predictive marker, discuss well-established and novel potential approaches to improve patient selection and clinical outcome and summarize current indications for anti-PD1/PD-L1 immunotherapy.

A recent approach of hematopoietic stem cell (HSC) transplantation from haploidentical donors "mobilized" with G-CSF is based on the selective depletion of αβ T and B lymphocytes from the graft. Through this approach, the patient receives both HSC and mature donor-derived effector cells (including NK cells), which exert both anti-leukemia activity and protection against infections. We previously showed that donor HSC mobilization with G-CSF results in accumulation in the graft of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs), capable of inhibiting in vitro the anti-leukemia activity of allogeneic NK cells. Here, we performed a detailed gene expression analysis on NK cells and PMN-MDSCs both derived from mobilized graft. Cytotoxicity assays and real time PCR arrays were performed in NK cells. Microarray technology followed by bioinformatics analysis was used for gene expression profiling in PMN-MDSCs. Results indicate that NK cells from the graft have a lower cytolytic activity as compared to those from non-mobilized samples. Further, mobilized PMN-MDSCs displayed a peculiar transcriptional profile distinguishing them from non-mobilized ones. Differential expression of pro-proliferative and immune-modulatory genes was detected in mobilized PMN-MDSCs. These data strengthen the concept that G-CSF-mobilized PMN-MDSCs present in the graft display unique molecular characteristics, in line with the strong inhibitory effect on donor NK cells., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Pelosi, Besi, Tumino, Merli, Quatrini, Li Pira, Algeri, Moretta and Vacca.)

Myeloid derived suppressor cells (MDSC) are heterogeneous populations that through the release of soluble factors and/or by cell-to-cell interactions suppress both innate and adaptive immune effector cells. In pathological conditions, characterized by the presence of inflammation, a partial block in the differentiation potential of myeloid precursors causes an accumulation of these immunosuppressive cell subsets both in peripheral blood and in tissues. On the contrary, NK cells represent a major player of innate immunity able to counteract tumor growth. The anti-tumor activity of NK cells is primarily related to their cytolytic potential and to the secretion of soluble factors or cytokines that may act on tumors either directly or indirectly upon the recruitment of other cell types. NK cells have been shown to play a fundamental role in haploidentical hemopoietic stem cell transplantation (HSCT), for the therapy of high-risk leukemias. A deeper analysis of MDSC functional effects demonstrated that these cells are capable, through several mechanisms, to reduce the potent GvL activity exerted by NK cells. It is conceivable that, in this transplantation setting, the MDSC-removal or -inactivation may represent a promising strategy to restore the anti-leukemia effect mediated by NK cells. Thus, a better knowledge of the cellular interactions occurring in the tumor microenvironment could promote the development of novel therapeutic strategies for the treatment of solid and hematological malignances., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Tumino, Di Pace, Besi, Quatrini, Vacca and Moretta.)

Glucocorticoids (GCs) are endogenous hormones that are crucial for the homeostasis of the organism and adaptation to the external environment. Because of their anti-inflammatory effects, synthetic GCs are also extensively used in clinical practice. However, almost all cells in the body are sensitive to GC regulation. As a result, these mediators have pleiotropic effects, which may be undesirable or detrimental to human health. Here, we summarize the recent findings that contribute to deciphering the molecular mechanisms downstream of glucocorticoid receptor activation. We also discuss the complex role of GCs in infectious diseases such as sepsis and COVID-19, in which the balance between pathogen elimination and protection against excessive inflammation and immunopathology needs to be tightly regulated. An understanding of the cell type- and context-specific actions of GCs from the molecular to the organismal level would help to optimize their therapeutic use.

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., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

In the last years, immunotherapy with antibodies against programmed cell death protein 1 (PD-1) and programmed death-ligand 1 (PD-L1) has shown remarkable efficacy in the treatment of different types of tumours, representing a true revolution in oncology. While its efficacy has initially been attributed only to unleashing T cell responses, responsivity to PD-1/PD-L1 blockade was observed in some tumours with low Human Leukocyte Antigen (HLA) I expression and increasing evidence has revealed PD-1 surface expression and inhibitory function also in natural killer (NK) cells. Thus, the contribution of anti-PD-1/PD-L1 therapy to the recovery of NK cell anti-tumour response has recently been appreciated. Here, we summarize the studies investigating PD-1 expression and function in NK cells, together with the limitations and perspectives of immunotherapies. A better understanding of checkpoint biology is needed to design next-generation therapeutic strategies and to improve the clinical protocols of current therapies.

Helper Innate Lymphoid Cells (hILCs), including ILC1s, ILC2s, and ILC3s, are mainly localized at the mucosal barriers where they play an important role in tissue regeneration and homeostasis through the secretion of specific sets of cytokines. The recent identification of a circulating ILC precursor able to generate all ILC mature subsets in physiological conditions, suggests that "ILC-poiesis" may be important in the context of hematopoietic stem cell transplantation (HSCT). Indeed, in HSCT the conditioning regimen (chemotherapy and radiotherapy) and Graft vs Host Disease (GvHD) may cause severe damages to mucosal tissues. Therefore, it is conceivable that rapid reconstitution of the hILC compartment may be beneficial in HSCT, by promoting mucosal tissue repair/regeneration and providing protection from opportunistic infections. In this review, we will summarize the evidence for a role of hILCs in allogenic HSCT for the treatment of hematological malignancies in all its steps, from the preparative regimen to the immune reconstitution in the recipient. The protective properties of hILCs at the mucosal barrier interfaces make them an attractive target to exploit in future cellular therapies aimed at improving allogenic HSCT outcome., (Copyright © 2020 Quatrini, Tumino, Moretta, Besi, Vacca and Moretta.)

The highly destructive mechanisms by which the immune system faces microbial infections is under the control of a series of inhibitory receptors. While most of these receptors prevent unwanted/excessive responses of individual effector cells, others play a more general role in immunity, acting as true inhibitory checkpoints controlling both innate and adaptive immunity. Regarding human NK cells, their function is finely regulated by HLA-class I-specific inhibitory receptors which allow discrimination between HLA-I + , healthy cells and tumor or virus-infected cells displaying loss or substantial alterations of HLA-I molecules, including allelic losses that are sensed by KIRs. A number of non-HLA-specific receptors have been identified which recognize cell surface or extracellular matrix ligands and may contribute to the physiologic control of immune responses and tolerance. Among these receptors, Siglec 7 (p75/AIRM-1), LAIR-1 and IRp60, recognize ligands including sialic acids, extracellular matrix/collagen or aminophospholipids, respectively. These ligands may be expressed at the surface of tumor cells, thus inhibiting NK cell function. Expression of the PD-1 checkpoint by NK cells requires particular cytokines (IL-15, IL-12, IL-18) together with cortisol, a combination that may occur in the microenvironment of different tumors. Blocking of single or combinations of inhibitory receptors unleashes NK cells and restore their anti-tumor activity, with obvious implications for tumor immunotherapy., (Copyright © 2020 Sivori, Della Chiesa, Carlomagno, Quatrini, Munari, Vacca, Tumino, Mariotti, Mingari, Pende and Moretta.)

The liver harbors two main innate lymphoid cell (ILC) populations: conventional NK (cNK) cells and tissue-resident NK (trNK) cells. Using the MCMV model of infection, we find that, in contrast to liver cNK cells, trNK cells initially undergo a contraction phase followed by a recovery phase to homeostatic levels. The contraction is MCMV independent because a similar phenotype is observed following poly(I:C)/CpG or α-GalCer injection. The rapid contraction phase is due to apoptosis, whereas the recovery phase occurs via proliferation in situ. Interestingly, trNK cell apoptosis is not mediated by fratricide and not induced by liver lymphocytes or inflammatory cytokines. Instead, we find that trNK cell apoptosis is the consequence of an increased sensitivity to lactic acid. Mechanistic analysis indicates that trNK cell sensitivity to lactate is linked to impaired mitochondrial function. These findings underscore the distinctive properties of the liver-resident NK cell compartment., Competing Interests: Conflicts of Interest E.V. is an employee of Innate-Pharma., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

In humans, psychological stress has been associated with a higher risk of infectious illness. However, the mechanisms by which the stress pathway interferes with host response to pathogens remain unclear. We demonstrate here a role for the β2-adrenergic receptor (β2-AR), which binds the stress mediators adrenaline and noradrenaline, in modulating host response to mouse cytomegalovirus (MCMV) infection. Mice treated with a β2-AR agonist were more susceptible to MCMV infection. By contrast, β2-AR deficiency resulted in a better clearance of the virus, less tissue damage, and greater resistance to MCMV. Mechanistically, we found a correlation between higher levels of IFN-γ production by liver natural killer (NK) cells and stronger resistance to MCMV. However, the control of NK cell IFN-γ production was not cell intrinsic, revealing a cell-extrinsic downregulation of the antiviral NK cell response by adrenergic neuroendocrine signals. This pathway reduces host immune defense, suggesting that the blockade of the β2-AR signaling could be used to increase resistance to infectious diseases., Competing Interests: Disclosures: The authors declare no competing interests exist., (© 2020 Wieduwild et al.)

Innate lymphoid cells (ILCs) were found to be developmentally related to natural killer (NK) cells. In humans, they are mostly located in "barrier" tissues where they contribute to innate defenses against different pathogens. ILCs are heterogeneous and characterized by a high degree of plasticity. ILC1s are Tbet + , produce interferon gamma and tumor necrosis factor alpha, but, unlike NK cells, are non-cytolytic and are Eomes independent. ILC2 (GATA-3+) secrete type-2 cytokines, while ILC3s secrete interleukin-22 and interleukin-17. The cytokine signatures of ILC subsets mirror those of corresponding helper T-cell subsets. The ILC role in defenses against pathogens is well-documented, while their involvement in tumor defenses is still controversial. Different ILCs have been detected in tumors. In general, the conflicting data reported in different tumors on the role of ILC may reflect the heterogeneity and/or differences in tumor microenvironment. The remarkable plasticity of ILCs suggests new therapeutic approaches to induce differentiation/switch toward ILC subsets more favorable in tumor control., (Copyright © 2020 Tumino, Vacca, Quatrini, Munari, Moretta, Pelosi, Mariotti and Moretta.)

Neuroendocrine hormones are recognized as important mediators of inflammation that participate in the regulation of the magnitude and length of the immune response. It was demonstrated that endogenous glucocorticoids control the function of innate lymphoid cells (ILCs), and this regulatory mechanism is both cell type- and tissue-specific and is required for host protection during infections. We describe here how to analyze in vitro the effects of corticosterone on murine ILCs, using flow cytometry. The protocols described allow for the identification of the specific combination of stimuli with which glucocorticoids cooperate to regulate the function of ILCs. These methods are instrumental to understanding the molecular mechanisms downstream of glucocorticoid receptor activation and can explain the tissue specificity of ILC response to glucocorticoids.

These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Different studies have evaluated the comparability of various immunohistochemical assays for PD-L1 expression evaluation, with contrasting results. Besides the important issues related to analytic performance and comparability of validated assays, not all platforms are available in all laboratories; moreover, standardized assays are very expensive, and funding for PD-L1 testing is hard to obtain, especially in the research setting. One of the most widely used and inexpensive PD-L1 clones is E1L3N (Cell Signaling Technology, Danvers, MA), which is labeled for research use only. In this work, we wanted to further study and validate in a larger cohort the analytical performance of E1L3N clone on Ventana platform (Ventana Medical Systems, Tucson, AZ) and its comparability with assays SP263 and 22C3 run onto their dedicated platforms. Serial sections of tissue microarrays built from 165 cases of resected lung cancer were stained for E1L3N onto Ventana platform following a previously reported protocol and for 22C3 and SP263 assays onto their respective platforms following manufacturer's instructions. Overall, we found very high concordance when comparing E1L3N with SP263 at both 1% and 50% cutoffs. Lower concordance was found between E1L3N and 22C3 at both cutoffs; however, 100% sensitivity was found for E1L3N compared with both SP263 and 22C3 at 50% cutoff. Given the 100% sensitivity at 50% cutoff demonstrated by E1L3N in comparison with both SP263 and 22C3 and therefore the lack of false-negative cases, we propose an algorithm for PD-L1 testing in NSCLC when considering pembrolizumab as first-line therapy., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Innate lymphoid cells (ILCs) belong to a family of immune cells. Recently, ILCs have been classified into five different groups that mirror the function of adaptive T cell subsets counterparts. In particular, NK cells mirror CD8 + cytotoxic T cells while ILC1, ILC2, ILC3, and Lymphoid tissue inducer (LTi)-like cells reflect the function of CD4 + T helper (Th) cells (Th1, Th2, and Th17 respectively). ILCs are involved in innate host defenses against pathogens and tumors, in lymphoid organogenesis, and in tissue remodeling/repair. In recent years, important molecular inducible checkpoints (PD-1, TIM3, and TIGIT) were shown to control/inactivate different immune cell types. The expression of many of these receptors has been detected on NK cells and subsets of tissue-resident ILCs in both physiological and pathological conditions, including cancer. In particular, it has been demonstrated that the interaction between PD-1 + immune cells and PD-L1/PD-L2+ tumor cells may compromise the anti-tumor effector function leading to tumor immune escape. However, while the effector function of NK cells in tumor is well-established, limited information exists on the other ILC subsets. We will summarize what is known to date on the expression and function of these checkpoint receptors on NK cells and ILCs, with a particular focus on the recent data that reveal an essential contribution of the blockade of PD-1 and TIGIT on NK cells to the immunotherapy of cancer. A better information regarding the presence and the function of different ILCs and of the inhibitory checkpoints in pathological conditions may offer important clues for the development of new immune therapeutic strategies.

The activities of the immune system in repairing tissue injury and combating pathogens were long thought to be independent of the nervous system. However, a major regulatory role of immunomodulatory molecules released locally or systemically by the neuroendocrine system has recently emerged. A number of observations and discoveries support indeed the notion of the nervous system as an immunoregulatory system involved in immune responses. Innate lymphoid cells (ILCs), including natural killer (NK) cells and tissue-resident ILCs, form a family of effector cells present in organs and mucosal barriers. ILCs are involved in the maintenance of tissue integrity and homeostasis. They can also secrete effector cytokines rapidly, and this ability enables them to play early roles in the immune response. ILCs are activated by multiple pathways including epithelial and myeloid cell-derived cytokines. Their functions are also regulated by mediators produced by the nervous system. In particular, the peripheral nervous system, through neurotransmitters and neuropeptides, works in parallel with the hypothalamic-pituitary-adrenal and gonadal axis to modulate inflammatory events and maintain homeostasis. We summarize here recent findings concerning the regulation of ILC activities by neuroendocrine mediators in homeostatic and inflammatory conditions., (© 2018 The Authors Immunological Reviews Published by John Wiley & Sons Ltd.)

Controlling the balance between immunity and immunopathology is crucial for host resistance to pathogens. After infection, activation of the hypothalamic-pituitary-adrenal (HPA) axis leads to the production of glucocorticoids. However, the pleiotropic effects of these steroid hormones make it difficult to delineate their precise role(s) in vivo. Here we found that the regulation of natural killer (NK) cell function by the glucocorticoid receptor (GR) was required for host survival after infection with mouse cytomegalovirus (MCMV). Mechanistically, endogenous glucocorticoids produced shortly after infection induced selective and tissue-specific expression of the checkpoint receptor PD-1 on NK cells. This glucocorticoid-PD-1 pathway limited production of the cytokine IFN-γ by spleen NK cells, which prevented immunopathology. Notably, this regulation did not compromise viral clearance. Thus, the fine tuning of NK cell functions by the HPA axis preserved tissue integrity without impairing pathogen elimination, which reveals a novel aspect of neuroimmune regulation.

Upon infection, the immune system produces inflammatory mediators important for pathogen clearance. However, inflammation can also have deleterious effect on the host and is tightly regulated. Immune system-derived cytokines stimulate the hypothalamic-pituitary-adrenal (HPA) axis, triggering endogenous glucocorticoid production. Through interaction with ubiquitously expressed glucocorticoid receptors (GRs), this steroid hormone has pleiotropic effects on many cell types. Using a genetic mouse model in which the gene encoding the GR is selectively deleted in NKp46 + innate lymphoid cells (ILCs), we demonstrated a major role for the HPA pathway in host resistance to endotoxin-induced septic shock. GR expression in group 1 ILCs is required to limit their IFN-γ production, thereby allowing the development of IL-10-dependent tolerance to endotoxin. These findings suggest that neuroendocrine axes are crucial for tolerization of the innate immune system to microbial endotoxin exposure through direct corticosterone-mediated effects on NKp46-expressing innate cells, revealing a novel strategy of host protection from immunopathology., (© 2017 Quatrini et al.)

Natural Killer cells are innate lymphocytes involved in tumor immunosurveillance. They express activating receptors able to recognize self-molecules poorly expressed on healthy cells but up-regulated upon stress conditions, including transformation. Regulation of ligand expression in tumor cells mainly relays on transcriptional mechanisms, while the involvement of ubiquitin or ubiquitin-like modifiers remains largely unexplored. Here, we focused on the SUMO pathway and demonstrated that the ligand of DNAM1 activating receptor, PVR, undergoes SUMOylation in multiple myeloma. Concurrently, we found that PVR is preferentially located in intracellular compartments in human multiple myeloma cell lines and malignant plasma cells and that inhibition of the SUMO pathway promotes its translocation to the cell surface, increasing tumor cell susceptibility to NK cell-mediated cytolysis. Our findings provide the first evidence of an innate immune activating ligand regulated by SUMOylation, and confer to this modification a novel role in impairing recognition and killing of tumor cells.

Natural-killer receptor group 2, member D (NKG2D) is a well characterized natural killer (NK) cell activating receptor that recognizes several ligands poorly expressed on healthy cells but up-regulated upon stressing stimuli in the context of cancer or viral infection. Although NKG2D ligands represent danger signals that render target cells more susceptible to NK cell lysis, accumulating evidence demonstrates that persistent exposure to ligand-expressing cells causes the decrease of NKG2D surface expression leading to a functional impairment of NKG2D-dependent NK cell functions. Upon ligand binding, NKG2D is internalized from the plasma membrane and sorted to lysosomes for degradation. However, receptor endocytosis is not only a mechanism of receptor clearance from the cell surface, but is also required for the proper activation of signalling events leading to the functional program of NK cells. This review is aimed at providing a summary of current literature relevant to the molecular mechanisms leading to NKG2D down-modulation with particular emphasis given to the role of NKG2D endocytosis in both receptor degradation and signal propagation. Examples of chronic ligand-induced down-regulation of NK cell activating receptors other than NKG2D, including natural cytotoxicity receptors (NCRs), DNAX accessory molecule-1 (DNAM1) and CD16, will be also discussed., Competing Interests: The authors declare no conflict of interest.