TLR2/4 are novel activating receptors for SARS-CoV-2 spike protein on NK cells.

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 CD56brightNK 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 CD56dimNK 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.

Transition to a mesenchymal state in neuroblastoma may be characterized by a high expression of GD2 and by the acquisition of immune escape from NK cells.

Background: Neuroblastoma (NB) is characterized by both adrenergic (ADRN) and undifferentiated mesenchymal (MES) subsets. The ganglioside sialic acid-containing glycosphingolipid (GD2) is widely overexpressed on tumors of neuroectodermal origin promoting malignant phenotypes. MES cells are greatly enriched in post-therapy and relapsing tumors and are characterized by decreased expression of GD2. This event may cause failure of GD2-based immunotherapy. NK cells represent a key innate cell subset able to efficiently kill tumors. However, the tumor microenvironment (TME) that includes tumor cells and tumor-associated (TA) cells could inhibit their effector function. Methods: We studied eight NB primary cultures that, in comparison with commercial cell lines, more faithfully reflect the tumor cell characteristics. We studied four primary NB-MES cell cultures and two pairs of MES/ADRN (691 and 717) primary cultures, derived from the same patient. In particular, in the six human NB primary cultures, we assessed their phenotype, the expression of GD2, and the enzymes that control its expression, as well as their interactions with NK cells, using flow cytometry, RT-qPCR, and cytotoxicity assays. Results: We identified mature (CD105+/CD133-) and undifferentiated (CD133+/CD105-) NB subsets that express high levels of the MES transcripts WWTR1 and SIX4. In addition, undifferentiated MES cells display a strong resistance to NK-mediated killing. On the contrary, mature NB-MES cells display an intermediate resistance to NK-mediated killing and exhibit some immunomodulatory capacities on NK cells but do not inhibit their cytolytic activity. Notably, independent from their undifferentiated or mature phenotype, NB-MES cells express GD2 that can be further upregulated in undifferentiated NB-MES cells upon co-culture with NK cells, leading to the generation of mature mesenchymal GD2bright neuroblasts. Concerning 691 and 717, they show high levels of GD2 and resistance to NK cell-mediated killing that can be overcome by the administration of dinutuximab beta, the anti-GD2 monoclonal antibody applied in the clinic. Conclusions: NB is a heterogeneous tumor representing a further hurdle in NB immunotherapy. However, different from what was reported with NB commercial cells and independent of their MES/ADRN phenotype, the expression of GD2 and its displayed sensitivity to anti-GD2 mAb ADCC indicated the possible effectiveness of anti-GD2 immunotherapy.

The anti-inflammatory cytokine IL-37 improves the NK cell-mediated anti-tumor response.

IL-37 is a member of the IL-1 superfamily exerting anti-inflammatory functions in a number of diseases. Extracellular IL-37 triggers the inhibitory receptor IL-1R8 that is known to regulate different NK cell pathways and functional activities including their anti-tumor effect. However, the effect of IL-37 on human NK cell functions is still to be unveiled. This study aimed to investigate the functional effect of IL-37 in human NK cells activated with IL-15. We found that IL-37 enhanced both NK cell cytotoxic activity against different tumor cell lines and cytokines production. These effects were associated with increased phosphorylation of ERK and NF-Kb. The improved NK cell activity was also strictly related to a time-dependent GSK3ß-mediated degradation of IL-1R8. The enhanced activation profile of IL-37 treated NK cells possibly due to IL-1R8 degradation was confirmed by the results with IL-1R8-silenced NK cells. Lastly, in line with these data, through the analysis of the TNM plot database of a large group of patients, IL-37 mRNA expression was found to be significantly lower in colon and skin cancers than in normal tissues. Colon adenocarcinoma and neuroblastoma patients with higher IL-37 mRNA levels had significantly higher overall survival, suggesting that the presence of IL-37 might be considered an independent positive prognostic factor for this tumor. Our results provide novel information on the mechanisms regulating IL-1R8 function in human NK cells, highlighting the IL-37-IL-1R8 axis as a potential new target to improve the anti-tumor immune response.

The roles of different forms of IL-15 in human melanoma progression.

Background: Melanoma is a lethal skin cancer, and the risk of developing it is increased by exposure to ultraviolet (UV) radiation. The production of cytokines such as interleukin-15 (IL-15), induced by the exposure of skin cells to UV rays, could also promote melanoma development. The aim of this study is to investigate the possible role of Interleukin-15/Interleukin-15 Receptor α (IL-15/IL-15Rα) complexes in melanoma development. Methods: The expression of IL-15/IL-15Rα complexes by melanoma cells was evaluated both ex vivo and in vitro by tissue microarray, PCR, and flow cytometry. The presence of the soluble complex (sIL-15/IL-15Rα) in the plasma of metastatic melanoma patients was detected using an ELISA assay. Subsequently, we investigated the impact of natural killer (NK) cell activation after rIL-2 starvation followed by exposure to the sIL-15/IL-15Rα complex. Finally, by analyzing public datasets, we studied the correlation between IL-15 and IL-15Rα expressions and melanoma stage, NK and T-cell markers, and overall survival (OS). Results: Analysis of a melanoma tissue microarray shows a significant increase in the number of IL-15+ tumor cells from the benign nevi to metastatic melanoma stages. Metastatic melanoma cell lines express a phorbol-12-myristate-13-acetate (PMA)-cleavable membrane-bound IL-15 (mbIL-15), whereas cultures from primary melanomas express a PMA-resistant isoform. Further analysis revealed that 26% of metastatic patients present with consistently high plasmatic levels of sIL-15/IL-15Rα. When the recombinant soluble human IL-15/IL-15Rα complex is added to briefly starved rIL-2-expanded NK cells, these cells exhibit strongly reduced proliferation and levels of cytotoxic activity against K-562 and NALM-18 target cells. The analysis of public gene expression datasets revealed that high IL-15 and IL-15Rα intra-tumoral production correlates with the high levels of expression of CD5+ and NKp46+ (T and NK markers) and significantly correlates with a better OS in stages II and III, but not in stage IV. Conclusions: Membrane-bound and secreted IL-15/IL-15Rα complexes are continuously present during progression in melanoma. It is notable that, although IL-15/IL-15Rα initially promoted the production of cytotoxic T and NK cells, at stage IV promotion of the development of anergic and dysfunctional cytotoxic NK cells was observed. In a subgroup of melanoma metastatic patients, the continuous secretion of high amounts of the soluble complex could represent a novel NK cell immune escape mechanism.

MicroRNA analysis of Natural Killer cell-derived exosomes: the microRNA let-7b-5p is enriched in exosomes and participates in their anti-tumor effects against pancreatic cancer cells.

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.

A systematic review of exercise protocols applied to athymic mice in tumor-related experiments.

Athymic mice are unable to produce T-cells and are then characterized as immunodeficient. This characteristic makes these animals ideal for tumor biology and xenograft research. New non-pharmacological therapeutics are required owing to the exponential increase in global oncology costs over the last 10 years and the high cancer mortality rate. In this sense, physical exercise is regarded as a relevant component of cancer treatment. However, the scientific community lacks information regarding the effect of manipulating training variables on cancer in humans, and experiments with athymic mice. Therefore, this systematic review aimed to address the exercise protocols used in tumor-related experiments using athymic mice. The PubMed, Web of Science, and Scopus databases were searched without restrictions on published data. A combination of key terms such as athymic mice, nude mice, physical activity, physical exercise, and training was used. The database search retrieved 852 studies (PubMed, 245; Web of Science, 390; and Scopus, 217). After title, abstract, and full-text screening, 10 articles were eligible. Based on the included studies, this report highlights the considerable divergences in the training variables adopted for this animal model. No studies have reported the determination of a physiological marker for intensity individualization. Future studies are recommended to explore whether invasive procedures can result in pathogenic infections in athymic mice. Moreover, time-consuming tests cannot be applied to experiments with specific characteristics such as tumor implantation. In summary, non-invasive, low-cost, and time-saving approaches can suppress these limitations and improve the welfare of these animals during experiments.

Remote and Unsupervised Exercise Strategies for Improving the Physical Activity of Colorectal Cancer Patients: A Meta-Analysis.

Colorectal cancer (CRC) burden across the world is expected to increase by ~2.2 million new cases and ~1.1 million deaths by 2030. Regular physical exercise is recommended to prevent CRC, but the myriad of protocols preclude further discussion on how to manage its variables for this population. Home-based exercise guided by remote monitoring provides an alternative to surpass the barriers of supervised exercise. However, no meta-analysis was conducted to verify the effectiveness of this intervention for improving physical activity (PA). We performed a systematic review of remote and unsupervised strategies imposed on CRC patients for improving PA and compared, via a meta-analysis, their effectiveness against CRC patients submitted to usual care or no intervention. The databases PubMed, Scopus, and Web of Science were searched on 20 September 2022. Eleven studies attained the criteria for eligibility in the qualitative approach, and seven were included in the meta-analysis. No significant effect (p = 0.06) of remote and unsupervised exercise intervention was observed. However, a sensitivity analysis including three studies that only considered CRC patients was performed, demonstrating a significant effect in favor of exercise (p = 0.008). Based on our sensitivity analysis, remote and unsupervised exercise strategies were effective to improve the PA of CRC patients.

Myeloid derived suppressor cells in tumor microenvironment: Interaction with innate lymphoid cells.

Human myeloid-derived suppressor cells (MDSC) represent a stage of immature myeloid cells and two main subsets can be identified: monocytic and polymorphonuclear. MDSC contribute to the establishment of an immunosuppressive tumor microenvironment (TME). The presence and the activity of MDSC in patients with different tumors correlate with poor prognosis. As previously reported, MDSC promote tumor growth and use different mechanisms to suppress the immune cell-mediated anti-tumor activity. Immunosuppression mechanisms used by MDSC are broad and depend on their differentiation stage and on the pathological context. It is known that some effector cells of the immune system can play an important role in the control of tumor progression and metastatic spread. In particular, innate lymphoid cells (ILC) contribute to control tumor growth representing a potential, versatile and, immunotherapeutic tool. Despite promising results obtained by using new cellular immunotherapeutic approaches, a relevant proportion of patients do not benefit from these therapies. Novel strategies have been investigated to overcome the detrimental effect exerted by the immunosuppressive component of TME (i.e. MDSC). In this review, we summarized the characteristics and the interactions occurring between MDSC and ILC in different tumors discussing how a deeper knowledge on MDSC biology could represent an important target for tumor immunotherapy capable of decreasing immunosuppression and enhancing anti-tumor activity exerted by immune cells.

Liquid chromatography coupled to high-resolution mass spectrometry metabolomics: A useful tool for investigating tumor secretome based on a three-dimensional co-culture model.

Three-dimensional (3D) cell culture technologies, which more closely mimic the complex microenvironment of tissue, are being increasingly evaluated as a tool for the preclinical screening of clinically promising new molecules, and studying of tissue metabolism. Studies of metabolites released into the extracellular space (secretome) allow understanding the metabolic dynamics of tissues and changes caused by therapeutic interventions. Although quite advanced in the field of proteomics, studies on the secretome of low molecular weight metabolites (< 1500 Da) are still very scarce. We present an untargeted metabolomic protocol based on the hybrid technique of liquid chromatography coupled with high-resolution mass spectrometry for the analysis of low-molecular-weight metabolites released into the culture medium by 3D cultures and co-culture (secretome model). For that we analyzed HT-29 human colon carcinoma cells and 3T3-L1 preadipocytes in 3D-monoculture and 3D-co-culture. The putative identification of the metabolites indicated a sort of metabolites, among them arachidonic acid, glyceric acid, docosapentaenoic acid and beta-Alanine which are related to cancer and obesity. This protocol represents a possibility to list metabolites released in the extracellular environment in a comprehensive and untargeted manner, opening the way for the generation of metabolic hypotheses that will certainly contribute to the understanding of tissue metabolism, tissue-tissue interactions, and metabolic responses to the most varied interventions. Moreover, it brings the potential to determine novel pathways and accurately identify biomarkers in cancer and other diseases. The metabolites indicated in our study have a close relationship with the tumor microenvironment in accordance with the literature review.

IL-1R8 silencing improves the anti-tumor function of freshly isolated human NK cells.

The inhibitory receptor interleukin-1 receptor 8 (IL-1R8) has been recently recognized to be expressed also by human natural killer (NK) cells. This study was aimed to design and optimize IL-1R8 silencing conditions in human NK cells to precisely establish the activity of such receptor in these cells. Electroporation of freshly isolated or IL-2-cultured NK cells with small interfering RNA (siRNA), resulted in a marked, even though variable, IL-1R8-silencing. Although the expression profile revealed downregulation of most genes involved in several intracellular pathways, some genes related to proliferation, expression of some chemokine receptors, antibody-dependent cell cytotoxicity and cytotoxic activity were upregulated in IL-1R8-silenced NK cells. Furthermore, upon IL-15 activation, the majority of genes involved in NK cell function were upregulated in IL-1R8-siRNA-compared with control-siRNA-transfected NK cells. More importantly, in agreement with these findings, the reduction of IL-1R8 gene expression levels resulted in enhanced expression of NK cell activation markers, production of cytokines and chemokines, and cytotoxic activity against several NK cell targets with different susceptibility to NK-mediated lysis. Similar results were obtained following stimulation with IL-18. All together these data, deeply impacting on the main effector functions of human NK cells, can lead to a better understanding of IL-1R8-mediated regulation on these cells and to the design of new strategies for improving NK cell-mediated anti-tumor responses.

Home-based, supervised, and mixed exercise intervention on functional capacity and quality of life of colorectal cancer patients: a meta-analysis.

This systematic review and meta-analysis of randomized controlled trials tested the effects of home-based, supervised, or mixed exercise interventions on the functional capacity (FC) and quality of life (QoL) in colorectal cancer patients. A literature search was performed using the PubMed, Embase, Cochrane, and Medline databases. Two reviewers screened the literature through March 10, 2021 for studies related to exercise and colorectal cancer. Of the 1161 screened studies in the initial search, 13 studies met the eligibility criteria (home-based = 6 studies; supervised or mixed = 7 studies). Overall, 706 patients were enrolled in the trials, and 372 patients were submitted to home-based, supervised, or mixed exercise intervention. The overall results from the main meta-analysis showed a significant effect regarding supervised or mixed intervention (6 studies; p = 0.002; I2 = 43%; PI 0.41-1.39); however, no significant effect was observed for home-based intervention (5 studies; p = 0.05; I2 = 25%; PI - 0.34-0.76). A sensitivity analysis based on studies with intervention adherence ≥ 80% (home-based = 3 studies; supervised or mixed = 4 studies) revealed that home-based intervention or intervention entirely supervised or with some level of supervision (mixed) are effective in improving the QoL and FC of CRC patients. In summary, this meta-analysis verified that supervised and home-based exercise can modify QoL and FC when intervention adherence ≥ 80%. Regardless of the supervision characteristics, future RCTs are strongly encouraged to provide a detailed description of the exercise variables in physical interventions for CRC prescription. This perspective will allow a refined exercise prescription for patients with CRC, mainly according to their clinical status.

Toll-like receptor 8 agonists improve NK-cell function primarily targeting CD56brightCD16- subset.

BACKGROUND: Toll-like receptors (TLRs) are pattern-recognition sensors mainly expressed in innate immune cells that directly recognize conserved pathogen structures (pathogen-associated molecular patterns-PAMPs). Natural killer (NK) cells have been described to express different endosomal TLRs triggered by RNA and DNA sequences derived from both viruses and bacteria. This study was addressed to establish which endosomal TLR could directly mediate NK activation and function after proper stimuli. It was also important to establish the most suitable TLR agonist to be used as adjuvant in tumor vaccines or in combined cancer immunotherapies. METHODS: We assessed endosomal TLR expression in total NK cells by using RT-qPCR and western blotting technique. In some experiments, we purified CD56brightCD16- and CD56dimCD16+ cells subsets by using NK Cell Isolation Kit Activation marker, cytokine production, CD107a expression and cytotoxicity assay were evaluated by flow cytometry. Cytokine release was quantified by ELISA. NK cells obtained from ovarian ascites underwent the same analyses. RESULTS: Although the four endosomal TLRs (TLR3, TLR7/8, and TLR9) were uniformly expressed on CD56brightCD16- and CD56dimCD16+ cell subsets, the TLR7/8 (R848), TLR3 (polyinosinic-polycytidylic acid, Poly I:C) and TLR9 (ODN2395) ligands promoted NK-cell function only in the presence of suboptimal doses of cytokines, including interleukin (IL)-2, IL-12, IL-15, and IL-18, produced in vivo by other environmental cells. We showed that R848 rather than TLR3 and TLR9 agonists primarily activated CD56brightCD16- NK cells by increasing their proliferation, cytokine production and cytotoxic activity. Moreover, we demonstrated that R848, which usually triggers TLR7 and TLR8 on dendritic cells, macrophages and neutrophils cells, activated CD56brightCD16- NK-cell subset only via TLR8. Indeed, specific TLR8 but not TLR7 agonists increased cytokine production and cytotoxic activity of CD56brightCD16- NK cells. Importantly, these activities were also observed in peritoneal NK cells from patients with metastatic ovarian carcinoma, prevalently belonging to the CD56brightCD16- subset. CONCLUSION: These data highlight the potential value of TLR8 in NK cells as a new target for immunotherapy in patients with cancer.

Neuroblastoma Tumor-Associated Mesenchymal Stromal Cells Regulate the Cytolytic Functions of NK Cells.

Neuroblastoma tumor-associated mesenchymal stromal cells (NB-TA-MSC) have been extensively characterized for their pro-tumorigenic properties, while their immunosuppressive potential, especially against NK cells, has not been thoroughly investigated. Herein, we study the immune-regulatory potential of six primary young and senescent NB-TA-MSC on NK cell function. Young cells display a phenotype (CD105+/CD90+/CD73+/CD29+/CD146+) typical of MSC cells and, in addition, express high levels of immunomodulatory molecules (MHC-I, PDL-1 and PDL-2 and transcriptional-co-activator WWTR1), able to hinder NK cell activity. Notably, four of them express the neuroblastoma marker GD2, the most common target for NB immunotherapy. From a functional point of view, young NB-TA-MSC, contrary to the senescent ones, are resistant to activated NK cell-mediated lysis, but this behavior is overcome using anti-CD105 antibody TRC105 that activates antibody-dependent cell-mediated cytotoxicity. In addition, proliferating NB-TA-MSC, but not the senescent ones, after six days of co-culture, inhibit proliferation, expression of activating receptors and cytolytic activity of freshly isolated NK. Inhibitors of the soluble immunosuppressive factors L-kynurenine and prostaglandin E2 efficiently counteract this latter effect. Our data highlight the presence of phenotypically heterogeneous NB-TA-MSC displaying potent immunoregulatory properties towards NK cells, whose inhibition could be mandatory to improve the antitumor efficacy of targeted immunotherapy.

MiR-22, a serum predictor of poor outcome and therapy response in diffuse large B-cell lymphoma patients.

Diffuse large B-cell lymphoma (DLBCL) is an aggressive, heterogeneous neoplasm where prognostication and therapeutic decision are challenging. The available prognostic tools are not able to identify all patients refractory to treatment. MicroRNAs, small RNAs frequently deregulated in cancer, stably circulate in biofluids, representing interesting candidates for non-invasive biomarkers. Here we validated serum miR-22, an evolutionarily conserved microRNA, as a prognostic/predictive biomarker in DLBCL. Moreover, we found that its expression and release from DLBCL cells are related to therapy response and adversely affect cell proliferation. These results suggest that miR-22 is a promising complementary or even independent non-invasive biomarker for DLBCL management.

Pediatric Tumors-Mediated Inhibitory Effect on NK Cells: The Case of Neuroblastoma and Wilms' Tumors.

Natural killer (NK) cells play a key role in the control of cancer development, progression and metastatic dissemination. However, tumor cells develop an array of strategies capable of impairing the activation and function of the immune system, including NK cells. In this context, a major event is represented by the establishment of an immunosuppressive tumor microenvironment (TME) composed of stromal cells, myeloid-derived suppressor cells, tumor-associated macrophages, regulatory T cells and cancer cells themselves. The different immunoregulatory cells infiltrating the TME, through the release of several immunosuppressive molecules or by cell-to-cell interactions, cause an impairment of the recruitment of NK cells and other lymphocytes with effector functions. The different mechanisms by which stromal and tumor cells impair NK cell function have been particularly explored in adult solid tumors and, in less depth, investigated and discussed in a pediatric setting. In this review, we will compare pediatric and adult solid malignancies concerning the respective mechanisms of NK cell inhibition, highlighting novel key data in neuroblastoma and Wilms' tumor, two of the most frequent pediatric extracranial solid tumors. Indeed, both tumors are characterized by the presence of stromal cells acting through the release of immunosuppressive molecules. In addition, specific tumor cell subsets inhibit NK cell cytotoxic function by cell-to-cell contact mechanisms likely controlled by the transcriptional coactivator TAZ. These findings could lead to a more performant diagnostic approach and to the development of novel immunotherapeutic strategies targeting the identified cellular and molecular targets.

NK Cells and PMN-MDSCs in the Graft From G-CSF Mobilized Haploidentical Donors Display Distinct Gene Expression Profiles From Those of the Non-Mobilized Counterpart.

A recent approach of hematopoietic stem cell (HSC) transplantation from haploidentical donors "mobilized" with G-CSF is based on the selective depletion of αß 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.

HCMV-controlling NKG2C+ NK cells originate from novel circulating inflammatory precursors.

BACKGROUND: There is limited knowledge on the origin and development from CD34+ precursors of the ample spectrum of human natural killer (NK) cells, particularly of specialized NK subsets. OBJECTIVE: This study sought to characterize the NK-cell progeny of CD34+DNAM-1brightCXCR4+ and of other precursors circulating in the peripheral blood of patients with chronic viral infections (eg, HIV, hepatitis C virus, cytomegalovirus reactivation). METHODS: Highly purified precursors were obtained by flow cytometric sorting and cultured in standard NK-cell differentiation media (ie, SCF, FLT3, IL-7, IL-15). Phenotypic and functional analyses on progenies were performed by multiparametric cytofluorimetric assays. Transcriptional signatures of NK-cell progenies were studied by microarray analysis. Inhibition of cytomegalovirus replication was studied by PCR. RESULTS: Unlike conventional CD34+ precursors, Lin-CD34+DNAM-1brightCXCR4+ precursors from patients with chronic infection, rapidly differentiate into cytotoxic, IFN-γ-secreting CD94/NKG2C+KIR+CD57+ NK-cell progenies. An additional novel subset of common lymphocyte precursors was identified among Lin-CD34-CD56-CD16+ cells and characterized by expression of CXCR4 and lack of perforin and CD94. Lin-CD34-CD56-CD16+Perf-CD94-CXCR4+ precursors are also endowed with generation potential toward memory-like NKG2C+NK cells. Maturing NK-cell progenies mediated strong human cytomegalovirus-inhibiting activity. Microarray analysis confirmed a transcriptional signature compatible with NK-cell progenies and with maturing adaptive NK cells. CONCLUSIONS: During viral infections, precursors of adaptive NK cells are released and circulate in the peripheral blood.

Identification of neuroblastoma cell lines with uncommon TAZ+/mesenchymal stromal cell phenotype with strong suppressive activity on natural killer cells.

BACKGROUND: Neuroblastoma (NB) is the most common, extracranial childhood solid tumor arising from neural crest progenitor cells and is a primary cause of death in pediatric patients. In solid tumors, stromal elements recruited or generated by the cancer cells favor the development of an immune-suppressive microenvironment. Herein, we investigated in NB cell lines and in NB biopsies, the presence of cancer cells with mesenchymal phenotype and determined the immune-suppressive properties of these tumor cells on natural killer (NK) cells. METHODS: We assessed the mesenchymal stromal cell (MSC)-like phenotype and function of five human NB cell lines and the presence of this particular subset of neuroblasts in NB biopsies using flow-cytometry, immunohistochemistry, RT-qPCR, cytotoxicity assays, western blot and silencing strategy. We corroborated our data consulting a public gene-expression dataset. RESULTS: Two NB cell lines, SK-N-AS and SK-N-BE(2)C, exhibited an unprecedented MSC phenotype (CD105+/CD90+/CD73+/CD29+/CD146+/GD2+/TAZ+). In these NB-MSCs, the ectoenzyme CD73 and the oncogenic/immune-regulatory transcriptional coactivator TAZ were peculiar markers. Their MSC-like nature was confirmed by their adipogenic and osteogenic differentiation potential. Immunohistochemical analysis confirmed the presence of neuroblasts with MSC phenotype (CD105+/CD73+/TAZ+). Moreover, a public gene-expression dataset revealed that, in stage IV NB, a higher expression of TAZ and CD105 strongly correlated with a poorer outcome.Among the NB-cell lines analyzed, only NB-MSCs exhibited multifactorial resistance to NK-mediated lysis, inhibition of activating NK receptors, signal adaptors and of NK-cell cytotoxicity through cell-cell contact mediated mechanisms. The latter property was controlled partially by TAZ, since its silencing in NB cells efficiently rescued NK-cell cytotoxic activity, while its overexpression induced opposite effects in non-NB-MSC cells. CONCLUSIONS: We identified a novel NB immunoregulatory subset that: (i) displayed phenotypic and functional properties of MSC, (ii) mediated multifactorial resistance to NK-cell-induced killing and (iii) efficiently inhibited, in coculture, the cytotoxic activity of NK cells against target cells through a TAZ-dependent mechanism. These findings indicate that targeting novel cellular and molecular components may disrupt the immunomodulatory milieu of the NB microenvironment ameliorating the response to conventional treatments as well as to advanced immunotherapeutic approaches, including adoptive transfer of NK cells and chimeric antigen receptor T or NK cells.

Wilms' Tumor Primary Cells Display Potent Immunoregulatory Properties on NK Cells and Macrophages.

The immune response plays a crucial defensive role in cancer growth and metastasis and is a promising target in different tumors. The role of the immune system in Wilm's Tumor (WT), a common pediatric renal malignancy, is still to be explored. The characterization of the immune environment in WT could allow the identification of new therapeutic strategies for targeting possible inhibitory mechanisms and/or lowering toxicity of the current treatments. In this study, we stabilized four WT primary cultures expressing either a blastematous (CD56+/CD133-) or an epithelial (CD56-/CD133+) phenotype and investigated their interactions with innate immune cells, namely NK cells and monocytes. We show that cytokine-activated NK cells efficiently kill WT cells. However, after co-culture with WT primary cells, NK cells displayed an impaired cytotoxic activity, decreased production of IFNγ and expression of CD107a, DNAM-1 and NKp30. Analysis of the effects of the interaction between WT cells and monocytes revealed their polarization towards alternatively activated macrophages (M2) that, in turn, further impaired NK cell functions. In conclusion, we show that both WT blastematous and epithelial components may contribute directly and indirectly to a tumor immunosuppressive microenvironment that is likely to play a role in tumor progression.

Polymorphonuclear Myeloid-Derived Suppressor Cells Are Abundant in Peripheral Blood of Cancer Patients and Suppress Natural Killer Cell Anti-Tumor Activity.

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.