B7-H3 in Pediatric Tumors: Far beyond Neuroblastoma.

B7-H3 is a 4Ig transmembrane protein that emerged as a tumor-associated antigen in neuroblastoma. It belongs to the B7 family, shows an immunoregulatory role toward NK and T cells, and, therefore, has been included in the growing family of immune checkpoints. Besides neuroblastoma, B7-H3 is expressed by many pediatric cancers including tumors of the central nervous system, sarcomas, and acute myeloid leukemia. In children, particularly those affected by solid tumors, the therapeutic protocols are aggressive and cause important life-threatening side effects. Moreover, despite the improved survival observed in the last decade, a relevant number of patients show therapy resistance and fatal relapses. Immunotherapy represents a new frontier in the cure of cancer patients and the targeting of tumor antigens or immune checkpoints blockade showed exciting results in adults. In this encouraging scenario, researchers and clinicians are exploring the possibility to use immunotherapeutics targeting B7-H3; these include mAbs and chimeric antigen receptor T-cells (CAR-T). These tools are rapidly evolving to improve the efficacy and decrease the unwanted side effects; drug-conjugated mAbs, bi-tri-specific mAbs or CAR-T, and, very recently, NK cell engagers (NKCE), tetra-specific molecules engaging a tumor-associated antigen and NK cells, have been generated. Preclinical data are promising, and clinical trials are ongoing. Hopefully, the B7-H3 targeting will provide important benefits to cancer patients.

Monocyte and Macrophage in Neuroblastoma: Blocking Their Pro-Tumoral Functions and Strengthening Their Crosstalk with Natural Killer Cells.

Over the past decade, immunotherapy has represented an enormous step forward in the fight against cancer. Immunotherapeutic approaches have increasingly become a fundamental part of the combined therapies currently adopted in the treatment of patients with high-risk (HR) neuroblastoma (NB). An increasing number of studies focus on the understanding of the immune landscape in NB and, since this tumor expresses low or null levels of MHC class I, on the development of new strategies aimed at enhancing innate immunity, especially Natural Killer (NK) cells and macrophages. There is growing evidence that, within the NB tumor microenvironment (TME), tumor-associated macrophages (TAMs), which mainly present an M2-like phenotype, have a crucial role in mediating NB development and immune evasion, and they have been correlated to poor clinical outcomes. Importantly, TAM can also impair the antibody-dependent cellular cytotoxicity (ADCC) mediated by NK cells upon the administration of anti-GD2 monoclonal antibodies (mAbs), the current standard immunotherapy for HR-NB patients. This review deals with the main mechanisms regulating the crosstalk among NB cells and TAMs or other cellular components of the TME, which support tumor development and induce drug resistance. Furthermore, we will address the most recent strategies aimed at limiting the number of pro-tumoral macrophages within the TME, reprogramming the TAMs functional state, thus enhancing NK cell functions. We also prospectively discuss new or unexplored aspects of human macrophage heterogeneity.

Strategies for Potentiating NK-Mediated Neuroblastoma Surveillance in Autologous or HLA-Haploidentical Hematopoietic Stem Cell Transplants.

High-risk neuroblastomas (HR-NB) still have an unacceptable 5-year overall survival despite the aggressive therapy. This includes standardized immunotherapy combining autologous hemopoietic stem cell transplantation (HSCT) and the anti-GD2 mAb. The treatment did not significantly change for more than one decade, apart from the abandonment of IL-2, which demonstrated unacceptable toxicity. Of note, immunotherapy is a promising therapeutic option in cancer and could be optimized by several strategies. These include the HLA-haploidentical αßT/B-depleted HSCT, and the antibody targeting of novel NB-associated antigens such as B7-H3, and PD1. Other approaches could limit the immunoregulatory role of tumor-derived exosomes and potentiate the low antibody-dependent cell cytotoxicity of CD16 dim/neg NK cells, abundant in the early phase post-transplant. The latter effect could be obtained using multi-specific tools engaging activating NK receptors and tumor antigens, and possibly holding immunostimulatory cytokines in their construct. Finally, treatments also consider the infusion of novel engineered cytokines with scarce side effects, and cell effectors engineered with chimeric antigen receptors (CARs). Our review aims to discuss several promising strategies that could be successfully exploited to potentiate the NK-mediated surveillance of neuroblastoma, particularly in the HSCT setting. Many of these approaches are safe, feasible, and effective at pre-clinical and clinical levels.

A multi-organ-on-chip to recapitulate the infiltration and the cytotoxic activity of circulating NK cells in 3D matrix-based tumor model.

The success of immunotherapeutic approaches strictly depends on the immune cells interaction with cancer cells. While conventional in vitro cell cultures under-represent the complexity and dynamic crosstalk of the tumor microenvironment, animal models do not allow deciphering the anti-tumor activity of the human immune system. Therefore, the development of reliable and predictive preclinical models has become crucial for the screening of immune-therapeutic approaches. We here present an organ-on-chip organ on chips (OOC)-based approach for recapitulating the immune cell Natural Killer (NK) migration under physiological fluid flow, infiltration within a 3D tumor matrix, and activation against neuroblastoma cancer cells in a humanized, fluid-dynamic environment. Circulating NK cells actively initiate a spontaneous "extravasation" process toward the physically separated tumor niche, retaining their ability to interact with matrix-embedded tumor cells, and to display a cytotoxic effect (tumor cell apoptosis). Since NK cells infiltration and phenotype is correlated with prognosis and response to immunotherapy, their phenotype is also investigated: most importantly, a clear decrease in CD16-positive NK cells within the migrated and infiltrated population is observed. The proposed immune-tumor OOC-based model represents a promising approach for faithfully recapitulating the human pathology and efficiently employing the immunotherapies testing, eventually in a personalized perspective. An immune-organ on chip to recapitulate the tumor-mediated infiltration of circulating immune cells within 3D tumor model.

miR-24-3p down-regulates the expression of the apoptotic factors FasL and BIM in human natural killer cells.

MicroRNAs are involved in the regulation of different functions in immune and non-immune cells. Here we show that miR-24-3p functionally interacts with FASLG mRNA and down-regulates its expression. This interaction occurs in human natural killer cells (NK), leading to the modulation of FasL surface expression. Moreover, miR-24-3p also modulates the mRNA and protein expression of BIM in NK cells. Thus, it likely contributes to the control of both the extrinsic and intrinsic apoptotic pathways. In line with this hypothesis, inhibition of miR-24-3p improves both initiator caspase-8 and effector caspase-3 and -7 activities, increases cell apoptosis, and reduces cell viability. Our data suggest that miR-24-3p can act as a survival factor in NK cells, affecting the FasL-mediated killing of Fas expressing cells and the BIM-dependent cell death. More generally, miR-24-3p may condition the level of cell apoptosis, which increases at the contraction phase of the immune response when the clearance of various expanded effector cells is needed.

Epitope characterization of a monoclonal antibody that selectively recognizes KIR2DL1 allotypes.

Killer immunoglobulin-like receptor (KIR) genes code for a family of inhibitory and activating receptors, finely tuning NK cell function. Numerous studies reported the relevance of KIR allelic polymorphism on KIR expression, ligand affinity, and strength in signal transduction. Although KIR variability, including gene copy number and allelic polymorphism, in combination with HLA class I polymorphism, impacts both KIR expression and NK cell education, only a precise phenotypic analysis can define the size of the different KIRpos NK cell subsets. In this context, reagents recognizing a limited number of KIRs is essential. In this study, we have characterized the specificity of an anti-KIR mAb termed HP-DM1. Testing its binding to HEK-293T cells transfected with plasmids coding for different KIRs, we demonstrated that HP-DM1 mAb exclusively reacts with KIR2DL1. Using site-directed mutagenesis, we identified the four amino acids relevant for HP-DM1 recognition: M44, S67, R68, and T70. HP-DM1 mAb binds to a conformational epitope including M44, the residue crucial for HLA-C K80 recognition by KIR2DL1. Based on the HP-DM1 epitope characterization, we could extend its reactivity to all KIR2DL1 allotypes identified except for KIR2DL1*022 and, most likely, KIR2DL1*020, predicting that it does not recognize any other KIR with the only exception of KIR2DS1*013. Moreover, by identifying the residues relevant for HP-DM1 binding, continuously updating of its reactivity will be facilitated.

Tumor Microenvironment and Hydrogel-Based 3D Cancer Models for In Vitro Testing Immunotherapies.

In recent years, immunotherapy has emerged as a promising novel therapeutic strategy for cancer treatment. In a relevant percentage of patients, however, clinical benefits are lower than expected, pushing researchers to deeply analyze the immune responses against tumors and find more reliable and efficient tools to predict the individual response to therapy. Novel tissue engineering strategies can be adopted to realize in vitro fully humanized matrix-based models, as a compromise between standard two-dimensional (2D) cell cultures and animal tests, which are costly and hardly usable in personalized medicine. In this review, we describe the main mechanisms allowing cancer cells to escape the immune surveillance, which may play a significant role in the failure of immunotherapies. In particular, we discuss the role of the tumor microenvironment (TME) in the establishment of a milieu that greatly favors cancer malignant progression and impact on the interactions with immune cells. Then, we present an overview of the recent in vitro engineered preclinical three-dimensional (3D) models that have been adopted to resemble the interplays between cancer and immune cells and for testing current therapies and immunotherapeutic approaches. Specifically, we focus on 3D hydrogel-based tools based on different types of polymers, discussing the suitability of each of them in reproducing the TME key features based on their intrinsic or tunable characteristics. Finally, we introduce the possibility to combine the 3D models with technological fluid dynamics platforms, reproducing the dynamic complex interactions between tumor cells and immune effectors migrated in situ via the systemic circulation, pointing out the challenges that still have to be overcome for setting more predictive preclinical assays.

Inhibitory axes impacting on the activity and fate of Innate Lymphoid Cells.

In neoplastic patients, an effective immune response ideally should be achieved by the coordinated action of different immune cells with tumor-suppressive functions. These include the more cytolytic members of the Innate Lymphoid Cells (ILCs) family represented by the Natural Killer (NK) cells, whose activities in cancer patients, however, can be hampered by several inhibitory signals. These are generated by membrane-bound and soluble molecules that, interacting with specific inhibitory receptors, create inhibitory axes impacting the NK cell differentiation and effector functions. These breaks, which now represent major immunotherapeutic targets, may be sensitive to interferon (IFN)-γ, whose source, in vivo, is represented by different cell types including the NK and ILC1. Since also ILCs can express receptors of the inhibitory axes like PD-1 and TIGIT, their therapeutic blockade might further amplify the IFN-γ release that, as an unwanted side effect, would promote the onset of NK cell-resistant tumor variants (NKRTV) expressing ligands involved in inhibitory axes. These variants might also arise from the activity of other cytokines such as IL-27, which can increase the expression of HLA class I and PD-Ls in different cell types, including tumor cells. Besides the amplification of membrane-bound inhibitory axes, tumors can reduce the number of infiltrating cytolytic ILCs, promote the recruitment of poorly cytolytic NK cell subsets, and manipulate to their advantage the infiltrating immune cells, which acquire tumor-promoting activities. This occurs thanks to the production of soluble factors including TGF-ß1 and IL-18 that, alone or in combination, modify the activating and chemokine receptor repertoire of NK cells, and induce the ILCs differentiation towards cells ineffective in fighting cancer or, even worse, with tumor-promoting functions. The present review aims to present and discuss major inhibitory axes impacting on ILCs functions, migration, and differentiation with a major focus on tumor context.

Multiparametric flow cytometry highlights B7-H3 as a novel diagnostic/therapeutic target in GD2neg/low neuroblastoma variants.

BACKGROUND: High-risk neuroblastomas (HR-NBs) are rare, aggressive pediatric cancers characterized by resistance to therapy and relapse in more than 30% of cases, despite using an aggressive therapeutic protocol including targeting of GD2. The mechanisms responsible for therapy resistance are unclear and might include the presence of GD2neg/low NB variants and/or the expression of immune checkpoint ligands such as B7-H3. METHOD: Here, we describe a multiparametric flow cytometry (MFC) combining the acquisition of 106 nucleated singlets, Syto16pos CD45neg CD56pos cells, and the analysis of GD2 and B7-H3 surface expression. 41 bone marrow (BM) aspirates from 25 patients with NB, at the onset or relapse, are analyzed, comparing results with cytomorphological analysis (CA) and/or immunohistochemistry (IHC). Spike in experiments assesses the sensitivity of MFC. Kaplan-Meier analysis on 498 primary NBs selects novel prognostic markers possibly integrating the MFC panel. RESULTS: No false positive are detected, and MFC shows high sensitivity (0.0005%). Optimized MFC identifies CD45negCD56pos NB cells in 11 out of 12 (91.6%) of BM indicated as infiltrated by CA, 7 of which coexpress high levels of GD2 and B7-H3. MFC detects CD45negCD56posGD2neg/low NB variants expressing high surface levels of B7-H3 in two patients with HR-NB (stage M) diagnosed at 53 and 139 months of age. One of them has a non-MYCN amplified tumor with unusual THpos PHOX2Bneg phenotype, which relapsed 141 months post-diagnosis with BM infiltration and a humerus lesion. All GD2neg/low NB variants are detected in patients at relapse. Kaplan-Meier analysis highlights an interesting dichotomous prognostic value of MML5, ULBPs, PVR, B7-H6, and CD47, ligands involved in NB recognition by the immune system. CONCLUSIONS: Our study validates a sensitive MFC analysis providing information on GD2 and B7-H3 surface expression and allowing fast, specific and sensitive evaluation of BM tumor burden. With other routinely used diagnostic and prognostic tools, MFC can improve diagnosis, prognosis, orienting novel personalized treatments in patients with GD2low/neg NB, who might benefit from innovative therapies combining B7-H3 targeting.

Stromal-like Wilms tumor cells induce human Natural Killer cell degranulation and display immunomodulatory properties towards NK cells.

The similarity of stromal-like Wilms tumor (str-WT) cells with mesenchymal stem cells (MSC), suggests their relevant role in the interplay with immune cells in the tumor microenvironment. We investigated the interaction between str-WT cells and NK cells. We observed that str-WT cells expressed some major ligands for activating and inhibitory NK cell receptors. Moreover, they expressed inhibitory checkpoint molecules involved in the negative regulation of anti-tumor immune response. The analysis of the interaction between str-WT cells and NK lymphocytes revealed that activated NK cells could efficiently degranulate upon interaction with str-WT cells. On the other hand, str-WT cells could exert potent inhibitory effects on cytokine-induced activation of NK cell proliferation and phenotype, which were mediated by the production of IDO and PGE2 inhibitory factors. Our data provide insight into the molecular interactions between str-WT cells and NK lymphocytes that may result in different outcomes possibly occurring in the WT microenvironment.

Circulating exosomal microRNAs as potential biomarkers of hepatic injury and inflammation in a murine model of glycogen storage disease type 1a.

Most patients affected by glycogen storage disease type 1a (GSD1a), an inherited metabolic disorder caused by mutations in the enzyme glucose-6-phosphatase-α (G6Pase-α), develop renal and liver complications, including the development of hepatocellular adenoma/carcinoma. The purpose of this study was to identify potential biomarkers of the pathophysiology of the GSD1a-affected liver. To this end, we used the plasma exosomes of a murine model of GSD1a, the LS-G6pc-/- mouse, to uncover the modulation in microRNA expression associated with the disease. The microRNAs differentially expressed between LS-G6pc-/- and wild-type mice, LS-G6pc-/- mice with hepatocellular adenoma and LS-G6pc-/- mice without adenoma, and LS-G6pc-/- mice with amyloidosis and LS-G6pc-/- mice without amyloidosis were identified. Pathway analysis demonstrated that the target genes of the differentially expressed microRNA were significantly enriched for the insulin signaling pathway, glucose and lipid metabolism, Wnt/ß-catenin, telomere maintenance and hepatocellular carcinoma, and chemokine and immune regulation signaling pathways. Although some microRNAs were common to the different pathologic conditions, others were unique to the cancerous or inflammatory status of the animals. Therefore, the altered expression of several microRNAs is correlated with various pathologic liver states and might help to distinguish them during the progression of the disease and the development of late GSD1a-associated complications.

NK Cell Function Regulation by TGF-ß-Induced Epigenetic Mechanisms.

TGF-ß is a potent immunosuppressive cytokine that severely affects the function of NK cells. Tumor cells can take advantage of this ability, enriching their surrounding microenvironment with TGF-ß. TGF-ß can alter the expression of effector molecules and of activating and chemokine receptors, influence metabolism, induce the NK cell conversion toward the less cytolytic ILC1s. These and other changes possibly occur by the induction of complex gene expression programs, involving epigenetic mechanisms. While most of these programs are at present unexplored, the role of certain transcription factors, microRNAs and chromatin changes determined by TGF-ß in NK cells start to be elucidated in human and/or mouse NK cells. The deep understanding of these mechanisms will be useful to design therapies contributing to restore the full NK function.

Cell-Laden Hydrogel as a Clinical-Relevant 3D Model for Analyzing Neuroblastoma Growth, Immunophenotype, and Susceptibility to Therapies.

High risk Neuroblastoma (NB) includes aggressive, metastatic solid tumors of childhood. The survival rate improved only modestly, despite the use of combination therapies including novel immunotherapies based on the antibody-mediated targeting of tumor-associated surface ligands. Treatment failures may be due to the lack of adequate in vitro models for studying, in a given patient, the efficacy of potential therapeutics, including those aimed to enhance anti-tumor immune responses. We here propose a 3D alginate-based hydrogel as extracellular microenvironment to evaluate the effects of the three-dimensionality on biological and immunological properties of NB cells. NB cell lines grown within the 3D alginate spheres presented spheroid morphology, optimal survival, and proliferation capabilities, and a reduced sensitivity to the cytotoxic effect of imatinib mesylate. 3D cultured NB cells were also evaluated for the constitutive and IFN-γ-induced expression of surface molecules capable of tuning the anti-tumor activity of NK cells including immune checkpoint ligands. In particular, IFN-γ induced de novo expression of high amounts of HLA-I molecules, which protected NB cells from the attack mediated by KIR/KIR-L matched NK cells. Moreover, in the 3D alginate spheres, the cytokine increased the expression of the immune checkpoint ligands PD-Ls and B7-H3 while virtually abrogating that of PVR, a ligand of DNAM-1 activating receptor, whose expression correlates with high susceptibility to NK-mediated killing. Our 3D model highlighted molecular features that more closely resemble the immunophenotypic variants occurring in vivo and not fully appreciated in classical 2D culture conditions. Thus, based on our results, 3D alginate-based hydrogels might represent a clinical-relevant cell culture platform where to test the efficacy of personalized therapeutic approaches aimed to optimize the current and innovative immune based therapies in a very systematic and reliable way.

Novel Immunoregulatory Functions of IL-18, an Accomplice of TGF-ß1.

TGF-ß1 is a pleiotropic factor exerting a strong regulatory role in several cell types, including immune cells. In NK cells it profoundly alters the surface expression of crucial activating and chemokine receptors. To understand which soluble signals might better contrast these effects, we cultured human NK cells in the presence of TGF-ß1 and different innate and adaptive cytokines, generally referred as "immunostimulatory". These included IL-2, IL-15, IL-21, IL-27, and IL-18. Unexpectedly, IL-18 strengthened rather than contrasting important TGF-ß1-mediated functions. In particular, IL-18 further reduced the expression of CX3CR1 and NKp30, leading to the virtual abrogation of the triggering capability of this activating receptor. Moreover, IL-18 further increased the expression of CXCR4. The IL-18-mediated additive effect on NKp30 and CXCR4 expression involved transcriptional regulation and activation of MEK/ERK and/or p38MAPK. A proteomic approach quantified both surface and intracellular proteins significantly modified in cytokine-treated NK cells, thus giving global information on the biological processes involving TGF-ß1 and IL-18. Our data support the concept that IL-18 may have a different behavior depending on the type of soluble factors characterizing the microenvironment. In a TGF-ß1 rich milieu such as tumors, it may contribute to the impairment of both NK cells recruitment and killing capability.

2B4 dysfunction in XLP1 NK cells: More than inability to control EBV infection.

X-linked lymphoproliferative disease 1 (XLP1) is a monogenic disorder caused by mutations in SH2D1A, resulting in the absence/dysfunction of the signaling lymphocyte activation molecule (SLAM)-associated protein (SAP). Consequently, SLAM receptors as 2B4 (CD244) and NTB-A (SLAMF6), upon ligand engagement, exert inhibitory instead of activating function. This causes an immune dysfunction that is worsened by the selective inability of NK and T cells to kill EBV-infected B cells with dramatic clinical sequelae (e.g. fulminant mononucleosis, hyperinflammation, lymphoma). Here we outline recent findings on the interplay between inhibitory 2B4 and the various activating receptors in NK cells. 2B4 engagement selectively blocks ITAM-dependent activating receptors as NCR and CD16, while it does not affect NKG2D and DNAM-1. Furthermore, inhibitory 2B4 participates to NK cell education, as highlighted by the existence in XLP1 patients of a large subset of fully functional NK cells that lack self-HLA specific inhibitory receptors and exert autoreactivity against mature dendritic cells.

Molecular Mechanisms Directing Migration and Retention of Natural Killer Cells in Human Tissues.

A large body of data shows that Natural Killer (NK) cells are immune effectors exerting a potent cytolytic activity against tumors and virus infected cells. The discovery and characterization of several inhibitory and activating receptors unveiled most of the mechanisms allowing NK cells to spare healthy cells while selectively attacking abnormal tissues. Nevertheless, the mechanisms ruling NK cell subset recirculation among the different compartments of human body have only lately started to be investigated. This is particularly true for pathological settings such as tumors or infected tissues but also for para-physiological condition like pregnant human uterine mucosa. It is becoming evident that the microenvironment associated to a particular clinical condition can deeply influence the migratory capabilities of NK cells. In this review we describe the main mechanisms and stimuli known to regulate the expression of chemokine receptors and other molecules involved in NK cell homing to either normal or pathological/inflamed tissues, including tumors or organs such as lung and liver. We will also discuss the role played by the chemokine/chemokine receptor axes in the orchestration of physiological events such as NK cell differentiation, lymphoid organ retention/egress and recruitment to decidua during pregnancy.

Updated clinical and biological information from the two-stage phase II study of imatinib mesylate in subjects with relapsed/refractory neuroblastoma.

Several studies support the notion that the kinase inhibitor Imatinib mesylate exerts off-target effects on cells of the immune system. After our first report of continuous daily oral administration in subjects with relapsed/refractory neuroblastoma (NB, EudraCT: 2005-005778-63), here we update the clinical information and report additional information on potential surrogate markers for prediction of efficacy. Peripheral blood (PB) samples collected at study entry and after the first and second cycle of Imatinib mesylate treatment were tested for IFN-γ, TNF-α, TGF-ß, IL-10, CXCL12 and soluble (s) B7-H6 plasma levels. In addition, paired PB and bone marrow (BM) samples collected at study entry and after the second Imatinib cycle were evaluated for CXCL12, CXCR4 and NKp30 isoform mRNA levels. Correlation between each parameter level and response/outcome was then evaluated. Out of the six subjects still alive at the time of the first report, thee died after additional therapy, two for NB progression and one for a second malignancy. Three are presently alive and cured from NB at 10 years after the first Imatinib cycle. Of these, one achieved complete response (CR) during Imatinib treatment and never relapsed, one had a local relapse removed by surgery and the third received TVD as rescue therapy. Response and outcome were associated with low Imatinib exposure, whereas none of the tested immunological and molecular parameters was predictive of response/outcome. However, after Imatinib treatment NKp30 isoform mRNA levels significantly increase in BM samples, indicating that Imatinib mesylate exerted an off-target effect on NK cells in vivo. Imatinib mesylate efficacy in relapsed/refractory NB has been confirmed at a longer follow-up, supporting its inclusion in new Phase II trials for these subjects, that should envisage collection of samples to evaluate the predictive power of other potential surrogate markers of efficacy.

Nidogen-1 is a novel extracellular ligand for the NKp44 activating receptor.

The release of soluble ligands of activating Natural Killer (NK) cell receptors may represent a regulatory mechanism of NK cell function both in physiologic and in pathologic conditions. Here, we identified the extracellular matrix protein Nidogen-1 (NID1) as a ligand of NKp44, an important activating receptor expressed by activated NK cells. When released as soluble molecule, NID1 regulates NK cell function by modulating NKp44-induced IFN-γ production or cytotoxicity. In particular, it also modulates IFN-γ production induced by Platelet-Derived Growth Factor (PDGF)-DD following NKp44 engagement. We also show that NID1 may be present at the cell surface. In this form or when bound to a solid support (bNID1), NID1 fails to induce NK cell cytotoxicity or cytokine release. However, analysis by mass spectrometry revealed that exposure to bNID1 can induce in human NK cells relevant changes in the proteomic profiles suggesting an effect on different biological processes.