Modern scientific research increasingly views olive leaf extract (OLE) not merely as a simple supplement, but as a sophisticated chemical orchestra where a wide array of phytochemicals works in natural harmony to provide therapeutic benefits. While olive oil is the most famous product of the Olea europaea tree, it is important to emphasize that the leaves are actually a far richer and more concentrated reservoir of bioactive molecules, often containing phenolic levels several times higher than those found in the fruit or oil. This whole plant extract often proves more biologically effective than isolated compounds because its components target multiple cellular pathways simultaneously. Many beneficial effects have been ascribed to OLE including anti-inflammatory, anti-oxidant, anti-microbial, anti-viral, neuroprotective, and anti-tumoral effects. In this review, we focused on the latter activity, especially in the field of pediatric tumors such as acute leukemias and neuroblastoma. This issue was discussed starting from the definition of OLE and its components describing the main biological activities, passing through the OLE roles on the immune system, moving on to the anti-cancer functions, and ending with future perspectives.

It is now clearly established that extracellular vesicles (EV)s represent important players in the regulation of bone marrow (BM) microenvironment during cancer progression, and anti-tumor responses. Indeed, healthy cells and neoplastic cells crosstalk through a dynamic transfer of extracellular vesicles in the tumor microenvironment including the BM. The mechanisms underlying reflect the peculiar features of EVs that carry bioactive cargo like proteins and non-coding RNAs on short and long distance to reprogram tumor microenvironment and modulate the immune response. In this context, we and others reported that tumor-derived EVs are equipped on the surface with immune-checkpoint and immunosuppressive molecules such as HLA-G. These EVs are often associated with disease progression, tumor metastases, and poor clinical outcome and their role in neuroblastoma (NB) and multiple myeloma (MM) is here reported. Although NB and MM are very different tumors in terms of origin and age of diagnosis, they share the common feature that grow up into the BM causing a dysregulation of the BM microenvironment. EVs take part in this process.

Background:Anti-GD2 monoclonal antibody effectively treats high-risk neuroblastoma (HR-NB) by recruiting NK cells for antibody-dependent cellular cytotoxicity (ADCC). We recently developed a cell product containing mature, cytotoxic γδ T and NK cells (GADEKILL), and its potential use as a novel immunotherapy for HR-NB has been investigated. Methods:The GADEKILL γδ T and NK cells were analyzed by flow cytometry for the expression of activating and inhibitory receptors and for cytotoxicity against NB, both with and without dinutuximab-β, at a 1:1 effector-to-target ratio. NB cell lines with high and low/absent GD2 expression, as well as patient-derived 3D tumor spheres, all GD2-expressing, were used as targets. Comparative analyses were performed between GADEKILL NK and purified NK cells obtained from the same donor leukapheresis. Furthermore, a panel of NB cell lines was tested for the expression of B7H6 (i.e., NKp30 ligand), Human influenza hemagglutinin-tag (HA-TAG) and calreticulin (i.e., NKp46 ligands), and butyrophilin (BTN)2A1 and BTN3A1/2/3 (i.e., TCRVδ2 ligands), and the impact on GADEKILL cytotoxicity was assessed. Results:Compared to their purified counterparts, GADEKILL NK cells showed: (i) higher expression of NKp30 and NKp44 and lower expression of CD16 and NKG2D, (ii) greater cytotoxicity (CD107a+) against GD2- NB cells, (iii) stronger induction of lysis in low GD2-expressing NB cells and patient-derived 3D tumor spheres, and (iv) comparable ADCC. In addition, both γδ T and NK cells degranulated and consistently induced lysis in a panel of NB cell lines and patient-derived 3D tumor spheres expressing B7H6, calreticulin, HA-TAG, BTN2A1, and BTN3A1/2/3 consistently. Finally, NB cell lysis positively correlated with B7H6 and BTN2A1, and B7H6-blocking experiments revealed a significant decrease in target cell lysis when cells highly expressing B7H6 were used as targets. Conclusions:Our study demonstrated the potential antineuroblastoma activity of the GADEKILL, supporting its therapeutic use, particularly in the context of relapsed/refractory R/R HR-NB with low GD2 expression.

γδ T lymphocytes and NK cells are effective to kill tumors or viral-infected cells avoiding graft versus host disease (GvHD), thus they have attracted high interest as potential tool for adoptive cell therapy. We generated an advanced therapy medicinal product (ATMP) composed of mature γδ T and NK cells to provide an innovative tool to protect patients against tumor relapse and life-threatening infection after haploidentical hematopoietic stem cell transplantation. The ATMP was manufactured and validated in a GMP facility and was obtained from leukapheresis stimulated with zoledronic acid and IL-2, afterward depleted of αβ T lymphocytes using the CliniMACS Prodigy. The ATMP is characterized by high homogeneity, cell viability, cytotoxic abilities, stability after cryogenic preservation, and it was virtually free of αβ T and B lymphocytes. Both NK and γδ T cells were activated and characterized by high expression of cytotoxic and activating receptors including NKG2D, CD16, NKp30, NKp44, and NKp46. Furthermore, γδ T lymphocytes and NK cells were cytotoxic against myeloid leukemia or neuroblastoma cells. In conclusion, we implemented a novel ATMP to be shortly translated into clinical practice, which may be used in the post-transplant phase as efficacious immunotherapy in neuroblastoma and leukemic pediatric patients.

IntroductionExtracellular vesicles (EVs) are critical mediators of intercellular communication and contribute to cancer progression and immune regulation.MethodsWe characterized EVs isolated from bone marrow (BM) plasma harvested from healthy donors and patients affected by Multiple Myeloma (MM) by Nano Tracking Analysis and by flow cytometry.ResultsEVs from MM patients were significantly more abundant and enriched in CD138, supporting their partial origin from malignant plasma cells, with additional input from BM resident cells, including monocytes and NK cells. Phenotypic profiling revealed increased expression of immune checkpoint molecules HLA-G, PD-1, and PD-L1 on MM-derived EVs compared to healthy controls. Functionally, MM-EVs suppressed Staphylococcal enterotoxin B (SEB)-induced T cell activation, as evidenced by reduced IFN-γ production and CD4+ T cell proliferation. Such effects were partially reversed by HLA-G blockade. Moreover, MM-derived EVs modulated cytokine secretion profiles suppressing IL-2, IFN-α, TNF-α, and IL-6, and enhancing GM-CSF, with some changes attributed to HLA-G and PD-L1 activity. Transcriptomic analysis showed higher HLA-G expression in patients with gain of chromosome 1q, suggesting a link between high-risk cytogenetics and EV-driven immune suppression. While clinical correlations were not observed, likely due to limited sample size, these findings underscore the immunosuppressive role of MM-derived EVs.DiscussionHLA-G+, PD-1+, and PD-L1+ EVs contribute to immune dysfunction in MM and represent promising targets to restore anti-tumor immunity.

Background/Objectives: Several studies reported that olive leaf extract (OLE) may exert potent anti-cancer activities against human solid and hematological tumors. Such effects are mostly related to the polyphenol oleuropein and its derivatives, which are highly concentrated in OLE. Here, we investigated the anti-tumor effects of OLE in vitro against human acute leukemia and lymphoma cells. Methods: Cell proliferation and apoptosis have been evaluated by flow cytometry (using CFSE and Annexin-V/7AAD, respectively) in the presence or absence of OLE at different concentrations and in combination with or without chemotherapeutic drugs. Cellular pathways have been analyzed using antibody arrays. Results: OLE inhibited cell proliferation and induced apoptosis in B-acute lymphoblastic leukemia (B-ALL) and, to a lesser extent, in lymphomas and acute myeloid leukemia (AML) cell lines. Notably, OLE-induced apoptosis also occurs in primary leukemic blasts from B-ALL patients, both at diagnosis and at relapse, but only marginally in primary AML blasts. The expression and phosphorylation of proteins involved in the induction of apoptosis were modulated by OLE in B-ALL, whereas modest effects were observed in AML. Interestingly, some proteins were modulated in opposite ways in B-ALL and AML, potentially explaining their different responses to OLE. Finally, a synergistic and additive effect was observed for OLE in combination with cytarabine, but not with cyclophosphamide. Conclusions: We may envisage that OLE may be used as a food supplement in B-ALL patients treated with cytarabine, taking advantage of the potentiated effect of chemotherapy, without additional side effects.

The role of extracellular vesicles has been extensively studied in physiological and pathological conditions, and growing evidence has pinpointed them as key players in tumor progression, regulation of the metastatic niche, and modulation of anti-tumor immune responses. Indeed, a dynamic transfer of extracellular vesicles between cancer cells and immunological or non-immunological cells homing in the tumor microenvironment exists, and the balance between their release by cancer cells and by normal cells determines cancer progression. Here, we focused on the role of extracellular vesicles in the dysregulation of the bone marrow environment in pediatric tumors such as acute leukemias and neuroblastomata, whose poor prognosis is strictly related to the involvement of such anatomical site. Acute leukemias arise from bone marrow progenitors, whereas approximately 50% of neuroblastoma patients have bone marrow metastases at diagnosis. Thus, here, we discuss the mechanisms underlying the bone marrow dysregulation in pediatric acute leukemias and neuroblastomata with particular emphasis on the involvement of extracellular vesicles.

IntroductionQuality and safety of a cell product, essential to guarantee the health of patients, depends on many factors including an appropriate environmental monitoring of the manufacturing rooms. Nonetheless, the maintenance of a controlled environment is requested to minimize the risk of contamination. Thus, a timely detection of changes in microbiological trends is important to adopt promptly effective measures against resistant strains that, in turn, may invalidate not only the sanitization procedures but also the safety of the cell product.MethodsWe analyzed microbes found in our cell processing clean room over the last 5 years. We used 10.147 plates for air sampler, passive air monitoring and for checking instruments and operators of the production unit.ResultsFrom these plates, 747 colonies were subjected to identification by the MALDI-TOF Vitek® MS system and the large majority of them was gram positive (97.8%) as witnessed by the finding that the most represented genera harvested from the classified areas were Staphylococcus (65%), Micrococcus (13%), Kocuria (8%) and Bacillus (5%). We never detected fungi. Most microbes found in the operators (both from class A and B) were collected from forearms and resulted of the Staphylococcus genus.ConclusionsThe observed microbial contamination is to be attributed to the personnel and no substantial microbial pitfalls in our Cell Factory has been detected.

The use of γδ T lymphocytes as advanced therapeutic medicinal product has attracted much interest in the last years. Indeed, such cells are an ideal tool for the reconstitution of the immune system in patients receiving hematopoietic stem cell transplantation, due to their MHC-independent anti-tumor and anti-viral activities. We have here setup a protocol for the production of pure and functional γδ T lymphocytes, expanded from healthy donors’ mononuclear cells, and validated the analytical methods to identify them and to analyze their potency. Next, we performed stability studies to ensure that the cell product (γδ T cells) can be used after freezing and thawing. Notably, such protocol can be promptly translated to GMP-facility, since it has been designed using only clinical grade reagents.

Background & AimPlacenta is a non-controversial and readily available source of cells for regenerative medicine. Human amnion epithelial cells (hAEC) from term placenta, once transplanted in immune-competent recipients, have been reported to engraft and survive, boosting the innate capacity of regeneration or correcting congenital disorders. Recently, it has been proposed that hAEC therapeutic potential is not mediated by intact cells only, but efficiently supported by hAEC secretome. Extracellular vesicles are secreted by all cells, but their protein and nucleic acid cargo varies significantly according to cell of origin.We coupled complete profiling of surface molecules and enzymes on intact hAEC and hAEC-derived EVs (hAEV), with cargo analysisMethods, Results & ConclusionhAEC from 20 full-term placentae were isolated according to cGMP procedures, and flow cytometric evaluation validated both hAEC and hAEV identity and surface enzymes. We purified hAEV and sequenced non-coding and microRNA cargo (Illumina MiSeq technology). Finally, we quantified and screened soluble factors (by Luminex and OLINK technologies)We identified several mediators and enzymes on the surface of intact hAEC, transferred to hAEVs. Amnion cells characteristically lack HLA class 2 expression and express both class 1a and non-polymorphic class 1b. We measured the constitutive presence of membrane-bound HLA-G on hAEC and hAEV, in addition to soluble isoforms. Both hAEC and hAEV modulate immune cells, in a dose-dependent matter. hAEC/hAEV purinergic mediators modulated immune effector cells (T-, B- and NK-cells), while soluble mediators induced the macrophage switch from M1 to M2. Finally, we qualified and sequenced biomolecules contained in small and large hAEV, revealing anti-fibrotic effects as well as a potential role in oncological treatment.The updated paradigm is that hAEC do not necessarily need to mature into adult cells, but they can rescue native parenchymal cells via indirect paracrine. The ability to treat most common (chronic/congenital) diseases with allogeneic stem cells without the administration of immunosuppressive drugs will greatly expand the number of patients who could receive cellular therapy. Immune evasive capacity could be a “game changer”, and the modulation, rather than suppression, of innate and adaptive immune cells may result in enhanced cell treatments for regenerative purposes, autoimmune disorders, and tumors treated with augmented immune response

IntroductionExtracellular vesicles (EVs) can be released by any cell and are crucial for cell-to-cell communications. EVs have been characterized in patients with solid and hematological tumors, where they play an important role in tumor progression and metastasis. EVs may express different surface proteins derived from the parental cells, including immunomodulatory molecules, such as HLA-G and PDL1.MethodsWe isolated EV from bone marrow (BM) samples of patients with Neuroblastoma (NB) and healthy controls and we analyzed the expression of CD56, GD2 and immune checkpoints on EV by flow cytometry. Next, we analyzed the function of T cells in vitro in the presence or absence of NB patients' BM-derived EV, in terms of proliferation and cytokine production. Finally, we analyzed the correlation between the expression of immune checkpoints on EV and the clinical outcome of patients.ResultsWe found a higher expression of CD56 on EVs derived from BM of patients with NB than in those from healthy donors (HD). However, CD56 expression was not dependent on BM infiltration of NB cells. Moreover, the analysis of GD2 expression revealed that only a small fraction of EVs was released by infiltrating NB cells, whereas the majority may derive from BM-resident cells. BM-derived EVs from NB patients display a higher expression of HLA-G and PD-L1 than those derived from HD. Nonetheless, such EVs are able to modulate T cell immune responses. We measured a robust response, in vitro, towards a common bacterial antigen, including the release of GM-CSF and proinflammatory cytokines, like IFN-a and IL-6, from mononuclear cells. Some of these immunomodulatory features are dependent on the expression of HLA-G and PD-L1, whereas others may rely on other mechanism(s). Finally, a high expression of CD56, HLA-G and PD-L1 on BM-derived EVs may represent a good prognostic factor.ConclusionsWe described the presence of HLA-G and PDL1-bearing EVs in the BM of NB patients, which may represent a mechanism performed by resident BM cells to counteract the inflammation occurring in the BM microenvironment of NB patients.

Supplementary Figure S7 - PDF file 603K, Immunochemical analysis of EBI3 expression in EOC. Immunochemistry with an anti-EBI3 Ab shows EBI3 expression predominantly by reactive cells in both ascites (A) and tumor tissues (B). Arrows indicate examples of tumor cell nests. The sections were observed with a Nikon Eclipse 80i light microscope equipped with a color camera imaging head, using a 40x objective. Bar=100m

Supplementary Figure S6 - PDF file 185K, A: Analysis of the correlation between IL18BP and EBI3 mRNA levels in high grade (Type II) tumors in two microarray datasets of EOC. A significant correlation was found between EBI3 and IL18BP in both datasets, suggesting a relationship between the expression of EBI3 and IL18BP mRNA in EOC cell primary tumors. Pearson's correlation coefficients are shown (r). Lines represent the best fit linear regression analysis with the 95% Confidence Interval. B: Association between different EBI3 mRNA expression levels and Progression Free Survival (PFS) in Type II EOC of the Tothill microarray dataset. High EBI3 mRNA levels are associated to a shorter PFS time. Median PFS was 13 months for EBI3 levels higher than third quartile versus 21 months for EBI3 levels lower than first quartile (P=0.016). Solid line: cases with EBI3 levels lower than first quartile (n.52). Dashed line: cases with EBI3 levels higher than third quartile (n.49). P values were determined using log-rank test.

Supplementary Figure S1 - PDF file 744K, Receiver Operating Characteristic (ROC) curve for IL-18BP serum levels at diagnosis in patients with all types and stages of ovarian tumors (malignant n. 48 versus normal controls n. 13): the area under the curve (AUC) value is significant with the 95% confidence interval indicated in parentheses. SE=standard error

Supplementary Table S1. Antibodies used in immunostaining. Supplementary Table S2. Characteristics and outcomes by levels of IL-30 expression in tumor cells (primary tumor). Supplementary Table S3. Characteristics and outcomes by levels of IL-30 expression in leukocytes infiltrating the primary tumor. Supplementary Table S4. Multivariable regression models evaluating the association between cancer stage at baseline and IL-30 expression levels.

Supplementary Figure S3 - PDF file 9497K, A: Immunochemical analysis of IL-18BP expression in cells from EOC ascites. Double staining with anti-IL-18BP (red, rabbit mAb clone EP1088Y, Epitomics) and anti-macrophage (brown, mAb HAM-56, Ventana Medical Systems) antibodies is shown. Biotin-labeled goat anti-rabbit followed by alkaline phosphatase-conjugated streptavidin and peroxidase-conjugated anti-mouse (BioSpa) were used as secondary antibodies. Fast Red and DAB (Sigma) served as substrates. Bar=100m. Some of the cells stained by the anti-macrophage Ab were also stained by anti-IL-18BP Ab (see enlarged inset). However, the brightest IL-18BP positive cells were negative for the anti-macrophage Ab. B: Two-color immunofluorescence analysis of IL-18BP protein versus leukocyte surface markers expression in cells from EOC ascites. Numbers indicate the % of cells in each quadrant. IL-18BP positive cells were CD13 positive and CD14 low or negative (as all the CD14 positive cells were within the CD13 positive population: lower right panel). FITC: fluorescein isothiocyanate; APC: allophycocyanin; PE: phycoerythrin. CD14APC and CD14PE were from Miltenyi Biotec, CD13PE from BD Pharmingen and NKp46PE from Beckman Coulter. Cells were analyzed on a FACSCalibur (Becton Dickinson) flow cytometer

Supplementary Figure S2 - PDF file 2513K, A and B: Analysis of the correlation between IL-18 and IL-18BP protein levels in EOC sera (n. 47) (A) and ascites (n. 17) (B). No significant correlation (P=ns) was found by Pearson's test. C: Analysis of the correlation between IL18 and IL-18BP protein levels and IFN-? in the ascites of 15 EOC patients. IFN-? levels are low to undetectable and show no correlation with IL-18 or IL-18BP levels (P=ns). Pearson's correlation coefficients are shown (r). Lines represent the best-fit linear regression analysis with the 95% Confidence Interval

Supplementary Figure S4 - PDF file 640K, Immunochemical analysis of IL-18BP expression in EOC cell lines and xenotransplants. Immunochemistry with an anti-IL-18BP Ab shows virtually no reactivity in EOC cell lines (A upper left panel, B left panel), whereas IL-27-cultured A2774 cells (B middle panel) and areas of orthotopic SKOV3 (A) and A2774 (B right panel) xenotransplants express IL-18BP. The sections were observed with a Nikon Eclipse 80i light microscope equipped with a color camera imaging head, using a 40x objective. Bar=100micron