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.

Nutlin-3a Enhances Natural Killer Cell-Mediated Killing of Neuroblastoma by Restoring p53-Dependent Expression of Ligands for NKG2D and DNAM-1 Receptors.

In this study, we explored whether Nutlin-3a, a well-known, nontoxic small-molecule compound antagonizing the inhibitory interaction of MDM2 with the tumor suppressor p53, may restore ligands for natural killer (NK) cell-activating receptors (NK-AR) on neuroblastoma cells to enhance the NK cell-mediated killing. Neuroblastoma cell lines were treated with Nutlin-3a, and the expression of ligands for NKG2D and DNAM-1 NK-ARs and the neuroblastoma susceptibility to NK cells were evaluated. Adoptive transfer of human NK cells in a xenograft neuroblastoma-bearing NSG murine model was assessed. Two data sets of neuroblastoma patients were explored to correlate p53 expression with ligand expression. Luciferase assays and chromatin immunoprecipitation analysis of p53 functional binding on PVR promoter were performed. Primary neuroblastoma cells were also treated with Nutlin-3a, and neuroblastoma spheroids obtained from one high-risk patient were assayed for NK-cell cytotoxicity. We provide evidence showing that the Nutlin-3a-dependent rescue of p53 function in neuroblastoma cells resulted in (i) increased surface expression of ligands for NK-ARs, thus rendering neuroblastoma cell lines significantly more susceptible to NK cell-mediated killing; (ii) shrinkage of human neuroblastoma tumor masses that correlated with overall survival upon adoptive transfer of NK cells in neuroblastoma-bearing mice; (iii) and increased expression of ligands in primary neuroblastoma cells and boosting of NK cell-mediated disaggregation of neuroblastoma spheroids. We also found that p53 was a direct transcription factor regulating the expression of PVR ligand recognized by DNAM-1. Our findings demonstrated an immunomodulatory role of Nutlin-3a, which might be prospectively used for a novel NK cell-based immunotherapy for neuroblastoma.

The BET-bromodomain inhibitor JQ1 renders neuroblastoma cells more resistant to NK cell-mediated recognition and killing by downregulating ligands for NKG2D and DNAM-1 receptors.

Low expression of ligands for NK cell-activating receptors contributes to neuroblastoma (NB) aggressiveness. Recently, we demonstrated that the expression of MYCN, a poor prognosis marker in NB, inversely correlates with that of activating ligands. This indicates that MYCN expression level can predict the susceptibility of NB cells to NK cell-mediated immunotherapy and that its downregulation can be exploited as a novel therapeutic strategy to induce the expression of activating ligands. Here we evaluated the effect of the BET-bromodomain inhibitor JQ1 on the expression of ligands for NK cell-activating receptors in NB cell lines. Although downmodulating MYCN, JQ1 impaired the expression of ligands for NK cell-activating receptors, rendering NB cell lines more resistant to NK cell-mediated killing. The downregulation of activating ligands was due to JQ1-mediated impaired functions of both c-MYC and p53, two transcription factors known to regulate the expression of ULBP1-3 ligands for NKG2D activating receptor. Moreover JQ1 strongly downregulated the levels of ROS, a stress-induced signaling event associated with the induction of ligands for NK cell-activating receptors. These results suggest that the use of JQ1 should be discourage in combination with NK cell-based immunotherapy in a perspective chemotherapeutic treatment of NB. Thus, further investigations, exploiting molecular strategies aimed to boost the NK cell-mediated killing of NB cells, are warranted.

Bone Marrow CX3CL1/Fractalkine is a New Player of the Pro-Angiogenic Microenvironment in Multiple Myeloma Patients.

C-X3-C motif chemokine ligand 1 (CX3CL1)/fractalkine is a chemokine released after cleavage by two metalloproteases, ADAM metallopeptidase domain 10 (ADAM10) and ADAM metallopeptidase domain 17 (ADAM17), involved in inflammation and angiogenesis in the cancer microenvironment. The role of the CX3CL1/ C-X3-C motif chemokine receptor 1(CX3CR1) axis in the multiple myeloma (MM) microenvironment is still unknown. Firstly, we analyzed bone marrow (BM) plasma levels of CX3CL1 in 111 patients with plasma cell disorders including 70 with active MM, 25 with smoldering myeloma (SMM), and 16 with monoclonal gammopathy of undetermined significance (MGUS). We found that BM CX3CL1 levels were significantly increased in MM patients compared to SMM and MGUS and correlated with BM microvessel density. Secondly, we explored the source of CX3CL1 in MM and BM microenvironment cells. Primary CD138⁺ cells did not express CXC3L1 but up-regulated its production by endothelial cells (ECs) through the involvement of tumor necrosis factor alpha (TNFα). Lastly, we demonstrated the presence of CX3CR1 on BM CD14⁺CD16⁺ monocytes of MM patients and on ECs, but not on MM cells. The role of CX3CL1 in MM-induced angiogenesis was finally demonstrated in both in vivo chick embryo chorioallantoic membrane and in vitro angiogenesis assays. Our data indicate that CX3CL1, present at a high level in the BM of MM patients, is a new player of the MM microenvironment involved in MM-induced angiogenesis.

Tumor-infiltrating T cells and PD-L1 expression in childhood malignant extracranial germ-cell tumors.

Although pediatric malignant extracranial germ-cell tumors (meGCTs) are among the most chemosensitive solid tumors, a group of patients relapse and die of disease. To identify new markers predicting clinical outcome, we examined the prognostic relevance of tumor-infiltrating T lymphocytes (TILs) and the expression of PD-1 and PD-L1 in a cohort of pediatric meGCTs by in situ immunohistochemistry. MeGCTs were variously infiltrated by T cell-subtypes according to the tumor subtype, tumor location and age at diagnosis. We distinguished three different phenotypes: i) tumors not infiltrated by T cells (immature teratomas and half of the yolk sac tumors), ii) tumors highly infiltrated by CD8+ T cells expressing PD-1, which identifies activated tumor-reactive T cells (seminomas and dysgerminomas), iii) tumors highly infiltrated by CD8+ T cells within an immunosuppressive tumor microenvironment characterized by CD4+FOXP3+ Treg cells and PD-L1-expressing tumor cells (embryonal carcinomas, choriocarcinomas and the remaining yolk sac tumors). Tumor subtypes belonging mixed meGCTs were variously infiltrated, suggesting the coexistence of multiple immune microenvironments either facilitating or precluding the entry of T cells. These findings support the hypothesis that TILs influence the development of meGCTs and might be of clinical relevance to improve risk stratification and the treatment of pediatric patients.

CD38: A Target for Immunotherapeutic Approaches in Multiple Myeloma.

Multiple Myeloma (MM) is a hematological cancer characterized by proliferation of malignant plasma cells in the bone marrow (BM). MM represents the second most frequent hematological malignancy, accounting 1% of all cancer and 13% of hematological tumors, with ~9,000 new cases per year. Patients with monoclonal gammopathy of undetermined significance (MGUS) and asymptomatic smoldering MM (SMM) usually evolve to active MM in the presence of increased tumor burden, symptoms and organ damage. Despite the role of high dose chemotherapy in combination with autologous stem cell transplantation and the introduction of new treatments, the prognosis of MM patients is still poor, and novel therapeutic approaches have been tested in the last years, including new immunomodulatory drugs, proteasome inhibitors and monoclonal antibodies (mAbs). CD38 is a glycoprotein with ectoenzymatic functions, which is expressed on plasma cells and other lymphoid and myeloid cell populations. Since its expression is very high and uniform on myeloma cells, CD38 is a good target for novel therapeutic strategies. Among them, immunotherapy represents a promising approach. Here, we summarized recent findings regarding CD38-targeted immunotherapy of MM in pre-clinical models and clinical trials, including (i) mAbs (daratumumab and isatuximab), (ii) radioimmunotherapy, and (iii) adoptive cell therapy, using chimeric antigen receptor (CAR)-transfected T cells specific for CD38. Finally, we discussed the efficacy and possible limitations of these therapeutic approaches for MM patients.

Human γδ T-Cells: From Surface Receptors to the Therapy of High-Risk Leukemias.

γδ T lymphocytes are potent effector cells, capable of efficiently killing tumor and leukemia cells. Their activation is mediated by γδ T-cell receptor (TCR) and by activating receptors shared with NK cells (e.g., NKG2D and DNAM-1). γδ T-cell triggering occurs upon interaction with specific ligands, including phosphoantigens (for Vγ9Vδ2 TCR), MICA-B and UL16 binding protein (for NKG2D), and PVR and Nectin-2 (for DNAM-1). They also respond to cytokines undergoing proliferation and release of cytokines/chemokines. Although at the genomic level γδ T-cells have the potential of an extraordinary TCR diversification, in tissues they display a restricted repertoire. Recent studies have identified various γδ TCR rearrangements following either hematopoietic stem cell transplantation (HSCT) or cytomegalovirus infection, accounting for their "adaptive" potential. In humans, peripheral blood γδ T-cells are primarily composed of Vγ9Vδ2 chains, while a minor proportion express Vδ1. They do not recognize antigens in the context of MHC molecules, thus bypassing tumor escape based on MHC class I downregulation. In view of their potent antileukemia activity and absence of any relevant graft-versus-host disease-inducing effect, γδ T-cells may play an important role in the successful clinical outcome of patients undergoing HLA-haploidentical HSCT depleted of TCR αß T/CD19+ B lymphocytes to cure high-risk acute leukemias. In this setting, high numbers of both γδ T-cells (Vδ1 and Vδ2) and NK cells are infused together with CD34+ HSC and may contribute to rapid control of infections and leukemia relapse. Notably, zoledronic acid potentiates the cytolytic activity of γδ T-cells in vitro and its infusion in patients strongly promotes γδ T-cell differentiation and cytolytic activity; thus, treatment with this agent may contribute to further improve the patient clinical outcome after HLA-haploidentical HSCT depleted of TCR αß T/CD19+ B lymphocytes.

CHL1 gene acts as a tumor suppressor in human neuroblastoma.

Neuroblastoma is an aggressive, relapse-prone childhood tumor of the sympathetic nervous system that accounts for 15% of pediatric cancer deaths. A distal portion of human chromosome 3p is often deleted in neuroblastoma, this region may contain one or more putative tumor suppressor genes. A 2.54 Mb region at 3p26.3 encompassing the smallest region of deletion pinpointed CHL1 gene, the locus for neuronal cell adhesion molecule close homolog of L1. We found that low CHL1 expression predicted poor outcome in neuroblastoma patients. Here we have used two inducible cell models to analyze the impact of CHL1 on neuroblastoma biology. Over-expression of CHL1 induced neurite-like outgrowth and markers of neuronal differentiation in neuroblastoma cells, halted tumor progression, inhibited anchorage-independent colony formation, and suppressed the growth of human tumor xenografts. Conversely, knock-down of CHL1 induced neurite retraction and activation of Rho GTPases, enhanced cell proliferation and migration, triggered colony formation and anchorage-independent growth, accelerated growth in orthotopic xenografts mouse model. Our findings demonstrate unambiguously that CHL1 acts as a regulator of proliferation and differentiation of neuroblastoma cells through inhibition of the MAPKs and Akt pathways. CHL1 is a novel candidate tumor suppressor in neuroblastoma, and its associated pathways may represent a promising target for future therapeutic interventions.

Neuroblastoma Cell Lines Are Refractory to Genotoxic Drug-Mediated Induction of Ligands for NK Cell-Activating Receptors.

Neuroblastoma (NB), the most common extracranial solid tumor of childhood, causes death in almost 15% of children affected by cancer. Treatment of neuroblastoma is based on the combination of chemotherapy with other therapeutic interventions such as surgery, radiotherapy, use of differentiating agents, and immunotherapy. In particular, adoptive NK cell transfer is a new immune-therapeutic approach whose efficacy may be boosted by several anticancer agents able to induce the expression of ligands for NK cell-activating receptors, thus rendering cancer cells more susceptible to NK cell-mediated lysis. Here, we show that chemotherapeutic drugs commonly used for the treatment of NB such as cisplatin, topotecan, irinotecan, and etoposide are unable to induce the expression of activating ligands in a panel of NB cell lines. Consistently, cisplatin-treated NB cell lines were not more susceptible to NK cells than untreated cells. The refractoriness of NB cell lines to these drugs has been partially associated with the abnormal status of genes for ATM, ATR, Chk1, and Chk2, the major transducers of the DNA damage response (DDR), triggered by several anticancer agents and promoting different antitumor mechanisms including the expression of ligands for NK cell-activating receptors. Moreover, both the impaired production of reactive oxygen species (ROS) in some NB cell lines and the transient p53 stabilization in response to our genotoxic drugs under our experimental conditions could contribute to inefficient induction of activating ligands. These data suggest that further investigations, exploiting molecular strategies aimed to potentiate the NK cell-mediated immunotherapy of NB, are warranted.

IL-25 dampens the growth of human germinal center-derived B-cell non Hodgkin Lymphoma by curtailing neoangiogenesis.

Interleukin (IL)-25, a member of the IL-17 cytokine superfamily, is produced by immune and non-immune cells and exerts type 2 pro-inflammatory effects in vitro and in vivo. The IL-25 receptor(R) is composed of the IL-17RA/IL-17RB subunits. Previous work showed that germinal centre (GC)-derived B-cell non Hodgkin lymphomas (B-NHL) expressed IL-17AR, formed by IL-17RA and IL-17RC subunits, and IL-17A/IL-17AR axis promoted B-NHL growth by stimulating neoangiogenesis. Here, we have investigated expression and function of IL-25/IL-25R axis in lymph nodes from human GC-derived B-NHL, i.e. Follicular Lymphoma (FL,10 cases), Diffuse Large B Cell Lymphoma (6 cases) and Burkitt Lymphoma (3 cases). Tumor cells expressed IL-25R and IL-25 that was detected also in non-malignant cells by flow cytometry. Immunohistochemical studies confirmed expression of IL-25R and IL-25 in FL cells, and highlighted IL-25 expression in bystander elements of the FL microenvironment. IL-25 i) up-regulated phosphorylation of NFkBp65, STAT-1 and JNK in B-NHL cells; ii) inhibited in vitro proliferation of the latter cells; iii) exerted anti-tumor activity in two in vivo B-NHL models by dampening expression of pro-angiogenic molecules as VEGF-C, CXCL6 and ANGPT3. In conclusion, IL-25, that is intrinsically pro-angiogenic, inhibits B-NHL growth by reprogramming the angiogenic phenotype of B-NHL cells.

Boosting Natural Killer Cell-Based Immunotherapy with Anticancer Drugs: a Perspective.

Natural killer (NK) cells efficiently recognize and kill tumor cells through several mechanisms including the expression of ligands for NK cell-activating receptors on target cells. Different clinical trials indicate that NK cell-based immunotherapy represents a promising antitumor treatment. However, tumors develop immune-evasion strategies, including downregulation of ligands for NK cell-activating receptors, that can negatively affect antitumor activity of NK cells, which either reside endogenously, or are adoptively transferred. Thus, restoration of the expression of NK cell-activating ligands on tumor cells represents a strategic therapeutic goal. As discussed here, various anticancer drugs can fulfill this task via different mechanisms. We envision that the combination of selected chemotherapeutic agents with NK cell adoptive transfer may represent a novel strategy for cancer immunotherapy.

Interleukin-31 and thymic stromal lymphopoietin expression in plasma and lymph node from Hodgkin lymphoma patients.

Hodgkin Lymphoma (HL) is a tumor of B-cell origin characterized by Hodgkin and Reed-Stenberg (H/RS) cells embedded in an inflammatory tissue where numerous cytokines/chemokines contribute to shape the microenvironment, leading to the typical clinical symptoms. We investigated: i) the expression of Interleukin-IL-31 (IL-31) and Thymic Stromal Lymphopoietin (TSLP), two Th2-related cytokines with tumor-promoting and pruritogenic functions, and of the respective receptors in HL invaded lymph nodes by flow cytometry, and ii) the potential association of IL-31/TSLP plasma concentrations with clinical characteristics by ELISA. H/RS cells and the major immune cell types infiltrating HL lymph nodes expressed intracytoplasmic and surface IL-31/TSLP, and their receptors. A subgroup of patients showing at diagnosis elevated IL-31 and TSLP plasma levels had an International Prognostic Score>2, indicative of high risk of relapse, and a subsequent positive interim PET-scan, indicative of insufficient response to chemotherapy. No correlation was found between IL-31/TSLP plasma levels and overall or event-free survival. In conclusion, IL-31/TSLP and their receptors are expressed in HL cells and in immune cells infiltrating affected lymph nodes, where both cytokines may contribute to local immune suppression. The clinical impact of IL-31 and TSLP plasma levels has to be further defined in larger patient cohorts.

Altered erythropoiesis and decreased number of erythrocytes in children with neuroblastoma.

Neuroblastoma (NB) is a pediatric tumor presenting at diagnosis either as localized or metastatic disease, which mainly involves the bone marrow (BM). The physical occupancy of BM space by metastatic NB cells has been held responsible for impairment of BM function. Here, we investigated whether localized or metastatic NB may alter hematopoietic lineages' maturation and release of mature cells in the periphery, through gene expression profiling, analysis of BM smears, cell blood count and flow cytometry analysis. Gene ontology and disease-associated analysis of the genes significantly under-expressed in BM resident cells from children with localized and metastatic NB, as compared to healthy children, indicated anemia, blood group antigens, and heme and porphyrin biosynthesis as major functional annotation clusters. Accordingly, in children with NB there was a selective impairment of erythrocyte maturation at the ortho-chromic stage that resulted in reduced erythrocyte count in the periphery, regardless of the presence of metastatic cells in the BM. By considering all NB patients, low erythrocyte count at diagnosis associated with worse survival. Moreover, in the subset of metastatic patients, low erythrocyte count, hemoglobin and hematocrit and high red cell distribution width at follow-up also associated with worse outcome. These observations provide an alternative model to the tenet that infiltrating cells inhibit BM functions due to physical occupancy of space and may open a new area of research in NB to understand the mechanism(s) responsible for such selective impairment.

Use of luciferase probes to measure ATP in living cells and animals.

ATP, the energy exchange factor that connects anabolism and catabolism, is required for major reactions and processes that occur in living cells, such as muscle contraction, phosphorylation and active transport. ATP is also the key molecule in extracellular purinergic signaling mechanisms, with an established crucial role in inflammation and several additional disease conditions. Here, we describe detailed protocols to measure the ATP concentration in isolated living cells and animals using luminescence techniques based on targeted luciferase probes. In the presence of magnesium, oxygen and ATP, the protein luciferase catalyzes oxidation of the substrate luciferin, which is associated with light emission. Recombinantly expressed wild-type luciferase is exclusively cytosolic; however, adding specific targeting sequences can modify its cellular localization. Using this strategy, we have constructed luciferase chimeras targeted to the mitochondrial matrix and the outer surface of the plasma membrane. Here, we describe optimized protocols for monitoring ATP concentrations in the cytosol, mitochondrial matrix and pericellular space in living cells via an overall procedure that requires an average of 3 d. In addition, we present a detailed protocol for the in vivo detection of extracellular ATP in mice using luciferase-transfected reporter cells. This latter procedure may require up to 25 d to complete.

MYCN is an immunosuppressive oncogene dampening the expression of ligands for NK-cell-activating receptors in human high-risk neuroblastoma.

Neuroblastoma (NB) is the most common extracranial solid tumor occurring in childhood. Amplification of the MYCN oncogene is associated with poor prognosis. Downregulation on NB cells of ligands recognized by Natural Killer (NK) cell-activating receptors, involved in tumor cell recognition and lysis, may contribute to tumor progression and relapse. Here, we demonstrate that in human NB cell lines MYCN expression inversely correlates with that of ligands recognized by NKG2D and DNAM1 activating receptors in human NB cell lines. In the MYCN-inducible Tet-21/N cell line, downregulation of MYCN resulted in enhanced expression of the activating ligands MICA, ULBPs and PVR, which rendered tumor cells more susceptible to recognition and lysis mediated by NK cells. Conversely, a MYCN non-amplified NB cell line transfected with MYCN showed an opposite behavior compared with control cells. Consistent with these findings, an inverse correlation was detected between the expression of MYCN and that of ligands for NK-cell-activating receptors in 12 NB patient specimens both at mRNA and protein levels. Taken together, these results provide the first demonstration that MYCN acts as an immunosuppressive oncogene in NB cells that negatively regulates the expression of ligands for NKG2D and DNAM-1 NK-cell-activating receptors. Our study provides a clue to exploit MYCN expression levels as a biomarker to predict the efficacy of NK-cell-based immunotherapy in NB patients.

The IL-31/IL-31 receptor axis: general features and role in tumor microenvironment.

IL-31 is a recently identified cytokine with a well-defined role in the pathogenesis of pruritus. IL-31, whose production is induced by IL-4 and IL-33, binds a heterodimeric receptor (R) composed of the exclusive IL-31RA chain and the shared oncostatin M R. Signaling through the IL-31R involves the MAPK, PI3K/AKT and Jak/STAT pathways. Different variants and isoforms of IL-31RA with different signaling activities have been identified. IL-31 is produced predominantly by circulating Th2 lymphocytes and skin-homing CLA+CD45RO+ T cells. Studies in humans have demonstrated a pathogenic role for IL-31 in atopic dermatitis and allergic asthma. The first demonstration of the involvement of the IL-31/IL-31R axis in cancer came from studies in patients with mycosis fungoides/Sézary syndrome, the most frequent, cutaneous T cell lymphoma. Tumor cells were shown to produce IL-31, whose serum levels correlated with pruritus intensity. Follicular lymphoma (FL) B cells and their counterparts-germinal center B cells-produced IL-31 and expressed IL-31R, which signaled in the former, but not the latter, cells. IL-31 released in association with microvesicles promoted tumor growth through autocrine/paracrine loops. Malignant mast cells from patients with mastocytosis or Philadelphia-negative myeloproliferative disorder produced IL-31, which contributed to pruritus pathogenesis. Finally, patients with endometrial carcinoma displayed high serum levels of IL-31 and IL-33, which may represent promising disease biomarkers. Targeting strategies for the IL-31/IL-31R axis have been developed, including the CIMM331 humanized anti-human IL-31RA antibody recently tested in a phase I/Ib study.

Constitutional 3p26.3 terminal microdeletion in an adolescent with neuroblastoma.

BACKGROUND: Neuroblastoma (NB) is a common and often lethal cancer of early childhood that accounts for 10% of pediatric cancer mortality. Incidence peaks in infancy and then rapidly declines, with less than 5% of cases diagnosed in children and adolescents ≥ 10 y. There is increasing evidence that NB has unique biology and an chronic disease course in older children and adolescents, but ultimately dismal survival. METHODS: We describe a rare constitutional 3p26.3 terminal microdeletion which occurred in an adolescent with NB, with apparently normal phenotype without neurocognitive defects. We evaluated the association of expression of genes involved in the microdeletion with NB patient outcomes using R2 platform. We screened NB patient's tumor cells for CHL1 protein expression using immunofluorescence. RESULTS: Constitutional and tumor DNA were tested by array-comparative genomic hybridization and single nucleotide-polymorphism-array analyses. Peripheral blood mononuclear cells from the patient showed a 2.54 Mb sub-microscopic constitutional terminal 3p deletion that extended to band p26.3. The microdeletion 3p disrupted the CNTN4 gene and the neighboring CNTN6 and CHL1 genes were hemizygously deleted, each of these genes encode neuronal cell adhesion molecules. Low expression of CNTN6 and CNTN4 genes did not stratify NB patients, whereas low CHL1 expression characterized 417 NB patients having worse overall survival. CHL1 protein expression on tumor cells from the patient was weaker than positive control. CONCLUSION: This is the first report of a constitutional 3p26.3 deletion in a NB patient. Since larger deletions of 3p, indicative of the presence of one or more tumor suppressor genes in this region, occur frequently in neuroblastoma, our results pave the way to the identification of one putative NB suppressor genes mapping in 3p26.3.

Mesenchymal stromal cells and autoimmunity.

Mesenchymal stromal cells (MSCs) are committed progenitors of mesodermal origin that are found virtually in every organ and exhibit multilineage differentiation into osteocytes, adipocytes and chondrocytes. MSCs also mediate a wide spectrum of immunoregulatory activities that usually dampen innate and adaptive immune responses. These features have attracted interest in the perspective of developing novel cell therapies for autoimmune disease. However, depending on the microenvironmental conditions, MSCs may show a plastic behavior and switch to an immunostimulatory phenotype. After thorough characterization of the effects of MSCs on the immune system, MSC cell therapy has been tested in animal models of autoimmunity using different cell sources, protocols of in vitro expansion and routes and schedules of administration. The pre-clinical results have been encouraging in some models [e.g. Crohn's disease (CD), multiple sclerosis] and heterogeneous in others (e.g. graft-versus-host disease, systemic lupus erythematosus, rheumatoid arthritis). Clinical trials have been carried out and many are ongoing. As discussed, the results obtained are too preliminary to draw any conclusion, with the only exception of topical administration of MSCs in CD that has proven efficacious. The mechanism of action of infused MSCs is still under investigation, but the apparent paradox of a therapeutic effect achieved in spite of the very low number of cells reaching the target organ has been solved by the finding that MSC-derived extracellular vesicles (EVs) closely mimic the therapeutic activity of MSCs in pre-clinical models. These issues are critically discussed in view of the potential clinical use of MSC-derived EVs.

Bevacizumab-mediated tumor vasculature remodelling improves tumor infiltration and antitumor efficacy of GD2-CAR T cells in a human neuroblastoma preclinical model.

GD2-redirected chimeric antigen receptor (CAR) T lymphocytes represent a promising therapeutic option for immunotherapy of neuroblastoma (NB). However, despite the encouraging therapeutic effects observed in some hematological malignancies, clinical results of CAR T cell immunotherapy in solid tumors are still modest. Tumor driven neo-angiogenesis supports an immunosuppressive microenvironment that influences treatment responses and is amenable to targeting with antiangiogenic drugs. The latter agents promote lymphocyte tumor infiltration by transiently reprogramming tumor vasculature, and may represent a valid combinatorial approach with CAR T cell immunotherapy. In light of these considerations, we investigated the anti-NB activity of GD2-CAR T cells combined with bevacizumab (BEV) in an orthotopic xenograft model of human NB. Two weeks after tumor implantation, mice received BEV or GD2-CAR T cells or both by single intravenous administration. GD2-CAR T cells exerted a significant anti-NB activity only in combination with BEV, even at the lowest concentration tested, which per se did not inhibit tumor growth. When combined with BEV, GD2-CAR T cells massively infiltrated tumor mass where they produced interferon-γ (IFN-γ), which, in turn, induced expression of CXCL10 by NB cells. IFN-γ, and possibly other cytokines, upregulated NB cell expression of PD-L1, while tumor infiltrating GD2-CAR T cells expressed PD-1. Thus, the PD-1/PD-L1 axis can limit the anti-tumor efficacy of the GD2-CAR T cell/BEV association. This study provides a strong rationale for testing the combination of GD2-CAR T cells with BEV in a clinical trial enrolling NB patients. PD-L1 silencing or blocking strategies may further enhance the efficacy of such combination.