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The inflammatory tissue microenvironment that promotes the development of breast cancer is not fully understood. Here we report a role for elevated IL30 in supporting the breast cancer cell viability and invasive migration. IL30 was absent in normal mammary ducts, ductules, and acini of histologically normal breast and scanty in the few stromal infiltrating leukocytes. In contrast, IL30 was expressed frequently in breast cancer specimens where it was associated with triple-negative and HER2+ molecular subtypes. In stromal leukocytes found in primary tumors or tumor-draining lymph nodes, which included mainly CD14+ monocytes, CD68+ macrophages, and CD33+/CD11b+ myeloid cells, IL30 levels increased with disease stage and correlated with recurrence. A negative correlation was determined between IL30 expression by nodal stromal leukocytes and overall survival. In vitro studies showed that human recombinant IL30 upregulated expression of a pro-oncogenic program, including especially IL6 in both triple-negative and HER2+ breast cancer cells. In triple-negative breast cancer cells, IL30 boosted a broader program of proliferation, invasive migration, and an inflammatory milieu associated with KISS1-dependent metastasis. Silencing of STAT1/STAT3 signaling hindered the regulation of the primary growth and progression factors in breast cancer cells. IL30 administration in vivo fostered the growth of triple-negative breast cancer by promoting proliferation and vascular dissemination of cancer cells and the accumulation of intratumoral CD11b+/Gr1+ myeloid cell infiltrates. Overall, our results show how IL30 regulates breast cancer cell viability, migration, and gene expression to promote breast cancer growth and progression and its impact on patient outcome. Cancer Res; 76(21); 6218–29. ©2016 AACR.

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 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.

Purpose: Interleukin (IL)-18 is an immune-enhancing cytokine, which induces IFN-γ production, T-helper 1 responses, and antitumor effects. In turn, IFN-γ stimulates IL-18–binding protein production, which blocks IL-18 activity. In view of the potential use of IL-18 in epithelial ovarian cancer (EOC) immunotherapy, here, we studied IL-18BP expression and its regulation by cytokines in EOC cells in vitro and in vivo.Experimental Design: Expression and production of IL-18BP in EOC cell lines, primary ovarian carcinomas, and the corresponding normal tissues, patients' serum, and ascites were investigated by immunochemistry, ELISA, screening of gene expression profiles, and reverse-transcription PCR.Results: Analysis of gene expression profiles revealed that IL18BP mRNA is increased in EOC tumors compared with normal ovary cells. Release of IL-18BP was detectable in EOC sera and to a greater extent in the ascites, indicating production at the tumor site. Indeed, immunochemical analyses on cells isolated from the ascites and on tumor sections indicated that IL-18BP is expressed in both tumor cells and tumor-associated leukocytes, which displayed a CD3−CD20−NKp46−CD13+CD14low phenotype. EOC cell lines do not constitutively express IL-18BP. However, its release is inducible both by IFN-γ stimulation in vitro and by xenotransplantation of EOC cells in immune-deficient mice, suggesting a role for the microenvironment. In vitro experiments and immunochemistry indicated that IL-27 is also involved in IL-18BP upregulation in EOC cell lines and primary cells through STAT1 activation. Together, these data indicate that IL-18BP, which is produced in EOC in response to microenvironmental factors, may inhibit endogenous or exogenous IL-18 activity. Clin Cancer Res; 19(17); 4611–20. ©2013 AACR.

Background The oncolytic viruses have shown promising results for the treatment of multiple myeloma. However, the use of human viruses is limited by the patients’ antiviral immune response. In this study, we investigated an alternative oncolytic strategy using non-human pathogen viruses as the bovine viral diarrhea virus (BVDV) that were able to interact with CD46. Methods We treated several human myeloma cell lines and non-myeloma cell lines with BVDV to evaluate the expression of CD46 and to study the effect on cell viability by flow cytometry. The possible synergistic effect of bortezomib in combination with BVDV was also tested. Moreover, we infected the bone marrow mononuclear cells obtained from myeloma patients and we checked the BVDV effect on different cell populations, defined by CD138, CD14, CD3, CD19, and CD56 expression evaluated by flow cytometry. Finally, the in vivo BVDV effect was tested in NOD-SCID mice injected subcutaneously with myeloma cell lines. Results Human myeloma cells were selectively sensitive to BVDV treatment with an increase of cell death and, consequently, of apoptotic markers. Consistently, bone marrow mononuclear cells isolated from myeloma patients treated with BVDV, showed a significant selective decrease of the percentage of viable CD138+ cells. Interestingly, bortezomib pre-treatment significantly increased the cytotoxic effect of BVDV in myeloma cell lines with a synergistic effect. Finally, the in vitro data were confirmed in an in vivo myeloma mouse model showing that BVDV treatment significantly reduced the tumoral burden compared to the vehicle. Conclusions Overall, our data indicate, for the first time, a direct oncolytic effect of the BVDV in human myeloma cells suggesting its possible use as novel alternative anti-myeloma virotherapy strategy.

Neuroblastoma is the most common extracranial pediatric solid tumor with a heterogeneous clinical course, ranging from spontaneous regression to metastatic disease and death, irrespective of intensive chemotherapeutic regimen. On the basis of several parameters, children affected by neuroblastoma are stratified into low, intermediate and high risk. At present, more than 50% of high-risk patients with metastatic spread display an overall poor long-term outcome also complicated by devastating long-term morbidities. Thus, novel and more effective therapies are desperately needed to improve lifespan of high-risk patients. In this regard, adoptive cell therapy holds great promise and several clinical trials are ongoing, demonstrating safety and tolerability, with no toxicities. Starting from the immunological and clinical features of neuroblastoma, we here discuss the immunotherapeutic approaches currently adopted for high-risk patients and different innovative therapeutic strategies currently under investigation. The latter are based on the infusion of natural killer (NK) cells, as support of consolidation therapy in addition to standard treatments, or chimeric antigen receptor (CAR) T cells directed against neuroblastoma associated antigens (e.g., disialoganglioside GD2). Finally, future perspectives of adoptive cell therapies represented by γδ T lymphocyes and CAR NK cells are envisaged.

The inflammatory tissue microenvironment that promotes the development of breast cancer is not fully understood. Here we report a role for elevated IL30 in supporting the breast cancer cell viability and invasive migration. IL30 was absent in normal mammary ducts, ductules, and acini of histologically normal breast and scanty in the few stromal infiltrating leukocytes. In contrast, IL30 was expressed frequently in breast cancer specimens where it was associated with triple-negative and HER2+ molecular subtypes. In stromal leukocytes found in primary tumors or tumor-draining lymph nodes, which included mainly CD14+ monocytes, CD68+ macrophages, and CD33+/CD11b+ myeloid cells, IL30 levels increased with disease stage and correlated with recurrence. A negative correlation was determined between IL30 expression by nodal stromal leukocytes and overall survival. In vitro studies showed that human recombinant IL30 upregulated expression of a pro-oncogenic program, including especially IL6 in both triple-negative and HER2+ breast cancer cells. In triple-negative breast cancer cells, IL30 boosted a broader program of proliferation, invasive migration, and an inflammatory milieu associated with KISS1-dependent metastasis. Silencing of STAT1/STAT3 signaling hindered the regulation of the primary growth and progression factors in breast cancer cells. IL30 administration in vivo fostered the growth of triple-negative breast cancer by promoting proliferation and vascular dissemination of cancer cells and the accumulation of intratumoral CD11b+/Gr1+ myeloid cell infiltrates. Overall, our results show how IL30 regulates breast cancer cell viability, migration, and gene expression to promote breast cancer growth and progression and its impact on patient outcome. Cancer Res; 76(21); 6218–29. ©2016 AACR.

The importance of glutamine (Gln) metabolism in multiple myeloma (MM) cells and its potential role as a therapeutic target are still unknown, although it has been reported that human myeloma cell lines (HMCLs) are highly sensitive to Gln depletion. In this study, we found that both HMCLs and primary bone marrow (BM) CD138(+) cells produced large amounts of ammonium in the presence of Gln. MM patients have lower BM plasma Gln with higher ammonium and glutamate than patients with indolent monoclonal gammopathies. Interestingly, HMCLs expressed glutaminase (GLS1) and were sensitive to its inhibition, whereas they exhibited negligible expression of glutamine synthetase (GS). High GLS1 and low GS expression were also observed in primary CD138(+) cells. Gln-free incubation or treatment with the glutaminolytic enzyme l-asparaginase depleted the cell contents of Gln, glutamate, and the anaplerotic substrate 2-oxoglutarate, inhibiting MM cell growth. Consistent with the dependence of MM cells on extracellular Gln, a gene expression profile analysis, on both proprietary and published datasets, showed an increased expression of the Gln transporters SNAT1, ASCT2, and LAT1 by CD138(+) cells across the progression of monoclonal gammopathies. Among these transporters, only ASCT2 inhibition in HMCLs caused a marked decrease in Gln uptake and a significant fall in cell growth. Consistently, stable ASCT2 downregulation by a lentiviral approach inhibited HMCL growth in vitro and in a murine model. In conclusion, MM cells strictly depend on extracellular Gln and show features of Gln addiction. Therefore, the inhibition of Gln uptake is a new attractive therapeutic strategy for MM.

In a previous study, lack of IL-12 signaling in il12rb2 knock-out mice was found to predispose to lung adenocarcinoma (LAC). We asked whether specific polymorphisms of the human IL12RB2 gene may confer susceptibility to LAC. We studied IL12RB2 single nucleotide polymorphisms (SNPs) spanning from the promoter to the first untranslated exon of the gene. Genotypes of 49 individuals with LAC were compared with those of 93 healthy subjects. Two allele variants were found to be associated with increased susceptibility to LAC. One haplotype (hap), hap18, was more frequent in patients (18%) versus controls (6%) and significantly associated with increased probability of disease occurrence. Furthermore, IL-12 driven STAT4 phosphorylation in T cell blasts from healthy individuals was found to correlate with both single allele variants and haplotypes. In conclusion, genetically determined low signaling activity of IL-12R predisposes to the development of LAC.

Background & Aim Viral infections remain a leading cause of morbidity and mortality after transplantation. Although prophylactic pharmacotherapy is effective in reducing the risk for some viral infections, therapeutic options for breakthrough infections are still unsatisfactory. Conventional pharmacologic agents against Adenovirus (AdV) and Epstein-Barr virus (EBV) have limited efficacy and relevant toxicity, whereas in the case of cytomegalovirus (CMV) are documented better response rates, but toxicity and reactivation after treatment stop are frequent. T-cell reconstitution is a key requirement for effective antiviral control following transplantation, and factors that influence the speed of T-cell recovery impact also the risk of viral infection in this period. The use of lymphocyte infusions derived from seropositive donors is an effective salvage therapy for viral infections in transplanted recipients prior to T-cell recovery, but the risk of severe graft-versus-host disease (GVHD) is a critical concern. Thus, we propose a novel immunotherapeutic approach based on gd T cells specific for AdV, CMV and EBV. Many studies highlighted the importance of gd T cells in antiviral immunity, uniquely combining conventional adaptive features with rapid, innate-like responses. These lymphocytes are effector cells that: i) recognize target cells in a MHC-independent manner, thus do not cause GvHD; ii) participate CMV and EBV responses, particularly when conventional adaptive immunity is insufficient to clear the viral infection; iii) may develop memory responses, and iv) cross-talk with other immune cells, including NK lymphocytes, alfa beta T cells and dendritic cells. These features render gd T cells extremely appealing for clinical purposes. We believe that an ATMP containing Vd1 and Vd2 gd T cells will be the ideal immunotherapeutic approach to prevent and treat viral infections after transplantation. Methods, Results & Conclusion To this end, we are developing a new method to obtain a cell product containing expanded Vd1 and Vd2 gd T cells using exclusively GMP-graded reagents, including zoledronic acid, cytokines, viral peptides (related to AdV, CMV and EBV) and ab depletion techniques. Such cell product has been tested for i) phenotype, ii) allo-reactivity, iii) cytotoxicity and iv) specificity. Our preliminary results strongly support the feasibility for development of new immunotherapeutic protocols using gd T cells, with the aim of preventing and treating viral infections after transplantation.

Although the progression of chronic lymphocytic leukemia (CLL) requires the cooperation of the microenvironment, the exact cellular and molecular mechanisms involved are still unclear. We investigated the interleukin (IL)-23 receptor (IL-23R)/IL-23 axis and found that circulating cells from early-stage CLL patients with shorter time-to-treatment, but not of those with a more benign course, expressed a defective form of the IL-23R complex lacking the IL-12Rβ1 chain. However, cells from both patient groups expressed the complete IL-23R complex in tissue infiltrates and could be induced to express the IL-12Rβ1 chain when cocultured with activated T cells or CD40L+ cells. CLL cells activated in vitro in this context produced IL-23, a finding that, together with the presence of IL-23 in CLL lymphoid tissues, suggests the existence of an autocrine/paracrine loop inducing CLL cell proliferation. Interference with the IL-23R/IL-23 axis using an anti-IL-23p19 antibody proved effective in controlling disease onset and expansion in xenografted mice, suggesting potential therapeutic strategies.

// Silvia Esposito 1, 2, * , Marco V. Russo 1, 2, * , Irma Airoldi 4 , Maria Grazia Tupone 1, 2 , Carlo Sorrentino 1, 2, 3 , Giulia Barbarito 4 , Serena Di Meo 1, 2 , Emma Di Carlo 1, 2 1 Department of Medicine and Sciences of Aging, Section of Anatomic Pathology and Molecular Medicine, “G. d'Annunzio” University, Chieti, Italy 2 Ce.S.I. Aging Research Center, “G. d'Annunzio” University Foundation, Chieti, Italy 3 Specialisation School in Clinical Biochemistry, “G. d'Annunzio” University, Chieti, Italy 4 Laboratory of Oncology, Istituto Giannina Gaslini, Genova, Italy * These authors have contributed equally to this work Correspondence to: Emma Di Carlo, e-mail: edicarlo@unich.it Keywords: Prostate Cancer, Neuroendocrine Differentiation, Laser Capture Microdissection, SNAI2/Slug Received: August 25, 2014 Accepted: November 11, 2014 Published: February 05, 2015 ABSTRACT Prostate Cancer (PCa)-related deaths are mostly due to metastasization of poorly differentiated adenocarcinomas often endowed with neuroendocrine differentiation (NED) areas. The SNAI2/Slug gene is a major regulator of cell migration and tumor metastasization. We here assessed its biological significance in NED, and metastatic potential of PCa. SNAI2 expression was down-regulated in most PCa epithelia, in association with gene promoter methylation, except for cell clusters forming: a. the expansion/invasion front of high-grade PCa, b. NED areas, or c. lymph node metastasis. Knockdown of SNAI2 in PC3 cells down-regulated the expression of neural-tissue-associated adhesion molecules, Neural-Cadherin, Neural-Cadherin-2, Neuronal-Cell-Adhesion-Molecule, and of the NED marker Neuron-Specific Enolase, whereas it abolished Chromogranin-A expression. The metastasis-suppressor genes, Nm23-H1 and KISS1, were up-regulated, while the pluripotency genes SOX2, NOTCH1, CD44v6, WWTR1/TAZ and YAP1 were dramatically down-regulated. Over-expression of SNAI2 in DU145 cells substantiated its ability to regulate metastasis-suppressor, NED and pluripotency genes. In PCa and lymph node metastasis, expression of SOX2 and NOTCH1 was highly related to that of SNAI2. In conclusion, I. SNAI2 silencing in PCa may turn-off the expression of NED markers and pluripotency genes, while turning-on that of specific metastasis-suppressors, II. SNAI2 expression in selected PCa cells, by regulating their self-renewal, NED and metastatic potential, endows them with highly malignant properties. SNAI2 may thus constitute a key target for modern approaches to PCa progression.

// Irma Airoldi 1, * , Maria Grazia Tupone 2, 3, * , Silvia Esposito 2, 3 , Marco V. Russo 2, 3 , Giulia Barbarito 1 , Giuseppe Cipollone 4, 5 , Emma Di Carlo 2, 3 1 Laboratory of Oncology, Istituto “Giannina Gaslini”, Genova 16147, Italy 2 Anatomic Pathology and Molecular Medicine, Department of Medicine and Sciences of Aging, “G. d'Annunzio” University, Chieti 66100, Italy 3 Ce.S.I. Biotech, Aging Research Center, “G. d'Annunzio” University Foundation, Chieti 66100, Italy 4 Department of Experimental and Clinical Sciences, “G. d'Annunzio” University, Chieti 66100, Italy 5 General and Thoracic Surgery, “SS Annunziata” Hospital, Chieti 66100, Italy * These authors have contributed equally to this work Correspondence to: Emma Di Carlo, e-mail: edicarlo@unich.it Keywords: cytokines, lung cancer, tumor microenvironment, inflammation, immunotherapy Received: October 15, 2014 Accepted: November 20, 2014 Published: January 03, 2015 ABSTRACT Current therapies for Non-Small Cell Lung Cancer (NSCLC) still fail to significantly increase its survival rate. Here we asked whether Interleukin(IL)-27, which has revealed powerful antitumor activity and is toxicity-free in humans, is a promising therapeutic choice for NSCLC patients. IL-27's effects were tested on Adenocarcinoma (AC) and Squamous Cell Carcinoma (SCC) cell lines and xenograft models. IL-27Receptor(R) expression was assessed in lung tissues from 78 NSCLC patients. In vitro , IL-27 was ineffective on cancer cell proliferation or apoptosis, but fostered CXCL3/GROγ/MIP2β expression. In vitro and in vivo , IL-27 down-regulated stemness-related genes, namely SONIC HEDGEHOG in AC cells, and OCT4A , SOX2 , NOTCH1 , KLF4 along with Nestin , SNAI1/SNAIL , SNAI2/SLUG and ZEB1 , in SCC cells. In vivo , IL-27 hampered both AC and SCC tumor growth in association with a prominent granulocyte- and macrophage-driven colliquative necrosis, CXCL3 production, and a reduced pluripotency- and EMT-related gene expression. Myeloablation of tumor-bearing hosts mostly abolished IL-27's antitumor effects. In clinical samples, IL-27R expression was found in AC, SCC, pre-cancerous lesions and tumor infiltrating myeloid cells, and correlated with advanced stages of disease. Our data suggest that even immunocompromised or advancer NSCLC patients may benefit from IL-27's antitumor properties based on its ability to drive myeloid cells towards antitumor activities, and down-regulate stemness- and EMT-related genes in cancer cells.

We demonstrated that γδ T cells of patients given HLA-haploidentical HSCT after removal of αβ+ T cells and CD19+ B cells are endowed with the capacity of killing leukemia cells after ex vivo treatment with zoledronic acid (ZOL). Thus, we tested the hypothesis that infusion of ZOL in patients receiving this type of graft may enhance γδ T-cell cytotoxic activity against leukemia cells. ZOL was infused every 28 d in 43 patients; most were treated at least twice. γδ T cells before and after ZOL treatments were studied in 33 of these 43 patients, till at least 7 mo after HSCT by high-resolution mass spectrometry, flow-cytometry, and degranulation assay. An induction of Vδ2-cell differentiation, paralleled by increased cytotoxicity of both Vδ1 and Vδ2 cells against primary leukemia blasts was associated with ZOL treatment. Cytotoxic activity was further increased in Vδ2 cells, but not in Vδ1 lymphocytes in those patients given more than one treatment. Proteomic analysis of γδ T cells purified from patients showed upregulation of proteins involved in activation processes and immune response, paralleled by downregulation of proteins involved in proliferation. Moreover, a proteomic signature was identified for each ZOL treatment. Patients given three or more ZOL infusions had a better probability of survival in comparison to those given one or two treatments (86% vs. 54%, respectively, p = 0.008). Our data indicate that ZOL infusion in pediatric recipients of αβ T- and B-cell-depleted HLA-haploidentical HSCT promotes γδ T-cell differentiation and cytotoxicity and may influence the outcome of patients.

Purpose: Multiple myeloma (MM) is a hematologic malignancy characterized by a clonal expansion of plasma cells (PCs) in the bone marrow (BM). Since MM has so far remained incurable, further insights into its pathogenesis and the concomitant identification of new therapeutic targets are urgently needed. The tyrosine kinase receptor EphA3 is known to be involved in various cellular processes including cell viability, cell movement and cell-cell interactions. Recently, EphA3 has emerged as a potential therapeutic target in several hematologic and solid tumors. Here, we aimed to uncover the role of EphA3 in MM. Methods: EphA3 mRNA and protein expression in primary MM bone marrow plasma cells (BMPCs), in MM-derived cell lines and in healthy controls (HCs) was assessed using qRT-PCR, Western blotting and flow cytometry. The effects of siRNA-mediated EphA3 silencing and anti EphA3 antibody (EphA3mAb) treatment on MM PC trafficking and viability were evaluated using in vitro assays. The effects of EphA3mAb treatment were also assessed in two MM-derived mouse xenograft models. Results: We found that EphA3 was overexpressed in primary MM BMPCs and MM-derived cell lines compared to HCs. We also found that siRNA-mediated EphA3 silencing and EphA3mAb treatment significantly inhibited the ability of MM PCs to adhere to fibronectin and stromal cells and to invade in vitro, without affecting cell proliferation and viability. Gene expression profiling showed that EphA3 silencing resulted in expression modulation of several molecules that regulate adhesion, migration and invasion processes. Importantly, we found that EphA3mAb treatment significantly inhibited in vivo tumor growth and angiogenesis in two MM-derived mouse xenograft models. Conclusions: Our findings suggest that EphA3 plays an important role in the pathogenesis of MM and provide support for the notion that its targeting may represent a novel therapeutic opportunity for MM.

Purpose: The interleukin (IL)-27 cytokine subunit p28, also called IL-30, has been recognized as a novel immunoregulatory mediator endowed with its own functions. These are currently the subject of discussion in immunology, but completely unexplored in cancer biology. We set out to investigate the role of IL-30 in prostate carcinogenesis and its effects on human prostate cancer (hPCa) cells. Experimental Design: IL-30 expression, as visualized by immunohistochemistry and real-time reverse transcriptase PCR on prostate and draining lymph nodes from 125 patients with prostate cancer, was correlated with clinicopathologic data. IL-30 regulation of hPCa cell viability and expression of selected gene clusters was tested by flow cytometry and PCR array. Results: IL-30, absent in normal prostatic epithelia, was expressed by cancerous epithelia with Gleason ≥ 7% of 21.3% of prostate cancer stage I to III and 40.9% of prostate cancer stage IV. IL-30 expression by tumor infiltrating leukocytes (T-ILK) was higher in stage IV that in stage I to III prostate cancer (P = 0.0006) or in control tissue (P = 0.0011). IL-30 expression in prostate draining lymph nodes (LN)-ILK was higher in stage IV than in stage I to III prostate cancer (P = 0.0031) or in control nodes (P = 0.0023). The main IL-30 sources were identified as CD68+ macrophages, CD33+/CD11b+ myeloid cells, and CD14+ monocytes. In vitro, IL-30 stimulated proliferation of hPCa cells and also downregulated CCL16/LEC, TNFSF14/LIGHT, chemokine-like factor (CKLF), and particularly CKLF-like MARVEL transmembrane domain containing 3 (CMTM3) and greatly upregulated ChemR23/CMKLR. Conclusions: We provide the first evidence that IL-30 is implicated in prostate cancer progression because (i) its expression by prostate cancer or T- and LN-ILK correlates with advanced disease grade and stage; and (ii) IL-30 exerts protumor activity in hPCa cells. Clin Cancer Res; 20(3); 585–94. ©2013 AACR.

CD38 is a multifunctional cell surface protein endowed with receptor/enzymatic functions. The protein is generally expressed at low/intermediate levels on hematological tissues and some solid tumors, scoring the highest levels on plasma cells (PC) and PC-derived neoplasia. CD38 was originally described as a receptor expressed by activated cells, mainly T lymphocytes, wherein it also regulates cell adhesion and cooperates in signal transduction mediated by major receptor complexes. Furthermore, CD38 metabolizes extracellular NAD+, generating ADPR and cyclic ADPR. This ecto-enzyme controls extra-cellular nucleotide homeostasis and intra-cellular calcium fluxes, stressing its relevance in multiple physiopathological conditions (infection, tumorigenesis and aging). In clinics, CD38 was adopted as a cell activation marker and in the diagnostic/staging of leukemias. Quantitative surface CD38 expression by multiple myeloma (MM) cells was the basic criterion used for therapeutic application of anti-CD38 monoclonal antibodies (mAbs). Anti-CD38 mAbs-mediated PC depletion in autoimmunity and organ transplants is currently under investigation. This review analyzes different aspects of CD38’s role in regulatory cell populations and how these effects are obtained. Characterizing CD38 functional properties may widen the extension of therapeutic applications for anti-CD38 mAbs. The availability of therapeutic mAbs with different effects on CD38 enzymatic functions may be rapidly translated to immunotherapeutic strategies of cell immune defense.

Background & Aim Leukemia is the most common cancer in childhood, accounting for almost 1 out of 3 cases. Of these malignancies, most are acute lymphoblastic leukemia (ALL), with the remaining cases largely acute myeloid leukemia (AML). Despite remarkable advances in treatments, 50%-70% of all children with ALL and AML who relapse after allogeneic hematopoietic stem cell transplantation (HSCT), succumb to their disease. Thus, there is an urgent clinical need of novel therapies for improving prognosis. γδ T cells are a minor subset (1-5%) of CD3+ combining conventional adaptive features with rapid, innate-like responses that place them in the initiation phase of immune cascade. In addition, they recognize tumor cells in a major histocompatibility complex (MHC) independent manner, thereby bypassing the occurrence of Graft versus Host Disease (GvHD). gd T cells is mainly constituted of Vg9Vd2 and Vd1 subpopulations which has demonstrated a potent Graft versus Leukemia (GvL) effect. The Vg9Vd2 subset, which represents the main circulating proportion of gd T cells, recognizes phospoantigens presented by leukemic blasts. The Vd1 subset, conversely, residing within the epithelial tissues, especially at sites of cytomegalovirus (CMV) replication, exerts a protective anti-infections post-transplant role. Methods, Results & Conclusion Here we purpose to develop a novel strategy for ex vivo expanding both Vg9Vd2 and Vd1 gdT-cells in a clinical-scale fashion. The ultimate goal is to post-HSCT administer multiple infusions of expanded gd T cells in order to reduce the risk of leukemia recurrence. We have previously demonstrated that the in vitro exposure of gd T cells to zoledronic acid (ZOL) combined with IL-2, allows robust expansion and activation of the Vd2 subset alone. Herein, we developed a novel protocol for the expansion of both Vd1 and Vd2 by exploiting the mitogenic effect of Concanavalin-A (Con-A), together with ZOL and a cocktail of different cytokines (Table 1). Preliminary results obtained from 5 different healthy donors show that 1) we succeeded in expanding gd T cells up to 50 folds, 2) the use of Con-A is essential for the Vd1 subset expansion, 3) gd T cells ex vivo expanded acquire an effector memory phenotype (Fig 1) and are cytotoxic in vitro against pediatric acute leukemia cell lines. Therefore, these encouraging results lay the foundation for advancing the translational use of gd T cells.

// Emma Di Carlo 1,2 , Carlo Sorrentino 1,2 , Alessia Zorzoli 3 , Serena Di Meo 1,2 , Maria Grazia Tupone 1,2 , Emanuela Ognio 4 , Gabriella Mincione 2,5 and Irma Airoldi 3 1 Department of Medicine and Sciences of Aging, Section of Anatomic Pathology and Molecular Medicine, “G. d’Annunzio” University, Chieti, Italy 2 Ce.S.I. Aging Research Center, “G. d’Annunzio” University Foundation, Chieti, Italy 3 Laboratory of Oncology, Istituto Giannina Gaslini, Genova, Italy 4 Animal Facility, IRCCS AOU San Martino-IST Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy 5 Department of Experimental and Clinical Sciences, “G. d’Annunzio” University, Chieti, Italy Correspondence: Emma Di Carlo, email: // Keywords : prostate cancer, interleukin-27, cytokines, immunotherapy, anti-tumor activity, tumor microenvironment Received : September 19, 2013 Accepted : December 28, 2013 Published : December 28, 2013 Abstract Prostate cancer (PCa) is of increasing significance worldwide as a consequence of the population ageing. Fragile elderly patients may particularly benefit from noninvasive and well tolerable immunotherapeutic approaches. Preclinical studies have revealed that the immune-regulatory cytokine IL-27 may exert anti-tumor activities in a variety of tumor types without discernable toxicity. We, thus, investigated whether IL-27 may function as anti-tumor agent in human (h) PCa and analyzed the rationale for its clinical application. In vitro, IL-27 treatment significantly inhibited proliferation and reduced the angiogenic potential of hPCa cells by down-regulating the pro-angiogenesis-related genes fms-related tyrosine kinase (FLT)1, prostaglandin G/H synthase 1/cyclooxygenase-1 (PTGS1/COX-1) and fibroblast growth factor receptor (FGFR)3. In addition, IL-27 up-regulated the anti-angiogenesis-related genes such as CXCL10 and TIMP metallopeptidase inhibitor 3 (TIMP3). In vivo, IL-27 reduced proliferation and vascularization in association with ischemic necrosis of tumors developed after PC3 or DU145 cell injection in athymic nude mice. In patients’ prostate tissues, IL-27R was expressed by normal epithelia and low grade PCa and lost by high tumor grade and stages. Nevertheless, IL-27R was expressed by CD11c + , CD4 + and CD8 + leukocytes infiltrating the tumor and draining lymph nodes. These data lead to the conclusion that i) IL-27’s anti-PCa potential may be fully exploited in patients with well-differentiated, localized IL-27R positive PCa, since in this case it may act on both cancerous epithelia and the tumor microenvironment; ii) PCa patients bearing high grade and stage tumor that lack IL-27R may benefit, however, from IL-27’s immune-stimulatory properties.

Purpose: Interleukin (IL)-18 is an immune-enhancing cytokine, which induces IFN-γ production, T-helper 1 responses, and antitumor effects. In turn, IFN-γ stimulates IL-18–binding protein production, which blocks IL-18 activity. In view of the potential use of IL-18 in epithelial ovarian cancer (EOC) immunotherapy, here, we studied IL-18BP expression and its regulation by cytokines in EOC cells in vitro and in vivo. Experimental Design: Expression and production of IL-18BP in EOC cell lines, primary ovarian carcinomas, and the corresponding normal tissues, patients' serum, and ascites were investigated by immunochemistry, ELISA, screening of gene expression profiles, and reverse-transcription PCR. Results: Analysis of gene expression profiles revealed that IL18BP mRNA is increased in EOC tumors compared with normal ovary cells. Release of IL-18BP was detectable in EOC sera and to a greater extent in the ascites, indicating production at the tumor site. Indeed, immunochemical analyses on cells isolated from the ascites and on tumor sections indicated that IL-18BP is expressed in both tumor cells and tumor-associated leukocytes, which displayed a CD3−CD20−NKp46−CD13+CD14low phenotype. EOC cell lines do not constitutively express IL-18BP. However, its release is inducible both by IFN-γ stimulation in vitro and by xenotransplantation of EOC cells in immune-deficient mice, suggesting a role for the microenvironment. In vitro experiments and immunochemistry indicated that IL-27 is also involved in IL-18BP upregulation in EOC cell lines and primary cells through STAT1 activation. Together, these data indicate that IL-18BP, which is produced in EOC in response to microenvironmental factors, may inhibit endogenous or exogenous IL-18 activity. Clin Cancer Res; 19(17); 4611–20. ©2013 AACR.

International audience; The anti-tumoral effect of lenalidomide is increased in vivo by hypoxia-inducible factor (HIF)-1α inhibition in myeloma cells We investigated the effect of stable suppression of hypoxia inducible factor (HIF)-1α in myeloma cells on sensitivity to lenalidomide (LEN) in vivo. We found that the in vivo anti-tumoral effect of LEN is enhanced by HIF-1α suppression in myeloma cells. It has been reported that HIF-1α is over-expressed by myeloma cells 1-4 and that HIF-1α suppression significantly blocks myeloma-induced angiogenesis, and reduces both tumoral burden and bone destruction in vivo in multiple myeloma (MM) mouse models. 3 The potential effects of HIF-1α modulation on drug sensitivity in MM cells are not known and are currently under investigation. The immunomodulatory drugs (IMiDs®), including LEN, are a class of drugs derived from thalidomide 4 able to exert anti-myeloma effects by the selective Cereblon-dependent destruction of IKZF proteins, 5,6 either through a direct action on MM cell proliferation and survival, 7 or through indirect immunomodulatory and anti-angiogenic effects. 7 Previous data indicated that HIF-1α inhibition did not increase the anti-proliferative in vitro effect of bortezomib on MM cells; 2,3 this drug induces a strong downregula-tion of HIF-1α in MM cells. 2 We recently reported that HIF-1α knockdown in the human myeloma cell line (HMCL) JJN3 potentiated the in vitro effect of LEN treatment (48-72 h) on cell proliferation through a significant upregulation of p27 without changing cell viability. 3 It has been consistently reported that LEN only slightly down-regulated HIF-1α expression in MM cells. 2 Such evidence has provided the rationale to investigate the effect of stable suppression of HIF-1α in myeloma cells on LEN sensitivity in vivo. Therefore, in the present study, we first inhibited HIF-1α in three HMCLs (JJN3, OPM2 and H929) using an anti-HIF1α lentiviral shRNA pool, as previously described. 3 H929 showed a very high mortality rate after anti-HIF1α lentiviral infection (data not shown) and were not used for the subsequent in vivo experiments. We assessed the effect of LEN in combination with HIF-1α inhibition in a non-obese diabetic/severe combined immunodeficiency (NOD/SCID) subcutaneous in vivo mouse model. 3 Different groups of animals (5 animals in each group) of two sets of independent experiments were injected with JJN3 pLKO.1 (empty vector) or JJN3 anti-HIF1α. When tumors became palpable (approx. 7-10 days after injection) mice were treated with LEN 5 mg/kg (Selleckchem, Houston, TX, USA) or vehicle (DMSO) using the intraperitoneal route. The same mouse model, grouping strategy and treatment conditions were used for OPM2 pLKO.1 or OPM2 anti-HIF1α. After three weeks, we evaluated tumor volume and weight, as previously described, 3 and immunohisto-chemistry was used to evaluate the microvascular density (MVD) and checked by CD34 immunostaining (Santa Cruz, Dallas, TX, USA). 3 In addition, in the first set of mice experiments, the expression of p27 (Abcam, Cambridge, UK) was evaluated by immunohistochem-istry. The expression of S-phase kinase-associated protein 2 (SKP2), a p27 inhibitor, expression of the HIF-1α target key mediator of glycolysis and tumoral growth, Hexokinase II (HK2), and levels of pERK 1/2, and total Caspase-3 (Casp-3) were evaluated in the ex vivo plasma-cytoma lysates by western blot using the following anti-bodies: SKP2, Casp-3 (Santa Cruz, Dallas, TX, USA), HK2, pERK 1/2, (Cell Signaling, Danvers, MA, USA). b-actin was used as internal control (Millipore

IL-27 and TCRγδ+ T lymphocytes play critical roles in both innate and adaptive immune responses in health and disease, including infection and tumors. Although the activity of IL-27 is well characterized in different human immune cells, no information is available on the role of IL-27 in human TCRγδ+ T lymphocytes. Here, we provide the first evidence that TCRγδ+ T lymphocytes express both gp130 and WSX-1 chains of IL-27R, and that IL-27 may function in TCRγδ+ T cells by (i) inducing STAT1 and STAT3 phosphorylation, (ii) stimulating cytotoxicity against tumor cells through upregulation of cytotoxic granules production, (iii) reducing the release of Th2-related cytokines, such as IL-5 and IL-13, and inducing IFN-γ production, and (iv) upregulating the expression of CD62L. These results highlighted a novel immunoregulatory property of human IL-27 that may be relevant in the immune response against tumors. Our results may offer new perspectives for the development of future clinical trials using IL-27 and TCRγδ+ cells for cancer immunotherapy.

Review of data identifying IL-12 and IL-27 as potential therapeutic agents for pediatric AML by targeting leukemia initiating cells and/or blasts. AML is a hematologic malignancy that represents 15–20% of all childhood acute leukemias and is responsible for more than one-half of pediatric leukemic deaths. The bulk tumor is continuously regenerated and sustained by rare leukemic ICs that proliferate slowly, thus resulting refractory to chemotherapeutic agents targeting highly proliferating cells within the tumor. Therefore, a complete eradication of the bulk tumor may depend on efficacy of therapies that target IC. In spite of the improvements in the treatment of AML, the difficulty to eradicate completely the disease incites research for innovative therapeutic approaches. In this regard, the role of cytokines in the treatment of AML has been investigated for many years, and some of them have been tested in clinical trials as a result of their immunomodulatory properties. Furthermore, recent preclinical studies highlighted the ability of the IL-12 superfamily cytokines as potent antileukemic agents that act directly on tumor cells and on leukemic IC, thus opening new perspectives for leukemic patient treatment. Here, we review the current knowledge about the antileukemic effects of cytokines, documented in preclinical and clinical studies, discussing their potential clinical application.

Galectin-1 (Gal-1) is involved in tumoral angiogenesis, hypoxia and metastases. Actually the Gal-1 expression profile in multiple myeloma (MM) patients and its pathophysiological role in MM-induced angiogenesis and tumoral growth are unknown. In this study, we found that Gal-1 expression by MM cells was upregulated in hypoxic conditions and that stable knockdown of hypoxia inducible factor-1α significantly downregulated its expression. Therefore, we performed Gal-1 inhibition using lentivirus transfection of shRNA anti-Gal-1 in human myeloma cell lines (HMCLs), and showed that its suppression modified transcriptional profiles in both hypoxic and normoxic conditions. Interestingly, Gal-1 inhibition in MM cells downregulated proangiogenic genes, including MMP9 and CCL2, and upregulated the antiangiogenic ones SEMA3A and CXCL10. Consistently, Gal-1 suppression in MM cells significantly decreased their proangiogenic properties in vitro. This was confirmed in vivo, in two different mouse models injected with HMCLs transfected with anti-Gal-1 shRNA or the control vector. Gal-1 suppression in both models significantly reduced tumor burden and microvascular density as compared with the control mice. Moreover, Gal-1 suppression induced smaller lytic lesions on X-ray in the intratibial model. Overall, our data indicate that Gal-1 is a new potential therapeutic target in MM blocking angiogenesis.

Interleukin (IL)-23 and IL-27 are pro-inflammatory cytokines that share functional and structural similarities and may exert anti-tumor activities against solid and hematological malignancies. Here, we asked whether IL-23 and IL-27, alone or in combination, may act directly against human follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) cells. In this study, we demonstrated for the first time that human primary FL and DLBCL cells expressed complete and functional IL-23 and IL-27 receptors (R) and that IL-23 and IL-27 exerted anti-tumor activities in vitro and in vivo through different and complementary mechanisms. In vivo studies using severe combined immunodeficiency /non-obese diabetic mice-injected subcutaneously with human SU-DHL-4 cell line revealed that IL-23 inhibited directly tumor-cell proliferation, whereas IL-27 impaired the angiogenic program of lymphoma cells resulting in strong reduction of cell growth. In addition, combined treatment of IL-23 and IL-27 amplified the anti-tumor effects in vivo as compared with administration of each cytokine alone. These anti-tumor mechanisms were confirmed by in vitro experiments performed with primary lymphoma cells and cell lines. Our results strongly encourage the development of future clinical trials to evaluate the toxicity and efficacy of the IL-23 and IL-27 in lymphoma patients.

Childhood acute myeloid leukemia (AML) is a hematological malignancy in which tumor burden is continuously replenished by leukemic-initiating cells (ICs), which proliferate slowly and are refractory to chemotherapeutic agents. We investigated whether interleukin (IL)-12, an immuno-modulatory cytokine with anti-tumor activity, may target AML blasts (CD45(+)CD33(+)) and populations known to contain leukemia ICs (that is, CD34(+)CD38(-), CD33(+)CD38(+) and CD44(+)CD38(-) cells). We demonstrate for the first time that: i) AML blasts and their CD34(+)CD38(-), CD33(+)CD38(+), CD44(+)CD38(-) subsets express the heterodimeric IL-12 receptor (IL-12R), ii) AML cells injected subcutaneously into NOD/SCID/Il2rg(-/-) (NSG) mice developed a localized tumor mass containing leukemic ICs and blasts that were virtually eliminated by IL-12 treatment, iii) AML cells injected intravenously into NSG mice engrafted within the first month in the spleen, but not in bone marrow or peripheral blood. At this time, IL-12 dramatically dampened AML CD45(+)CD33(+), CD34(+)CD38(-), CD33(+)CD38(+) and CD44(+)CD38(-) populations, only sparing residual CD33(+)CD38(+) cells that did not express IL-12Rβ2. From 30 to 60 days after the initial inoculum, these IL-12-unresponsive cells expanded and metastasized in both control and IL-12-treated NSG mice. Our data indicate that the absence of IL-12Rβ2 in pediatric AML cells favours leukemia progression in NOD/SCID/IL2Rγc-deficient mice.

B-acute lymphoblastic leukemia (B-ALL) represents the most common pediatric hematological tumor that derives from the aberrant proliferation of early B lymphocytes in the bone marrow. Although most of the B-ALL children take advantage from current therapeutic protocols, some patients relapse and need alternative therapies. With this background, we investigated whether interleukin (IL)-27, an immunomodulatory cytokine with antitumor properties, may function as an antitumor agent against pediatric B-ALL cells. Here we show for the first time that pediatric B-ALL cells functional IL-27R and that IL-27 dampens directly tumor growth in vivo and in vitro through mechanisms elucidated in this study. The novelty of these results deals with the first demonstration that (1) B-ALL cells from pediatric patients injected intravenously (i.v.) into NOD/SCID/Il2rg(-/-) (NSG) mice gave rise to leukemic spreading that was severely hampered by IL-27; (2) IL-27-treated mice, compared with controls, showed significant reduction of putative B-ALL-initiating cells and blasts in the peripheral blood (PB), bone marrow (BM) and spleen; and that (3) IL-27 reduced in vitro B-ALL cell proliferation and angiogenesis, induced apoptosis and downregulated miR-155. Our results strongly encourage the development of future clinical trials to evaluate the toxicity and efficacy of IL-27 in childhood B-ALL patients.

B-acute lymphoblastic leukemia (ALL) is the most common hematologic tumor of pediatric age. Although patient survival has been improved, some cases still relapse and need alternative therapies. In this context, the role of microRNA in cancer is actually matter of investigation due to their regulatory function implicated in human tumorigenesis since the main target mRNA transcripts are involved in proliferation, apoptosis and differentiation. On the other hand, cytokines are actually used as adjuvants in B-ALL therapy, and recent studies reported that they may also function by acting directly against leukemic cells. Here we review the current knowledge about the role of miRNA and cytokines in B-ALL, highlighting the link between cytokine activity and miRNA expression. The translational relevance of these issues will be also discussed.

Interleukin (IL) -12 is a cytokine that has been extensively characterized for its immunoregulatory activities. IL-12 binds to a heterodimeric receptor composed of the β1 and β2 chains. In this review article, we discuss recent findings on the expression and function of IL-12 receptor (IL-12R) in malignant B cells frozen at various stages of differentiation and in their normal counterparts. These studies, together with others performed in Il12rb2 knockout mice, have established the concept that the IL-12Rβ2 gene is a gatekeeper from cancer. We will delineate three paradigms reflecting the differential expression of IL-12Rβ2 in different groups of malignant B cells and discuss the therapeutic perspectives stemming from these studies.

Metastatic melanoma is a poor prognosis skin cancer. Since conventional treatments including surgery and chemotherapy often fail, novel therapeutic strategies are needed. In particular, identification of melanoma associated antigen has fostered the progress of both active (vaccines) and adoptive immunotherapy. Some promising results have been obtained, but most melanoma patients are not yet cured possibly because of different immune-escape mechanisms operated by tumor cells. Several studies have addressed the use of interleukin (IL)-12 for melanoma therapy due to its immunoregulatory function and anti-tumor activity mediated by stimulation of T and NK effector cells. Unfortunately, IL-12 has shown considerable toxicity. We [1] have recently demonstrated that IL-12 exerts a direct anti-tumor activity on murine B16 melanoma cells expressing a functional IL-12 receptor (R). In our model low levels of endogenous IL-12 reduced proliferation, increased apoptosis, and defective microvessel formation of tumor cells. This review summarizes information about melanoma immunotherapy and highlights a novel mechanism of IL-12-mediated anti-tumor activity based upon the direct effect of the cytokine on IL-12R(+) tumor cells. In this view, new therapeutic approaches may be planned including: i) pre-screening of melanoma patients for IL-12Rbeta2 expression to identify potential responders, ii) administration of small and less frequent doses of IL-12 to avoid toxicity and iii) targeting of IL-12 to IL-12R(+) tumor cells, such as local administration in patients with skin tumors or injection of IL-12 fused to an antibody specific to tumor cells.

Up-regulation of some chemokine receptors on tumor cells is associated with increased metastatic potential. In this respect, limited information is available on chemokine receptor in human neuroblastoma (NB). Purpose of the study was to identify chemokines/chemokine receptors involved in bone marrow (BM) localization of metastatic NB cells in view of the development of targeted therapeutic strategies. CD45− metastatic NB cells were isolated from the BM of six patients by immunomagnetic bead manipulation. Some experiments were carried out using a panel of human neuroblastoma cell lines (GI-ME-N, GI-LI-N, LAN-5, HTLA-230, SH-SY-5Y and IMR-32). Immunophenotypic analyses were performed by flow cytometry. Cell migration assays were carried out using transwell systems. Calcium ion mobilization, chemokine receptor internalization and cell proliferation were investigated by flow cytometry. In all BM samples, CXCR5 was expressed by the majority of primary neuroblasts and mediated their chemotaxis in response to CXCL13. Primary metastatic NB cells from all BM samples expressed CXCR6, but were not attracted by soluble CXCL16. Studies performed with two CXCR6+ NB cell lines showed that the mechanism whereby neuroblasts did not migrate to CXCL16 was likely related to defective calcium ion mobilization. CXCR5 is the first chemokine receptor so far identified able to attract in vitro primary metastatic NB cells. CXCR6 may be involved in retention of metastatic neuroblasts in the BM through interaction with CXCL16 expressing stromal cells in the absence of signal transduction.

// Grazia Carbotti 1 , Gaia Barisione 1 , Irma Airoldi 2 , Delia Mezzanzanica 3 , Marina Bagnoli 3 , Simone Ferrero 4 , Andrea Petretto 5 , Marina Fabbi 1 and Silvano Ferrini 1 1 Department of Integrated Oncological Therapies, IRCCS AOU San Martino-IST Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy 2 Laboratory of Oncology, Istituto Giannina Gaslini, Genoa, Italy 3 Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 4 Department of Surgery, Unit of Obstetrics and Gynaecology, IRCCS AOU San Martino-IST Istituto Nazionale per la Ricerca sul Cancro, and DINOGMI, University of Genoa Genoa, Italy 5 Core Facilities, Istituto Giannina Gaslini, Genoa, Italy Correspondence to: Silvano Ferrini, email: // Marina Fabbi, email: // Keywords : IL-27, IDO, PD-L1, STAT, Immunology and Microbiology Section, Immune response, Immunity Received : July 08, 2015 Accepted : November 30, 2015 Published : December 09, 2015 Abstract IL-27 is a member of the IL-12 family that is produced by macrophages and dendritic cells. IL-27 inhibits the growth and invasiveness of different cancers and therefore represents a potential anti-tumor agent. By contrast, it may exert immune-regulatory properties in different biological systems. We reported that IL-27 induces the expression of the IL-18 inhibitor IL-18BP, in human Epithelial Ovarian Cancer (EOC) cells, thus potentially limiting the immune response. Here, we tested whether IL-27 may modulate other immune-regulatory molecules involved in EOC progression, including Indoleamine 2,3-dioxygenase (IDO) and Programmed Death-Ligand (PD-L)1. IDO and PD-L1 were not constitutively expressed by EOC cells in vitro , but IL-27 increased their expression through STAT1 and STAT3 tyrosine phosphorylation. Differently, cells isolated from EOC ascites showed constitutive activation of STAT1 and STAT3 and IDO expression. These findings, together with the expression of IL-27 in scattered leukocytes in EOC ascites and tissues, suggest a potential role of IL-27 in immune-regulatory networks of EOC. In addition, IL-27 induced IDO or PD-L1 expression in monocytes and in human PC3 prostate and A549 lung cancer cells. A current paradigm in tumor immunology is that tumor cells may escape from immune control due to “adaptive resistance” mediated by T cell-secreted IFN-γ, which induces PD-L1 and IDO expression in tumor cells. Our present data indicate that also IL-27 has similar activities and suggest that the therapeutic use of IL-27 as anti-cancer agent may have dual effects, in some tumors.

We prospectively assessed functional and phenotypic characteristics of γδ T lymphocytes up to 7 months after HLA-haploidentical hematopoietic stem cell transplantation (haplo-HSCT) depleted of αβ+ T cells and CD19+ B cells in 27 children with either malignant or nonmalignant disorders. We demonstrate that (1) γδ T cells are the predominant T-cell population in patients during the first weeks after transplantation, being mainly, albeit not only, derived from cells infused with the graft and expanding in vivo; (2) central-memory cells predominated very early posttransplantation for both Vδ1 and Vδ2 subsets; (3) Vδ1 cells are specifically expanded in patients experiencing cytomegalovirus reactivation and are more cytotoxic compared with those of children who did not experience reactivation; (4) these subsets display a cytotoxic phenotype and degranulate when challenged with primary acute myeloid and lymphoid leukemia blasts; and (5) Vδ2 cells are expanded in vitro after exposure to zoledronic acid (ZOL) and efficiently lyse primary lymphoid and myeloid blasts. This is the first detailed characterization of γδ T cells emerging in peripheral blood of children after CD19+ B-cell and αβ+ T-cell–depleted haplo-HSCT. Our results can be instrumental to the development of clinical trials using ZOL for improving γδ T-cell killing capacity against leukemia cells. This trial was registered at www.clinicaltrials.gov as #{"type":"clinical-trial","attrs":{"text":"NCT01810120","term_id":"NCT01810120"}}NCT01810120.

The interleukin-12 receptor beta2 (Il12rb2) gene is silenced in tumor cells from different human B-cell malignancies as opposed to their normal counterparts. It was hypothesized that this silencing allows neoplastic B cells to escape the control exerted by IL-12 on their growth. The aim of this study was to investigate whether targeted inactivation of the Il12rb2 gene in mice resulted into increased susceptibility to spontaneous tumor formation and immunopathology. Il12rb2 gene-deficient animals developed in the first year of life immune-complex mesangial glomerulonephritis with serum antinuclear antibodies. In older animals, multiorgan lymphoid infiltrates with features of vasculitis and Sjögren syndrome were detected in association with systemic B- and T-cell activation. In half of aged animals, lymph node plasmacytoma or lung carcinoma was observed. A mechanism for spontaneous development of autoimmune pathology and B-cell tumors is suggested by a strong IL-6 up-regulation detected in splenocytes and lymphoid infiltrates associated with oligoclonal B-cell expansion. The emergence of lung tumors may likely be attributed to an interferon-gamma (IFN-gamma) deficiency secondary to lack of IL-12 signaling. The development of autoimmunity, lymphoproliferation, and B-cell tumors in Il12rb2 knockout (KO) mice suggests that IL-12 functions physiologically to restrain aberrant B-cell activation.