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Simple Summary Malignant mesothelioma (MM), a rare and aggressive tumor, is related to asbestos exposure, which mediates a chronic inflammatory process involving the cytokine IL-6. Recent studies indicate that the PD-1/PD-L1 immune-suppressive axis is a clinically relevant target for therapy. The expression of PD-L1 in tumor cells is generally a cytokine-mediated effect. Here we show that the IL-6-related cytokine IL-27 is able to enhance PD-L1 expression and soluble (s)PD-L1 release by cultured MM cells, whereas IL-6 is ineffective. In agreement with previous findings, we found high IL-6 levels in pleural exudates from 77 MM patients which correlated with worse survival. More importantly, we also found sPD-L1 and IL-27 in the same samples. sPD-L1 and IL-27 levels showed a moderate albeit significant correlation and association with worse survival, which suggested a potential effect of IL-27 on PD-L1-mediated immune resistance in MM. Abstract Malignant mesothelioma (MM) is a rare tumor with an unfavorable prognosis. MM genesis involves asbestos-mediated local inflammation, supported by several cytokines, including IL-6. Recent data showed that targeting PD-1/PD-L1 is an effective therapy in MM. Here, we investigated the effects of IL-6 trans-signaling and the IL-6-related cytokine IL-27 on human MM cells in vitro by Western blot analysis of STAT1/3 phosphorylation. The effects on PD-L1 expression were tested by qRT-PCR and flow-cytometry and the release of soluble (s)PD-L1 by ELISA. We also measured the concentrations of sPD-L1 and, by multiplexed immunoassay, IL-6 and IL-27 in pleural fluids obtained from 77 patients in relation to survival. IL-27 predominantly mediates STAT1 phosphorylation and increases PD-L1 gene and surface protein expression and sPD-L1 release by human MM cells in vitro. IL-6 has limited activity, whereas a sIL-6R/IL-6 chimeric protein mediates trans-signaling predominantly via STAT3 phosphorylation but has no effect on PD-L1 expression and release. IL-6, IL-27, and sPD-L1 are present in pleural fluids and show a negative correlation with overall survival, but only IL-27 shows a moderate albeit significant correlation with sPD-L1 levels. Altogether these data suggest a potential role of IL-27 in PD-L1-driven immune resistance in MM.

Uveal melanoma (UM) is a rare tumor of the eye that leads to deadly metastases in about half of the patients. ADAM10 correlates with c-Met expression in UM and high levels of both molecules are related to the development of metastases. MiR122 and miR144 modulate ADAM10 and c-Met expression in different settings. We hypothesized a potential onco-suppressive role for miR122 and miR144 through modulation of ADAM10 and c-Met in UM. We analyzed the UM Cancer Genome Atlas data portal (TCGA) dataset, two other cohorts of primary tumors and five human UM cell lines for miR122 and miR144 expression by miR microarray, RT-qPCR, Western blotting, miR transfection and luciferase reporter assay. Our results indicate that miR122 and miR144 are expressed at low levels in the UM cell lines and in the TCGA UM dataset and were down-modulated in a cohort of seven UM samples, compared to normal choroid. Both miR122 and miR144 directly targeted ADAM10 and c-Met. Overexpression of miR122 and miR144 led to reduced expression of ADAM10 and c-Met in the UM cell lines and impaired cell proliferation, migration, cell cycle and shedding of c-Met ecto-domain. Our results show that miR122 and miR144 display an onco-suppressive role in UM through ADAM10 and c-Met modulation.

Background Recently, immunotherapy with anti-PD-1 antibodies has shown clinical benefit in recurrent Small Cell Lung Cancer (SCLC). Since anti-PD-1 re-activates anti-tumor Cytotoxic T Lymphocyte (CTL) responses, it is crucial to understand the mechanisms regulating HLA class I, and PD-L1 expression in HLA-negative SCLC. Here we addressed the role of IL-27, a cytokine related to both IL-6 and IL-12 families. Methods The human SCLC cell lines NCI-N592, -H69, -H146, -H446 and -H82 were treated in vitro with different cytokines (IL-27, IFN-γ, IL-6 or a soluble IL-6R/IL-6 chimera [sIL-6R/IL-6]) at different time points and analyzed for tyrosine-phosphorylated STAT proteins by Western blot, for surface molecule expression by immunofluorescence and FACS analyses or for specific mRNA expression by QRT-PCR. Relative quantification of mRNAs was calculated by the ΔΔCT method. The Student’s T test was used for the statistical analysis of experimental replicates. Results IL-27 triggered STAT1/3 phosphorylation and up-regulated the expression of surface HLA class I antigen and of TAP1 and TAP2 mRNA in four out of five SCLC cell lines tested. The IL-27-resistant NCI-H146 cells showed up-regulation of HLA class I by IFN-γ. IFN-γ also induced expression of PD-L1 in SCLC cells, while IL-27 was less potent in this respect. IL-27 failed to activate STAT1/3 phosphorylation in NCI-H146 cells, which display a low expression of the IL-27RA and GP130 receptor chains. As GP130 is shared in IL-27R and IL-6R complexes, we assessed its functionality in response to sIL-6R/IL-6. sIL-6R/IL-6 failed to trigger STAT1/3 signaling in NCI-H146 cells, suggesting low GP130 expression or uncoupling from signal transduction. Although both sIL-6R/IL-6 and IL-27 triggered STAT1/3 phosphorylation, sIL-6R/IL-6 failed to up-regulate HLA class I expression, in relationship to the weak activation of STAT1. Finally sIL-6R/IL-6 limited IL-27-effects, particularly in NCI-H69 cells, in a SOCS3-independent manner, but did not modify IFN-γ induced HLA class I up-regulation. Conclusions In conclusion, IL-27 is a potentially interesting cytokine for restoring HLA class I expression for SCLC combined immunotherapy purposes. However, the concomitant activation of the IL-6 pathway may limit the IL-27 effect on HLA class I induction but did not significantly alter the responsiveness to IFN-γ. Electronic supplementary material The online version of this article (10.1186/s13046-017-0608-z) contains supplementary material, which is available to authorized users.

Anti-PD-1 or anti-PD-L1 blocking monoclonal antibodies (mAbs) have shown potent anti-tumor effects in adult cancer patients and clinical studies have recently been started in pediatric cancers, including high-risk/relapsing neuroblastoma (NB). Therefore, we studied the effects of anti-PD-1/PD-L1 mAbs in two syngeneic models of disseminated NB generated by the injection of either Neuro2a or NXS2 cells, which express PD-L1. In addition, we tested the combination of these agents with the immune-enhancing cytokine IL-21, the Ecto-NTPDase inhibitor POM-1, an anti-CD25 mAb targeting Treg cells, or an anti-CD4 mAb. We previously showed that CD4-transient depletion removes CD4+CD25+ Treg cells and other CD4+CD25− regulatory subsets. Here we show that mono-therapy with anti-PD-1/PD-L1 mAbs had no effect on systemic NB progression in vivo, and also their combination with IL-21, POM-1 or anti-CD25 mAb was ineffective. The combined use of anti-PD-1 with an anti-CD4 mAb mediated a very potent, CD8-dependent, synergistic effect leading to significant elongation of tumor-free survival of mice, complete tumor regression and durable anti-NB immunity. Similar results were obtained by combining the anti-PD-L1 and anti-CD4 mAbs. These findings indicate that both PD-1/PD-L1 and CD4+ T cell-related immune-regulatory mechanisms must be simultaneously blocked to mediate therapeutic effects in these models.

// Fabio Morandi 1 , Sebastiano Barco 2 , Sara Stigliani 3 , Michela Croce 4 , Luca Persico 5 , Corrado Lagazio 5 , Francesca Scuderi 6 , Maria Luisa Belli 6 , Mariapina Montera 7 , Giuliana Cangemi 2 , Sarah Pozzi 8 , Valentina Rigo 4 , Paola Scaruffi 3 , Loredana Amoroso 6 , Giovanni Erminio 9 , Vito Pistoia 10 , Silvano Ferrini 4 and Maria Valeria Corrias 1 1 UOC Laboratory of Oncology, IRCCS Istituto Giannina Gaslini, Genoa, Italy 2 UOC Clinical Pathology Laboratory, IRCCS Istituto Giannina Gaslini, Genoa, Italy 3 UOS Physiopathology of Human Reproduction, IRCCS AOU SanMartino-IST, Genoa, Italy 4 UOC Biotherapy, IRCCS AOU SanMartino-IST, Genoa, Italy 5 Department of Economy, University of Genoa, Genoa, Italy 6 UOC Oncology, IRCCS Istituto Giannina Gaslini, Genoa, Italy 7 UOC Immune-Hematology and Transfusion Medicine, IRCCS Istituto Giannina Gaslini, Genoa, Italy 8 UOC Immune-Hematology and Transfusion Medicine, IRCCS AOU SanMartino-IST, Genoa, Italy 9 UOS Epidemiology, Biostatistics and Committees, IRCCS Istituto Giannina Gaslini, Genoa, Italy 10 Immunology Area, IRCCS Bambino Gesu, Rome, Italy Correspondence to: Fabio Morandi, email: fabiomorandi@gaslini.org Keywords: neuroblastoma, erythrocytes, bone marrow, survival, microenvironment Received: February 14, 2017 Accepted: May 09, 2017 Published: May 30, 2017 ABSTRACT 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.

// Andrea Petretto 1,* , Grazia Carbotti 2,* , Elvira Inglese 1 , Chiara Lavarello 1 , Maria Pia Pistillo 3 , Valentina Rigo 2 , Michela Croce 2 , Luca Longo 2 , Stefania Martini 2 , Paola Vacca 2,4 , Silvano Ferrini 2,* and Marina Fabbi 2,* 1 Core Facilities-Proteomics Laboratory, Istituto Giannina Gaslini, Genoa, Italy 2 Department of Integrated Oncological Therapies, IRCCS AOU San Martino-IST Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy 3 Tumor Epigenetics Unit, IRCCS AOU San Martino-IST Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy 4 Department of Experimental Medicine (DIMES), University of Genoa, Genoa, Italy * These authors have equally contributed to this work Correspondence to: Silvano Ferrini, email: // Marina Fabbi, email: // Keywords : IL-27, IFN-γ, proteomic analysis, HLA class I, cancer cells, Immunology and Microbiology Section, Immune response, Immunity Received : July 10, 2016 Accepted : September 17, 2016 Published : September 24, 2016 Abstract IL-27, a member of the IL-12-family of cytokines, has shown anti-tumor activity in several pre-clinical models due to anti-proliferative, anti-angiogenic and immune-enhancing effects. On the other hand, IL-27 demonstrated immune regulatory activities and inhibition of auto-immunity in mouse models. Also, we reported that IL-27, similar to IFN-γ, induces the expression of IL-18BP, IDO and PD-L1 immune regulatory molecules in human cancer cells. Here, a proteomic analysis reveals that IL-27 and IFN-γ display a broad overlap of functions on human ovarian cancer cells. Indeed, among 990 proteins modulated by either cytokine treatment in SKOV3 cells, 814 showed a concordant modulation by both cytokines, while a smaller number (176) were differentially modulated. The most up-regulated proteins were common to both IFN-γ and IL-27. In addition, functional analysis of IL-27-regulated protein networks highlighted pathways of interferon signaling and regulation, antigen presentation, protection from natural killer cell-mediated cytotoxicity, regulation of protein polyubiquitination and proteasome, aminoacid catabolism and regulation of viral protein levels. Importantly, we found that IL-27 induced HLA class I molecule expression in human cancer cells of different histotypes, including tumor cells showing very low expression. IL-27 failed only in a cancer cell line bearing a homozygous deletion in the B2M gene. Altogether, these data point out to a broad set of activities shared by IL-27 and IFN-γ, which are dependent on the common activation of the STAT1 pathway. These data add further explanation to the anti-tumor activity of IL-27 and also to its dual role in immune regulation.

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.

Although several genes have been associated to neuroblastoma (NB) predisposition and aggressiveness, further genes are likely involved in the overall risk of developing this pediatric cancer. We thus carried out whole-exome sequencing on germline DNA from two affected second cousins and two unlinked healthy relatives from a large family with hereditary NB. Bioinformatics analysis revealed 6999 variations that were exclusively shared by the two familial NB cases. We then considered for further analysis all unknown or rare missense mutations, which involved 30 genes. Validation and analysis of these variants led to identify a GALNT14 mutation (c.802C > T) that properly segregated in the family and was predicted as functionally damaging by PolyPhen2 and SIFT. Screening of 8 additional NB families and 167 sporadic cases revealed this GALNT14 mutation in the tumors of two twins and in the germline of one sporadic NB patient. Moreover, a significant association between MYCN amplification and GALNT14 expression was observed in both NB patients and cell lines. Also, GALNT14 higher expression is associated with a worse OS in a public dataset of 88 NB samples (http://r2.amc.nl). GALNT14 is a member of the polypeptide N-acetylgalactosaminyl-transferase family and maps closely to ALK on 2p23.1, a region we previously discovered in linkage with NB in the family here considered. The aberrant function of GALNTs can result in altered glycoproteins that have been associated to the promotion of tumor aggressiveness in various cancers. Although rare, the recurrence of this mutation suggests GALNT14 as a novel gene potentially involved in NB predisposition.

Conventional melanoma serum biomarkers (S100 and lactate dehydrogenase [LDH]) perform poorly in patients with uveal melanoma, and the search for new biomarkers is needed. A high expression of the oncoprotein c-Met in primary uveal melanoma is associated with metastatic progression, and c-Met is released as a soluble ectodomain through ADAM10- and ADAM17-mediated cleavage, suggesting a possible role as biomarker.To determine the potential role of soluble c-Met (sc-Met) as a biomarker of uveal melanoma progression in comparison with S100 and LDH.Soluble c-Met was studied in the conditioned medium of 9 uveal melanoma cell lines and in the blood serum samples of 24 mice with uveal melanoma xenografts, 57 patients with uveal melanoma (17 patients whose tumors metastasized and 40 patients whose tumors did not metastasize), and 37 healthy donors. We collected blood samples for as long as 5 years after treatment of the primary tumor. The concentration of sc-Met was measured using enzyme-linked immunosorbent assays, and the receiver operating characteristic curve was used to evaluate sensitivity and specificity in the identification of metastatic uveal melanoma. The study began on May 2, 2011, and the last samples were collected in January 2015.Levels of sc-Met in uveal melanoma cell cultures and in the blood serum samples of xenotransplanted mice, of healthy donors, and of patients with uveal melanoma during follow-up.The conditioned medium of uveal melanoma cell lines and the blood serum samples of mice with uveal melanoma xenografts contained significant levels of sc-Met. Patients with metastatic disease had significantly higher serum levels of sc-Met (median level, 590 ng/mL [range, 246-12,856 ng/mL]) than did patients without metastatic disease (median level, 296 ng/mL [range, 201-469 ng/mL]) (P .001) and healthy donors (median level, 285 ng/mL [range, 65-463 ng/mL]) (P .001). Analysis of receiver operating characteristic curves for sc-Met levels in patients with nonmetastatic uveal melanoma vs patients with metastatic uveal melanoma yielded an area under the curve of 0.82 (95% CI, 0.68-0.95) (P .001), which was superior to the areas under the curve achieved with S100 or LDH markers. Patients with progressive metastatic disease showed further increases in sc-Met level, whereas stable patients did not.The present pilot study suggests that sc-Met should be further exploited as a biomarker for monitoring of uveal melanoma.

The expression of the immunosuppressive moleculesIL-10and arginase 1 (ARG-1), and ofFOXP3andCD163, as markers of regulatory T cells (Treg) and macrophages, respectively, was evaluated in bone marrow (BM) and peripheral blood (PB) samples collected at diagnosis from patients with metastatic neuroblastoma (NB). IL-10 and ARG-1 plasma concentrations were measured and the association of each parameter with patients’ outcome was tested. The percentages of immunosuppressive Treg and type-1 regulatory (Tr1) cells were also determined. In both BM and PB samples,IL-10mRNA expression was higher in metastatic NB patients than in controls. IL-10 plasma concentration was higher in patients with NB regardless of stage. NeitherIL-10expression nor IL-10 plasma concentration significantly associated with patient survival. In PB samples from metastatic NB patients,ARG-1andCD163expression was higher than in controls but their expression did not associate with survival. Moreover, ARG-1 plasma concentration was lower than in controls, and no association with patient outcome was found. Finally, in metastatic NB patients, the percentage of circulating Treg was higher than in controls, whereas that of Tr1 cells was lower. In conclusion, although IL-10 concentration and Treg percentage were increased, their contribution to the natural history of metastatic NB appears uncertain.

// 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.

Activated leukocyte cell adhesion molecule (ALCAM) is involved in cell-cell interactions in cancer. Shedding of its ectodomain by the metalloprotease ADAM17/TACE generates a soluble form (sALCAM). Here, we show that serum sALCAM levels were significantly higher in epithelial ovarian cancer (EOC) (p < 0.005) than in controls. The performance of sALCAM as classifier, tested by receiver operating characteristic curve, resulted in an area under the curve (AUC) of 0.8067. Serum sALCAM levels showed direct correlation with Carbohydrate Antigen-125 (CA125/MUC16). Moreover, significantly higher levels were found in type II tumors, even in stage I/II, suggesting that elevated sALCAM is an early feature of aggressive EOC. In addition, sALCAM levels were higher in ascites than in sera, suggesting local processing of ALCAM in the peritoneal cavity. In immunodeficient mice, intraperitoneally implanted with a human EOC cell line, human sALCAM progressively increased in serum and was even higher in the ascites. The biochemical characterization of the sALCAM in EOC sera and ascites, showed two predominant forms of approximately 95 and 65 kDa but no EOC-specific isoform. In addition, full-length transmembrane ALCAM but no soluble form was detected in tumor-derived exosomes found in ascites. Finally, in vitro invasion assays showed that inhibition of ADAM17/TACE activity decreased EOC invasive properties, while opposite effects were mediated by a sALCAM-Fc chimera and by an antibody interfering with ALCAM/ALCAM interactions. Altogether these data suggest that sALCAM is a marker of EOC, which correlates with more aggressive type II tumors, and that ADAM17/TACE activity and sALCAM itself mediate enhanced invasiveness.

The in vitro and in vivo stability and anti-tumour efficacy of the anti-EGFR/anti-CD3 bispecific monoclonal antibody (biMAb), M26.1, were analysed. The interaction of the intact biMAb with Fc receptor I (Fc gamma RI) present on human leucocytes was not observed when the antibody was used as an F(ab')2 fragment. A CD8+ T-cell clone coated with M26.1 F(ab')2 was as effective as the intact biMAb in inducing IGROV1 target cell lysis when tested in a 51Cr-release assay. Variable levels of reduction of F(ab')2 to monovalent F(ab') were observed upon incubation with human ovarian cancer ascitic fluid (OCAF) or with human glioblastoma cavity fluid (GCF), but not with mouse or human sera. Activated lymphocytes coated with F(ab')2 and incubated in vitro with GCF or OCAF for 24 and 48 h respectively maintained their targeting. Thus, the F(ab')2, when present as a soluble molecule, but not when bound to T cells, might lose some functional activity as a consequence of partial reduction to F(ab'). In normal mice, M26.1 F(ab')2 retained full cytotoxic activity in the circulation, and clearance values were similar to those obtained with parental and other MAb F(ab')2. Treatment of IGROV1 tumour-bearing mice with activated human lymphocytes coated with the M26.1 F(ab')2 significantly prolonged survival of the animals compared with tumour-bearing untreated and control mice treated with lymphocytes or F(ab')2 alone. Together, these results suggest the clinical usefulness of bispecific M26.1 F(ab')2 as a targeting agent for local treatment of tumours such as glioma and ovarian cancers that express variable levels of epidermal growth factor receptor (EGFR).

Uveal melanoma is an aggressive cancer that metastasizes to the liver in about half of the patients, with a high lethality rate. Identification of patients at high risk of metastases may provide indication for a frequent follow-up for early detection of metastases and treatment. The analysis of the gene expression profiles of primary human uveal melanomas showed high expression of SDCBP gene (encoding for syndecan-binding protein-1 or mda-9/syntenin), which appeared higher in patients with recurrence, whereas expression of syndecans was lower and unrelated to progression. Moreover, we found that high expression of SDCBP gene was related to metastatic progression in two additional independent datasets of uveal melanoma patients. More importantly, immunohistochemistry showed that high expression of mda-9/syntenin protein in primary tumors was significantly related to metastatic recurrence in our cohort of patients. Mda-9/syntenin expression was confirmed by RT-PCR, immunofluorescence and immunohistochemistry in cultured uveal melanoma cells or primary tumors. Interestingly, mda-9/syntenin showed both cytoplasmic and nuclear localization in cell lines and in a fraction of patients, suggesting its possible involvement in nuclear functions. A pseudo-metastatic model of uveal melanoma to the liver was developed in NOD/SCID/IL2Rγ null mice and the study of mda-9/syntenin expression in primary and metastatic lesions revealed higher mda-9/syntenin in metastases. The inhibition of SDCBP expression by siRNA impaired the ability of uveal melanoma cells to migrate in a wound-healing assay. Moreover, silencing of SDCBP in mda-9/syntenin-high uveal melanoma cells inhibited the hepatocyte growth factor (HGF)-triggered invasion of matrigel membranes and inhibited the activation of FAK, AKT and Src. Conversely syntenin overexpression in mda-9/syntenin-low uveal melanoma cells mediated opposite effects. These results suggest that mda-9/syntenin is involved in uveal melanoma progression and that it warrants further investigation as a candidate molecular marker of metastases and a potential therapeutic target.

International audience; The ability of Interleukin-15 (IL-15) to activate many immune antitumor mechanisms renders the cytokine a good candidate for the therapy of solid tumors, particularly renal cell carcinoma. Although IL-15 is being currently used in clinical trials, the function of the cytokine on kidney's components has not been extensively studied; we thus investigated the role of IL-15 on normal and tumor renal epithelial cells. Herein, we analyzed the expression and the biological functions of IL-15 in normal renal proximal tubuli (RPTEC) and in their neoplastic counterparts, the renal clear cell carcinomas (RCC). This study shows that RPTEC express a functional heterotrimeric IL-15Rαβγc complex whose stimulation with physiologic concentrations of rhIL-15 is sufficient to inhibit epithelial mesenchymal transition (EMT) commitment preserving E-cadherin expression. Indeed, IL-15 is not only a survival factor for epithelial cells, but it can also preserve the renal epithelial phenotype through the cc-signaling pathway, demonstrating that the cytokine possess a wide range of action in epithelial homeostasis. In contrast, in RCC in vitro and in vivo studies reveal a defect in the expression of γc-receptor and JAK3 associated kinase, which strongly impacts IL-15 signaling. Indeed, in the absence of the cc/JAK3 couple we demonstrate the assembly of an unprecedented functional high affinity IL-15Rαβ heterodimer, that in response to physiologic concentrations of IL-15, triggers an unbalanced signal causing the down-regulation of the tumor suppressor gene E-cadherin, favoring RCC EMT process. Remarkably, the rescue of IL-15/γc-dependent signaling (STAT5), by co-transfecting γc and JAK3 in RCC, inhibits EMT reversion. In conclusion, these data highlight the central role of IL-15 and γc-receptor signaling in renal homeostasis through the control of E-cadherin expression and preservation of epithelial phenotype both in RPTEC (up-regulation) and RCC (down-regulation).

Arginase 1 (ARG1) inhibits T-cell proliferation by degrading extracellular arginine, which results in decreased responsiveness of T cells to CD3/TCR stimulation. In humans, ARG1 is stored in inactive form within granules of polymorphonuclear neutrophils (PMNs) and gets activated on release. We studied the role of PMNs-related ARG1 activity in nonsmall cell lung cancer (NSLC), in which tumor-infiltrating lymphocytes showed reduced proliferation in response to CD3/TCR triggering. Patients with NSCLC had increased ARG1 plasma levels as compared to healthy controls. Furthermore, immunohistochemistry showed that tumor-infiltrating PMNs display reduced intracellular ARG1, in comparison to intravascular or peritumoral PMNs, suggesting a role of tumor microenvironment in ARG1 release. Indeed, supernatants of NSCLC cell lines induced exocytosis of ARG1 from PMNs. All (4/4) NSCLC cell lines and all (7/7) CD14- cell samples from NSCLC expressed interleukin (IL)-8 mRNA, whereas TNFalpha mRNA was expressed by 1 cell line and by 2 tumor specimens. Furthermore, all NSCLC cell lines secreted immunoreactive IL-8, albeit at different levels. IL-8 was as effective as TNFalpha in triggering ARG1 release and the 2 cytokines acted synergistically. Secreted ARG1 was biologically active and catabolized extracellular arginine. The supernatant of IL-8 gene-silenced NSCLC cells did not mediate ARG1 release by PMNs. Altogether these findings demonstrate a role of IL-8 in ARG1 exocytosis by PMNs and indicate that, due at least in part to IL-8 secreted by NSCLC cells, PMNs infiltrating NSCLC release ARG1. This phenomenon could contribute to local immune suppression.

The surface phenotype CD3-NK1.1+DX5+CD11c(int)B220+GR1- has been recently ascribed to a novel subset of mouse leukocytes termed interferon (IFN)-producing killer dendritic cells (IKDCs) that shares functions with natural killer (NK) cells and DCs. Interleukin-15 (IL-15) is critical for NK cells but its relationship with IKDC remained unexplored. An expression cassette encoding human IL-15 (hIL-15) has been transferred by hydrodynamic injection into the liver of mice, resulting in transient expression of the cytokine that is detectable during the first 48 h. hIL-15 hydrodynamic gene transfer resulted in an expansion of NK cells and IKDCs. Relative expansions of IKDCs were more dramatic in the IL-15 gene-transferred hepatic tissue than in the spleen. Adoptively transferred DX5+ cells comprising both NK cells and IKDCs proliferated in response to hydrodynamic injection of hIL-15, indicating that quantitative increases are at least in part the result of proliferation from already differentiated cells. Expansion is accompanied by enhanced cytolytic activity and increased expression of TRAIL and CD137 (4-1BB), without augmenting interferon-gamma production. The effects of a single hydrodynamic injection surpassed those of two intraperitoneal doses of the recombinant protein. The novel functional link between circulating IL-15 and IKDCs opens new possibilities to study the biology and applications of this minority cell subset.

BACKGROUND: NK cells have been long time considered as cytotoxic lymphocytes competent in killing virus-infected cells and tumors. However, NK cells may also play essential immuno-regulatory functions. In this context, the real existence of a defined NK subset with negative regulatory properties has been hypothesized but never clearly demonstrated. METHODOLOGY/PRINCIPAL FINDINGS: Herein, we show the in vitro generation from human peripheral blood haematopoietic progenitors (PB-HP), of a novel subset of non-cytolytic NK cells displaying a mature phenotype and remarkable immuno-regulatory functions (NK-ireg). The main functional hallmark of these NK-ireg cells is represented by the surface expression/release of HLA-G, a major immunosuppressive molecule. In addition, NK-ireg cells secrete two powerful immuno-regulatory factors: IL-10 and IL-21. Through these factors, NK-ireg cells act as effectors of the down-regulation of the immune response: reconverting mature myeloid DC (mDC) into immature/tolerogenic DC, blocking cytolytic functions on conventional NK cells and inducing HLA-G membrane expression on PB-derived monocytes. The generation of "NK-ireg" cells is obtained, by default, in culture conditions favouring cell-to-cell contacts, and it is strictly dependent on reciprocal trans-presentation of membrane-bound IL-15 forms constitutively and selectively expressed by human CD34(+) PB-HP. Finally, a small subset of NKp46(+) HLA-G(+) IL-10(+) is detected within freshly isolated decidual NK cells, suggesting that these cells could represent an in vivo counterpart of the NK-ireg cells. CONCLUSIONS/SIGNIFICANCE: In conclusion, NK-ireg cells represent a novel truly differentiated non-cytolytic NK subset with a self-sustainable phenotype (CD56(+) CD16(+) NKp30(+) NKp44(+) NKp46(+) CD94(+) CD69(+) CCR7(+)) generated from specific pSTAT6(+) GATA3(+) precursors. NK-ireg cells could be employed to develop new immuno-suppressive strategies in autoimmune diseases, transplant rejection or graft versus host diseases. In addition, NK-ireg cells can be easily derived from peripheral blood of the patients and could constitute an autologous biotherapic tool to be used combined or in alternative to other immuno-regulatory cells.

We analyzed the recently defined ability of CD3-CD16+ cells to specifically recognize and lyse normal allogeneic target cells (PHA-induced blasts). The susceptibility to lysis by a given alloreactive natural killer (NK) clone ("1 anti-A") was expressed by PHA blasts derived from 9 of 38 random donors analyzed. In all instances, the specific lysis of "susceptible" target cells was greater than 35% while that of "nonsusceptible" targets was less than 6% at an E/T cell ratio of 5:1. In addition to 1 anti-A, A anti-1 specific CD3-CD16+ clones could also be isolated from the reverse MLC combination. The relationship existing between lysis of normal allogeneic cells or tumor cells by the same CD3-CD16+ effector cell has been investigated: 1 anti-A specific CD3-CD16+ clones lysed PHA blasts of three of six cancer patients, while they lysed fresh tumor cells (ovarian carcinoma) from all six patients. The type of inheritance of the character "susceptibility to lysis" was analyzed in representative families. This analysis revealed that the character is inherited in an autosomic recessive fashion, and it is therefore different from MHC. We further investigated the type of segregation of the opposite character "resistance to lysis" (which is inherited in a dominant mode). The finding that this character segregated in all donors expressing given MHC haplotypes indicated that the gene regulating the expression of the NK-defined alloantigen is present on chromosome 6.

Demonstrating the feasibility of magnetic resonance imaging (MRI) at 1.5 T of ultrasmall particle iron oxide (USPIO)-antibody bound to tumor cells in vitro and in a murine xenotransplant model.Human D430B cells or Raji Burkitt lymphoma cells were incubated in vitro with different amounts of commercially available USPIO-anti-CD20 antibodies and cell pellets were stratified in a test tube. For in vivo studies, D430B cells and Raji lymphoma cells were inoculated subcutaneously in immunodeficient mice. MRI at 1.5 T was performed with T1-weighted three-dimensional fast field echo sequences (17/4.6/13 degrees ) and T2-weighted three-dimensional fast-field echo sequences (50/12/7 degrees ). For in vivo studies MRI was performed before and 24 h after USPIO-anti-CD20 administration.USPIO-anti-CD20-treated D430B cells, showed a dose-dependent decrease in signal intensity (SI) on T2*-weighted images and SI enhancement on T1-weighted images in vitro. Raji cells showed lower SI changes, in accordance to the fivefold lower expression of CD20 on Raji with respect to D430B cells. In vivo 24 h after USPIO-anti-CD20 administration, both tumors showed an inhomogeneous decrease of SI on T2*-weighted images and SI enhancement on T1-weighted images.MRI at 1.5 T is able to detect USPIO-antibody conjugates targeting a tumor-associated antigen in vitro and in vivo.

IL-21 is an IL-2-like cytokine, signaling through a specific IL-21R and the IL-2R γ-chain. Because the TS/A mammary adenocarcinoma cells genetically modified to secrete IL-21 (TS/A-IL-21) are strongly immunogenic in syngeneic mice, we analyzed their application as vaccine. In mice bearing TS/A-parental cell (pc) micrometastases, vaccination with irradiated TS/A-IL-21 cells significantly increased the animal life span, but cured only 17% of mice. Spleen cells from cured mice developed CTL activity and produced IFN-γ in response to stimulation by the AH1 epitope of the gp70env Ag of TS/A-pc. We tested whether the low therapeutic outcome might be due to CD4+CD25+ regulatory T cells (Treg) present in TS/A-pc tumors and draining lymph nodes and whether IL-21 had any effect on these cells. Indeed, CD4+CD25+ cells suppressed IFN-γ production by splenocytes from immune mice in response to stimulation by the AH1 peptide. Low concentrations of IL-21 (10 ng/ml) failed to reverse the inhibitory activity of CD4+CD25+ cells in an allogeneic MLR, whereas 60 ng/ml rIL-21 partially restored responder T cell proliferation. IL-21R expression on CD25− lymphocytes suggested that IL-21 could be more effective in mice depleted of CD25+ cells. Depletion of Treg cells by a single dose of anti-CD25 mAb combined with TS/A-IL-21 cell vaccine cured >70% of mice bearing micrometastases, whereas anti-CD25 mAb treatment alone had no effect. Successful combined immunotherapy required NK cells, CD8+ T cells, and IFN-γ. In conclusion, immunotherapy of micrometastases by an IL-21-based cellular vaccine is strongly potentiated by CD25+ cell depletion.

Interleukin-21 (IL-21) is a member of the IL-2 cytokine family, which mediates proliferation or growth arrest and apoptosis of normal B cells, depending on their activation state. Here we demonstrate that surface IL-21 receptor (R) is expressed at variable levels by chronic lymphocytic leukemia (CLL) B cells freshly isolated from 33 different patients. IL-21R expression was up-regulated following cell stimulation via surface CD40. Therefore, IL-21 effects were more evident in CD40-activated CLL B cells. IL-21 induced an early signaling cascade in CLL B cells, which included JAK-1 and JAK-3 autophosphorylation and tyrosine phosphorylation of STAT-1, STAT-3, and STAT-5. IL-21 signaling failed to stimulate CLL B-cell proliferation, but induced their apoptosis. In addition, IL-21 counteracted the proliferative and antiapoptotic signals delivered by IL-15 to CLL B cells. IL-21-mediated apoptosis involved activation of caspase-8 and caspase-3, cleavage of Bid to its active form t-Bid, and cleavage of PARP and of p27Kip-1. Recent data indicate that CLL B cells require interaction with the microenvironment for their survival and expansion. The present findings thus provide a set of new mechanisms involved in the balance between cell-survival and apoptotic signals in CLL B cells.

Activated leukocyte cell adhesion molecule (ALCAM/CD166), a member of the immunoglobulin superfamily with five extracellular immunoglobulin-like domains, promotes heterophilic (ALCAM-CD6) and homophilic (ALCAM-ALCAM) cell-cell interactions. Here we describe a fully human single-chain antibody fragment (scFv) directed to ALCAM/CD166. We selected the I/F8 scFv from a phage display library of human V-gene segments by cell panning and phage internalization into IGROV-I human ovary carcinoma cells. The I/F8 specificity was identified as ALCAM/CD166 by matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) peptide mass fingerprinting of the I/F8-immunoprecipitated protein. The I/F8 scFv reacts with the human, monkey and murine ALCAM/CD166 molecule, indicating that the recognized epitope is highly conserved. The I/F8 scFv completely abolished binding of both ALCAM/Fc and CD6/Fc soluble ligands, whereas it did not compete with the anti-ALCAM/CD166 murine monoclonal antibodies J4-81 and 3A6 and therefore recognizes a different epitope. Engagement through I/F8 scFv, 3A6 monoclonal antibody or CD6/Fc ligand induced ALCAM/CD166 internalization, with a kinetics slower than that of transferrin in the same cells. Newly internalized I/F8-ALCAM complexes colocalized with clathrin but not with caveolin and we demonstrated, using surface biotinylation and recycling assays, that endocytosed ALCAM/CD166 recycles back to the cell surface. Such an endocytic pathway allows the efficient delivery of an I/F8 scFv-saporin immunotoxin into tumor cells, as the conjugates are able to selectively kill cell lines expressing ALCAM/CD166. Altogether these data provide evidence of the suitability of the I/F8 scFv for further functional analysis of ALCAM/CD166 and intracellular delivery of effector moieties.

The extracellular activities of the human immunodeficiency virus (HIV) transactivator protein (Tat) include induction of angiogenesis and stimulation of monocyte migration. Here it is shown that polymorphonuclear leukocytes (PMNL), mostly neutrophils, rapidly invade in response to Tat in vivo and initiate the formation of new vessels. In vitro, Tat was chemotactic for PMNL and induced calcium (Ca(2+)) mobilization. Tat proteins with inactivating substitutions in the arginine-glycine-aspartic acid or basic domain were still active in inducing PMNL migration, whereas Tat peptides mapped the migration and Ca(2+) mobilization activity to a cysteine-rich core domain, previously described as a Tat "chemokine-like" region (peptide CysL(24-51)). Tat and the CysL(24-51) peptide also induced PMNL superoxide production and the release of the angiogenic factors interleukin-8 and vascular endothelial growth factor from PMNL. CysL(24-51) did not induce endothelial cell migration but was angiogenic in vivo. These data indicate that the Tat activity on PMNL is mediated by its chemokine-like region and that PMNL recruitment by Tat is linked to angiogenesis.

Retrovirus-mediated gene therapy is a particularly attractive approach for glioblastoma multiforme (GBM), given the poor prognosis of this tumour and its localized proliferation in post-mitotic tissue. In this study we assessed, for the first time in humans, the therapeutic potential of a newly designed bicistronic Moloney vector (pLIL-2-TK), combining the expression of a suicide gene (thymidine kinase, tk) with an immunomodulatory gene (human interleukin 2, IL-2). Evidence of transgene activity in the treated tumours is presented.

Four patients with lymphoproliferative disease of granular lymphocytes (LDGL) coexpressing CD3 and the natural killer (NK)-related “p58” receptor for HLA-C alleles were studied. These CD3+p58+ LDGLs have been detected among a series of 44 CD3+ LDGLs analyzed. Two patients with LDGL (GI and BA) expressed only the p58 molecule defined by the GL-183 and CH-L monoclonal antibodies (MoAbs), while the cases of patients PU and MA also coexpressed the molecular form identified by EB6 anti-p58 MoAb. Three LDGL cases (GI, MA, and PU) displayed the CD8+4-CD16+ T- cell receptor (TCR)alpha/beta+ phenotype, while one patient (BA) was CD8+4+CD16+ TCRalpha/beta+. Freshly isolated granular lymphocytes (GL) from these cases displayed cytolytic activity in an anti-CD3 MoAb- triggered redirected killing assay against the Fcgamma-receptor+ (Fcgamma-R+) P815 target cell line. Lysis of P815 target cells, triggered by an anti-CD3 or by anti-CD16 MoAb, could be inhibited by the addition of anti-p58 MoAb in three fresh or interleukin (IL)-2- cultured GL tested (GI, MA, and PU). Triggering of cytotoxicity against the HLA-DR+ Fcgamma-R+ Daudi cell line induced by appropriate superantigens could also be inhibited by anti-p58 MoAb in patients PU and GI with LDGL. These data indicate that activation through the CD16, CD3, and TCR molecules can be modulated by p58 receptors in these LDGLs. On the contrary, IL-2-expanded cells of patient BA were induced to lyse P815 target cells by anti-p58 MoAb. In addition, anti-p58 MoAB enhanced anti-CD16 MoAb triggered lysis and did not inhibit activation via CD3. These data indicate that, in this particular patient with LDGL, p58 displays a stimulatory effect on cell triggering, rather than the typical inhibitory effect previously observed in p58+ T-cell clones derived from healthy donors. The anti-p58 MoAb did not induce CA++ mobilization in p58+ LDGLs and in a p58+CD3+ normal T-cell clone equipped with inhibitory p58 molecules, while Ca++ mobilization could be observed in cultured GL from patient BA, which could be activated by anti-58 MoAb. These findings suggest that stimulatory and inhibitory p58 molecules are equipped with different signal transducing properties, thus contributing to a better knowledge of the normal counterpart.

In this study we have analysed the CD3 and TCR transcript expression in three CD3- large granular lymphocyte (LGL) expansions. These LGL populations show a heterogenous attern of expression for CD2, CD8, CD16, CD56 and CD57 antigens. LGL1 is CD2+CD8-CD16+CD56+CD57+, while LGL2 is CD2-CD8+CD16-CD56-CD57-; LGL3 is similar to LGL1, except for CD8 antigen expression. Functional analysis has revealed a different behaviour of these LGL expansions in a cytotoxicity assay against the NK-sensitive K562 cell line. LGL1 and 3 display a significant NK-like activity, while LGL2 is inefficient against K562 target cells. TCR and CD3 transcript characterization of LGL expansions 1 and 3 showed that they expressed multiple TCR delta transcripts, a non-functional TCR beta transcript, CD3-zeta and -epsilon mRNA, but they lacked CD3 delta transcript and they lacked or expressed at very low levels of CD3 gamma transcript. On the other hand, LGL2 expressed TCR delta, CD3-gamma, -epsilon and -zeta transcripts, while it lacked CD3 delta mRNA. On the basis of these data, LGL1 and 3 seem to be closely related to peripheral blood mature natural killer (NK) cells, whereas LGL2 displays a pattern of TCR and CD3 expression similar to that found in CD1-2-3-4-8-16-thymocytes.

We investigated the role of the LFA-1/ICAM, VLA-4/VCAM-1 and CD2/LFA-3 adhesion pathways in the cytolysis of tumor cells mediated by an anti-EGF-R/anti-CD3 bispecific monoclonal antibody (biMAb). The biMAb induced efficient lysis of EGF-R+ tumor cells (A431, HT-29, IGROV-1 and MDA-MB468) by cytotoxic T lymphocytes (CTL) cultured in IL-2. Pretreatment of effector cells by anti-LFA-1 alpha (CD11a) and anti-LFA-1 beta (CD18) MAbs significantly inhibited cytolysis of all types of EGF-R+ tumor cells, while anti-CD2 and anti-VLA-4 MAbs were virtually ineffective. We investigated the expression of adhesion-molecule counter-receptors on tumor target cells by indirect immunofluorescence. HT-29, A431 and MDA-MB 468 tumor cells expressed an ICAM-1+2-3- VCAM-1- LFA-3+ phenotype, while IGROV-1 was ICAM-1-2+3- VCAM-1- LFA-3+. Pre-treatment of A431, HT-29 and MDA-MB468 with anti-ICAM-1 MAb inhibited cytolysis, further supporting the functional involvement of the LFA-1/ICAM adhesion pathway in biMAb-targeted tumor-cell lysis. In addition, treatment of target cells with TNF alpha or IFN gamma for 24 hr increased the expression of ICAM-1 in HT-29, A431 and MDA-MB468 (ICAM-2 was induced on IGROV-1) and also enhanced the sensitivity of these target cells to biMAb-targeted cytotoxicity. These data suggest that up-regulation of ICAM-molecule expression by inflammatory cytokines may increase susceptibility of tumor cells to biMAb-targeted lysis. Anti-LFA-1 MAbs did not significantly inhibit the formation of conjugates between biMAb-coated T lymphocytes and tumor cells. Co-aggregation of LFA-1 molecules with biMAb-bound CD3 molecules resulted in a more sustained and prolonged increase in the intracellular concentration of free Ca++ in CD8+ cultured CTL lines. These findings indicate that in T cells targeted by anti-CD3/anti-TAA biMAb LFA-1 may act as a co-receptor molecule which enhances signal transduction through the CD3/TCR complex.

Previously, the authors have described a molecule, identified by the LD6 monoclonal antibody (MoAb), present at the cell surface of long-term cultured T and Natural Killer (NK) cells which is involved in cell triggering. In the study described here the authors used biotin surface labelling and immunoprecipitation to show that LD6 MoAb recognizes a surface protein of approximately 65 kDa. In combination with submitogenic concentrations of phorbol esters (PMA); LD6 MoAb was able to induce accumulation of mRNA specific for GM-CSF, gamma-IFN and TNF-alpha and release of these cytokines by LD6+ T-cell lines. Both lymphokine production and lymphokine-specific mRNA accumulation induced by the LD6 MoAb were blocked totally by Cyclosporin A (CsA). To investigate the mechanism(s) of signal transduction through this activatory pathway, the authors performed Ca++ mobilization experiments. The results of these experiments suggested a role for Ca++ in signal transduction. The Ca++ mobilization induced by LD6 MoAb cross-linking could be inhibited totally by the use of pertussis toxin, indicating a possible role for G proteins in signalling through the LD6 MoAb-reactive molecule. Western blot analysis performed with an anti-phosphotyrosine antibody did not suggest that tyrosine kinase activation has a role.

A new anti-p58 monoclonal antibody (mAb), termed CH-L, has been used to characterize a minor subset of T lymphocytes co-expressing p58 and CD3 molecules. In two-color immunofluorescence analysis, CH-L+CD3+ cells represented 0.5 to 6% of the peripheral blood lymphocytes (in 20 healthy donors). Clonal analysis showed that most CD3+CH-L+ T cell clones expressed the CD8+4- T cell receptor (TcR) alpha/beta+ phenotype, while only a few were CD8-4+ TcR alpha/beta, CD8-4- TcR alpha/beta+ or CD8-4- TcR gamma/delta+. Western blot analysis indicated that the CH-L mAb identifies the same 56-58-kDa diffuse band in both T and natural killer cell (NK) clones. A minority of T cell clones also expressed other NK-related markers such as CD16, CD56 and CD94 and two clones also reacted with the anti-p58 mAb EB6. Interestingly, most clones displayed cytolytic activity in an anti-CD3 mAb-triggered redirected killing assay against the Fc gamma receptor+ P815 target cells and NK-like activity against K562 and Raji cells. In contrast, the IGROV-1 ovarian carcinoma cell line was resistant to cytolysis by all of these clones. Since p58 molecules have previously been shown to exert regulatory functions on NK-mediated lysis, we investigated whether anti-p58 mAb could also influence cytotoxicity mediated by CD3+p58+ T lymphocytes. Lysis of P815 target cells, triggered by anti-CD3 mAb, could be inhibited by anti-p58 mAb in 8 out of 12 cytolytic clones tested, while 4 clones were not inhibited. In addition, anti-p58 mAb enhanced the cytolytic activity of 3 clones against IGROV-1 and of 4 other clones against Raji target cells. Taken together, these data indicate that p58+ T cells express heterogeneous phenotypes and different forms of TcR and, in most instances, display cytolytic functions. Perhaps more importantly, the p58 molecule appears to modulate the cytolytic activity triggered via the CD3/TcR complex.

In this study we analyzed the T cell receptor(TcR) delta transcripts expressed by CD3-CD16+ cells and we investigated whether these cells expressed the hGATA-3 T cell transcription factor and the recombination-activating gene (RAG)-1. Multiple TcR delta transcripts deriving from an unrearranged TcR delta gene were detected in both polyclonal and clonal CD3-CD16+ natural killer(NK) cell lines. Two unrearranged TcR delta transcripts had a size similar to that of the functional TcR delta mRNA (2.3 and 1.3 kb) found in TcR gamma/delta+ T lymphocytes. Sequence analysis of nine different 2.3-kb cDNA clones obtained from NK-derived polyA+ RNA confirmed that they corresponded to an unrearranged TcR delta gene. These cDNA were 2343 bp long and their transcription initiation site was located 814 bp upstream from the J delta 1 segment. The sequence located upstream of the J delta 1 segment corresponded to the previously reported germ-line sequence. The J delta 1 segment was correctly spliced to C delta; in addition the four C delta exons were found to be already assembled. Two polyadenylation sites were present in the fourth C delta exon. However, only that located at the 3' end appeared to be utilized in the 2.3-kb cDNA. The expression of hGATA-3, a T cell-specific factor known to be involved in the regulation of the transcription of TcR delta locus, was analyzed by Northern blot, in cultured NK cell population and clones (but not in freshly derived cell populations). All NK clones and cell lines studied were found to express hGATA-3-specific mRNA, suggesting that hGATA-3 may be involved in the regulation of the unrearranged TcR delta gene expression in NK cells. Finally, no transcription of the RAG-1 gene could be detected in all NK cell lines or clones analyzed.