Simona Buccheri

Targeting human {gamma}delta} T cells with zoledronate and interleukin-2 for immunotherapy of hormone-refractory prostate cancer

The increasing evidence that gammadelta T cells have potent antitumor activity suggests their value in immunotherapy, particularly in areas of unmet need such as metastatic carcinoma. To this end, we initiated a phase I clinical trial in metastatic hormone-refractory prostate cancer to examine the feasibility and consequences of using the gammadelta T-cell agonist zoledronate, either alone or in combination with low-dose interleukin 2 (IL-2), to activate peripheral blood gammadelta cells. Nine patients were enlisted to each arm. Neither treatment showed appreciable toxicity. Most patients were treated with zoledronate + IL-2, but conversely only two treated with zoledronate displayed a significant long-term shift of peripheral gammadelta cells toward an activated effector-memory-like state (T(EM)), producing IFN-gamma and perforin. These patients also maintained serum levels of tumor necrosis factor-related apoptosis inducing ligand (TRAIL), consistent with a parallel microarray analysis showing that TRAIL is produced by gammadelta cells activated via the T-cell receptor and IL-2. Moreover, the numbers of T(EM) gammadelta cells showed a statistically significant correlation with declining prostate-specific antigen levels and objective clinical outcomes that comprised three instances of partial remission and five of stable disease. By contrast, most patients treated only with zoledronate failed to sustain either gammadelta cell numbers or serum TRAIL, and showed progressive clinical deterioration. Thus, zoledronate + IL-2 represents a novel, safe, and feasible approach to induce immunologic and clinical responses in patients with metastatic carcinomas, potentially providing a substantially increased window for specific approaches to be administered. Moreover, gammadelta cell phenotypes and possibly serum TRAIL may constitute novel biomarkers of prognosis upon therapy with zoledronate + IL-2 in metastatic carcinoma.

In vivo manipulation of Vgamma9Vdelta2 T cells with zoledronate and low-dose interleukin-2 for immunotherapy of advanced breast cancer patients.

The potent anti‐tumour activities of γδ T cells have prompted the development of protocols in which γδ‐agonists are administered to cancer patients. Encouraging results from small Phase I trials have fuelled efforts to characterize more clearly the application of this approach to unmet clinical needs such as metastatic carcinoma. To examine this approach in breast cancer, a Phase I trial was conducted in which zoledronate, a Vγ9Vδ2 T cell agonist, plus low‐dose interleukin (IL)‐2 were administered to 10 therapeutically terminal, advanced metastatic breast cancer patients. Treatment was well tolerated and promoted the effector maturation of Vγ9Vδ2 T cells in all patients. However, a statistically significant correlation of clinical outcome with peripheral Vγ9Vδ2 T cell numbers emerged, as seven patients who failed to sustain Vγ9Vδ2 T cells showed progressive clinical deterioration, while three patients who sustained robust peripheral Vγ9Vδ2 cell populations showed declining CA15‐3 levels and displayed one instance of partial remission and two of stable disease, respectively. In the context of an earlier trial in prostate cancer, these data emphasize the strong linkage of Vγ9Vδ2 T cell status to reduced carcinoma progression, and suggest that zoledronate plus low‐dose IL‐2 offers a novel, safe and feasible approach to enhance this in a subset of treatment‐refractory patients with advanced breast cancer.

CD90+ liver cancer cells modulate endothelial cell phenotype through the release of exosomes containing H19 lncRNA

BackgroundCD90+ liver cancer cells have been described as cancer stem-cell-like (CSC), displaying aggressive and metastatic phenotype. Using two different in vitro models, already described as CD90+ liver cancer stem cells, our aim was to study their interaction with endothelial cells mediated by the release of exosomes.MethodsExosomes were isolated and characterized from both liver CD90+ cells and hepatoma cell lines. Endothelial cells were treated with exosomes, as well as transfected with a plasmid containing the full length sequence of the long non-coding RNA (lncRNA) H19. Molecular and functional analyses were done to characterize the endothelial phenotype after treatments.ResultsExosomes released by CD90+ cancer cells, but not by parental hepatoma cells, modulated endothelial cells, promoting angiogenic phenotype and cell-to-cell adhesion. LncRNA profiling revealed that CD90+ cells were enriched in lncRNA H19, and released this through exosomes. Experiments of gain and loss of function of H19 showed that this LncRNA plays an important role in the exosome-mediated phenotype of endothelial cells.ConclusionsOur data indicate a new exosome-mediated mechanism by which CSC-like CD90+ cells could influence their tumor microenvironment by promoting angiogenesis. Moreover, we suggest the lncRNA H19 as a putative therapeutic target in hepatocellular carcinoma.

Design, synthesis, and biological evaluation of novel aminobisphosphonates possessing an in vivo antitumor activity through a gammadelta-T lymphocytes-mediated activation mechanism.

A small series of aminobisphosphonates (N-BPs) structurally related to zoledronic acid was synthesized with the aim of improving activity toward activation of human gammadelta T cells and in turn their in vivo antitumor activity. The absence of the 1-OH moiety, together with the position and the different basicity of the nitrogen, appears crucial for antitumor activity. In comparison to zoledronic acid, compound 6a shows a greater ability to activate gammadelta T cells expression (100 times more) and a proapoptotic effect that is better than zoledronic acid. The potent activation of gammadelta T cells, in addition to evidence of the in vivo antitumor activity of 6a, suggests it may be a new potential drug candidate for cancer treatment.

IL-4 depletion enhances host resistance and passive IgA protection against tuberculosis infection in BALB/c mice.

The influence of Th2 cytokines in tuberculosis has been a matter of dispute. Here we report that IL‐4 has a profound regulatory effect on the infection of BALB/c mice with Mycobacterium tuberculosis. Depletion of IL‐4 with a neutralizing mAb caused only evanescent reduction of lung infection, but when combined with i.n. inoculations of IgA anti‐mycobacterial α‐crystallin mAb and mouse rIFN‐γ, we observed a 40‐fold reduction of the bacterial counts in the lungs at 3 wks following i.n. infection (p<0.001). In genetically deficient IL‐4–/– BALB/c mice, infection in both lung and spleen was substantially reduced for up to 8 wks without further treatment. Reconstitution of IL‐4–/– mice with rIL‐4 increased bacterial counts to wild‐type levels and made the mice refractory to protection by IgA/IFN‐γ. Analysis of the lungs showed increased granulomatous infiltration and proinflammatory mediators in anti‐IL‐4/IgA/IFN‐γ‐treated and infected mice. We conclude that the action of IL‐4 in tuberculosis is targeted at macrophages and that it may include an antagonistic effect on their IgA/IFN‐γ‐induced activation and nitric oxide production. The described novel immunotherapy, combining treatments with anti‐IL‐4, IgA antibody and IFN‐γ, has potential for translation toward the passive immunoprophylaxis of tuberculosis.

Disulfiram, an old drug with new potential therapeutic uses for human hematological malignancies

Disulfiram (DSF) is an aldehyde dehydrogenase inhibitor currently used for the treatment of alcoholism. Here, we show that multiple myeloma (MM) cell lines and primary cells from newly diagnosed and relapsed/resistant patients affected by MM, acute myeloid and lymphoblastic leukemia are significantly sensitive to DSF alone and in combination with copper. These effects are present at doses lower than those achievable in vivo after DSF standard administration. The cytotoxic effect achieved by this treatment is comparable to that obtained by conventional chemotherapy and is absent in normal hematopoietic cells. In addition, we found that DSF plus copper induces loss of mitochondrial membrane potential, triggers reactive oxygen species (ROS) production and activates executioner caspases. DSF‐copper‐induced apoptosis and caspases activation are strongly reversed by antioxidant N‐acetylcysteine, thus indicating a critical role of ROS. These results might suggest the use of the old drug DSF, alone or in combination with copper, in the treatment of hematological malignancies.

Prevention of the post-chemotherapy relapse of tuberculous infection by combined immunotherapy

We report that a recently developed combined immunotherapy (CIT) has the capacity to prevent a spontaneous relapse of replicating Mycobacterium tuberculosis bacilli in the lungs of BALB/c, C57Bl/6 or C3H/HeJ strains of mice, following 4weeks of non-sterilising treatment with isoniazid and rifampicin. The CIT regimen, represented by recombinant IFNgamma, anti-alpha crystalline monoclonal IgA antibody and IL-4 neutralizing polyclonal antibody, reduced the 8-week relapse of viable bacterial counts in the lungs most significantly, when CIT was inoculated during the 5th week post infection, i.e. during the 3rd week of chemotherapy. Although CIT enhanced lung granuloma area, nitric oxide, cytokine and chemokine levels in lung washings significantly, these could not be directly associated with the beneficial effect of CIT on the degree of relapse in the lungs. These results represent a proof-of-principle, that the described CIT, when combined with chemotherapy, could have potential for future development of a shorter regimen of tuberculosis treatment.

CD200 expression in patients with Multiple Myeloma: another piece of the puzzle.

CD200 is a relatively ubiquitously expressed molecule that plays a role in cancer immune evasion through interaction with its receptors. High expression levels of CD200 have been described in different human malignancies. For example, CD200 has been shown to be targeted after RAS/RAF/MEK/ERK activation in melanoma. Here we present the analysis of CD200 expression in human Multiple Myeloma (MM) samples. We found that CD200-positive cells express ERK and p-ERK. Moreover, UO126, a MEK inhibitor, reduces CD200 expression. Furthermore, we observe that CD200-positive cells show reduced immunogenicity compared to normal lymphocytes and that such immunogenicity increases when UO126 is used. We therefore hypothesize that CD200 expression in MM could suppress antitumor response and that anti-CD200 treatment might be therapeutically beneficial in CD200-expressing tumors.

Increased phospho-mTOR expression in megakaryocytic cells derived from CD34+ progenitors of essential thrombocythaemia and myelofibrosis patients.

The mammalian target of rapamycin (mTOR) is a serine/ threonine kinase that forms different multiprotein complexes with diverse subunit compositions, downstream substrates and biological effects (Ma & Blenis, 2009). As mTOR is a key regulator of cell growth and metabolism, its improper activation has been previously linked to carcinogenesis (Meric-Bernstam & Gonzalez-Angulo, 2009). Polycythaemia vera (PV), essential thrombocythaemia (ET) and primary myelofibrosis (PMF) are BCR-ABL1-negative myeloproliferative neoplasms (MPNs). ET is characterized by megakaryocytic bone marrow hyperplasia and a sustained elevation of platelet count with a tendency for thrombosis and haemorrhage (Levine & Gilliland, 2008). PMF can arise ‘de novo’ or evolve from PV or ET. In PMF, the abnormal proliferation of an aberrant clone eventually leads to the replacement of the normal bone marrow with connective tissue fibres (Tefferi & Vainchenker, 2011). As the molecular culprit(s) of these disorders still need(s) to be elucidated, current treatment options are not curative and have yet to produce clear benefits in terms of increasing overall survival (Tefferi & Vainchenker, 2011). Mammalian target of rapamycin is a pivotal downstream target of the thrombopoietin receptor (Drayer et al, 2006). Thus, it is not surprising that several studies have previously demonstrated critical contributions of mTOR signalling in the proliferation and differentiation of normal megakaryocyte (MK) cultures (Drayer et al, 2006; Raslova et al, 2006). However, the role of mTOR in the megakaryopoiesis of ET and PMF cells remains to be investigated. As thrombocytosis is a common clinical feature of both ET and PMF, we wanted to establish whether mTOR activation was involved in the increased platelet counts observed in these MPNs. To this end, CD34-positive (CD34+) progenitors were employed to reproduce the different stages of human MK differentiation that generate morphologically and functionally mature platelets (Guerriero et al, 1995; Deutsch & Tomer, 2006). After acquiring informed consent, human CD34+ cells were isolated from healthy donors, ET and PMF patients using the midi-MACS immunomagnetic separation system (Miltenyi Biotec, Auburn, CA, USA) obtaining a >90% pure cell population. Megakaryocytic differentiation was then induced by growing these cells for 14 d in serum-free medium in the presence of bovine serum albumin, insulin, human transferrin, human low-density lipoprotein, and 100 ng/ml thrombopoietin (PeproTech, London, UK). The expression of the CD34 and CD61 surface markers was evaluated by flow cytometry, collecting cells at different time-points (days 0, 3, 12) and using a fluorescein isothiocyanate-labelled anti-CD34 antibody and an anti-CD61 antibody bound to phycoerythrin (Miltenyi Biotec). A gradual decrease in the CD34 signal associated with increased expression of the MK-specific CD61 marker confirmed the purity of the initial population and its progressive megakaryocytic differentiation (Figure S1A). Morphological observations after May–Grunwald Giemsa staining (Sigma, St Louis, MO, USA) correlated with the flow cytometry data, showing large polynucleated MK cells after 12 d of culture (Figure S1B). A unilineage system was subsequently employed to establish if MK cells isolated from patients with ET or PMF expressed higher levels of phosphorylated mTOR (p-mTOR) when compared to healthy individuals. We initially performed an immunofluorescence (IF) analysis in which cells were stained with an anti-p-mTOR (recognizing serine 2448) antibody (Cell Signaling, Danvers, MA, USA) after 3 and 12 d of culture. While we found no differences in mTOR phosphorylation after 3 d (Fig 1A, top panels), p-mTOR was increased in MKs from ET and PMF patients obtained after 12 d of culture (Fig 1A, bottom panels), but not in cells derived from healthy donors. To quantify the qualitative differences detected by IF, we analysed mTOR phosphorylation by flow cytometry, evaluating the xfold increase in median fluorescence intensity over the isotype control (D’Asaro et al, 2010). This analysis confirmed higher p-mTOR expression in ET and PMF-derived MKs generated after 12 d of culture (Fig 1B). To further confirm these data, we performed an anti-pmTOR immunohistochemical analysis on 35 bone marrow specimens derived from patients diagnosed with ET (n = 14) or PMF (n = 21). Three micrometre-thick sections were incubated with the same anti-p-mTOR antibody employed for the IF analyses. We found higher p-mTOR expression in 34 of 35 bone marrow samples as compared to the mostly negative staining observed in healthy individuals (Fig 2). The present study provides the first comparison of p-mTOR expression in MK cells derived from the CD34+ progenitors of healthy donors, ET and PMF patients. Recent evidence suggests that targeting mTOR with the oral inhibitor everolimus may be clinically useful for patients

Squamous Cell Tumors Recruit γδ T Cells Producing either IL17 or IFNγ Depending on the Tumor Stage

Tumor-infiltrating lymphocytes contain γδ T cells. In early-stage SCC tumors, γδ T cells had antitumor properties, such as production of IFNγ. However, clinically advanced tumors contained many more γδ T cells that produced IL-17 and promoted tumor growth. The identification of reciprocal interactions between tumor-infiltrating immune cells and the microenviroment may help us understand mechanisms of tumor growth inhibition or progression. We have assessed the frequencies of tumor-infiltrating and circulating γδ T cells and regulatory T cells (Treg) from 47 patients with squamous cell carcinoma (SCC), to determine if they correlated with progression or survival. Vδ1 T cells infiltrated SSC tissue to a greater extent than normal skin, but SCC patients and healthy subjects had similar amounts circulating. However, Vδ2 T cells were present at higher frequencies in circulation than in the tissue of either cancer patients or healthy donors. Tregs were decreased in the peripheral blood of SCC patients, but were significantly increased in the tumor compartment of these patients. Tumor-infiltrating γδ T cells preferentially showed an effector memory phenotype and made either IL17 or IFNγ depending on the tumor stage, whereas circulating γδ T cells of SCC patients preferentially made IFNγ. Different cell types in the tumor microenvironment produced chemokines that could recruit circulating γδ T cells to the tumor site and other cytokines that could reprogram γδ T cells to produce IL17. These findings suggest the possibility that γδ T cells in SCC are recruited from the periphery and their features are then affected by the tumor microenvironment. Elevated frequencies of infiltrating Vδ2 T cells and Tregs differently correlated with early and advanced tumor stages, respectively. Our results provide insights into the functions of tumor-infiltrating γδ T cells and define potential tools for tumor immunotherapy. Cancer Immunol Res; 5(5); 397–407. ©2017 AACR.