[Show abstract][Hide abstract] ABSTRACT: Background:
Vascular endothelial growth factor action in tumour angiogenesis is well characterised; nevertheless, it functions as a key element in the promotion of the immune system's evasion by tumours. We sought to investigate the possible direct effect of VEGF on T-cell activation and through which type of VEGF receptor it exerts this effect on cells isolated from ovarian cancer patients' ascites.
T cells isolated from the ascites of ovarian cancer patients were cultured with anti-CD3 and IL-2, with or without VEGF for 14 days and the number of viable T cells was counted. Cytotoxic activity of cultured T cells and expression of VEGF receptor-2 (VEGFR-2), was assayed.
The addition of VEGF in cultures significantly reduced the number and proliferation rate of T cells in a dose-dependent manner and CD3+ T cells expressed VEGFR-2 on their surface upon activation. Experiments with specific anti-VEGFR-2 antibodies revealed that the direct suppressive effect of VEGF on T-cell proliferation is mediated by VEGFR-2. We also showed that VEGF significantly reduced the cytotoxic activity of T cells.
Our study showed that ascites-derived T cells secrete VEGF and express VEGFR-2 upon activation. Vascular endothelial growth factor directly suppresses T-cell activation via VEGFR-2.
British Journal of Cancer 11/2012; 107(11):1869-75. DOI:10.1038/bjc.2012.468 · 4.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background
The combinations of methotrexate, vinblastine, Adriamycin, cisplatin (Pharmanell, Athens, Greece) (MVAC) or gemcitabine, cisplatin (GC) represent the standard treatment of advanced urothelial cancer (UC). Dose-dense (DD)-MVAC has achieved longer progression-free survival (PFS) than the conventional MVAC. However, the role of GC intensification has not been studied. We conducted a randomized, phase III study comparing a DD-GC regimen with DD-MVAC in advanced UC.Patients and methodsOne hundred and thirty patients were randomly assigned between DD-MVAC: 66 (M 30 mg/m(2), V 3 mg/m(2), A 30 mg/m(2), C 70 mg/m(2) q 2 weeks) and DD-GC 64 (G 2500 mg/m(2), C 70 mg/m(2) q 2 weeks). The median follow-up was 52.1 months (89 events).ResultsThe median overall survival (OS) and PFS were 19 and 8.5 months for DD-MVAC and 18 and 7.8 months for DD-GC (P = 0.98 and 0.36, respectively). Neutropenic infections were less frequent for DD-GC than for DD-MVAC (0% versus 8%). More patients on DD-GC received at least six cycles of treatment (85% versus 63%, P = 0.011) and the discontinuation rate was lower for DD-GC (3% versus 13%).Conclusions
Although DD-GC was not superior to DD-MVAC, it was better tolerated. DD-GC could be considered as a reasonable therapeutic option for further study in this patient population.Clinical Trial NumberACTRN12610000845033, www.anzctr.org.au.
Annals of Oncology 11/2012; 24(4). DOI:10.1093/annonc/mds583 · 7.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Introduction: Natural Killer (NK) cells represent important effectors against hematological and non-hematological malignances. They are highly efficient in killing ex-vivo-derived tumor cells and are effective against tumor cells with stem cell properties, the ultimate target of both conventional and innovative therapies. Clinical evidences however, show that, in vivo, tumors could avoid NK-mediated attack. Indeed it has been shown that NK cell cytolytic activity can be down-regulated by soluble mediators released by tumor cells and/or by immune cell types recruited at the tumor site. The observation that NK cells are often scarce within tumors, despite high local levels of chemokines prompted us to analyze whether tumor cells could avoid NK-mediated immunosurveillance by modulation of NK cell chemokine receptor repertoire. This study has been focused on immunomodulatory role of Neuroblastoma (NB), a poorly differentiated extracranial neuroectodermal tumor that accounts for 15% of all childhood cancer deaths. Material and Methods: NK cells were purified from peripheral blood mononuclear cells of healthy donors by Human NK Cell Isolation kits. NB cell lines utilized: SH-SY-5Y, HTLA-230, SK-NF-1, IMR32, GI-LI-N and SK-N-SH. For cytofluorimetric analysis cells were stained with PE-conjugated mAbs or with unconjugated mAbs followed by conjugated goat anti-mouse second reagent and analyzed by a cytometer. Neuroblastoma-conditioned supernatants were analyzed for the presence of 50 soluble factors using Multiplex Human Cytokine assays. Chemotaxis of neuroblastoma conditioned-NK cells was evaluated by standard assays. Results and Discussion: Freshly purified human NK cells were cultured under trans-well conditions in the presence of NB cell lines. After co-cultures, NK cells were analyzed for the expression of a panel of chemokine receptors known to regulate their migratory capacity. This analysis showed that NB-conditioning up regulates on NK cells CXCR3 and CXCR4 expression. On the contrary NB-conditioned NK cells were characterized by a significative decrease in CX 3 CR1 expression as compared to unconditioned NK cells. Thanks to the analysis of the soluble factors released by the different NB cell lines utilized and by the use of mAb-mediated blocking of specific soluble factors we identified the mediator responsible for the described effect. Interestingly the described immunomodulatory feature was shared only by some of the several NB cell lines utilized.: CD200 is an immunosuppressive molecule which over-expressed in some hematologic malignancies such as B-cell chronic lymphocytic leukemia (B-CLL) and multiple myeloma. It has been also shown to be an independent prognostic factor for patients with acute myeloid leukemia (AML). In this current study, simultaneous CD200 expression and foxp3+ regulatory T cells level were investigated in Iranian patients with AML. Material and Methods: CD200 and its receptor (CD200R) expression levels were examined on bone marrow and peripheral blood leukemic cells obtained from 43 AML patients by Flow cytometry. This technique was also used to determine the frequency of foxp3+ regulatory T cells in these patients. ELISA method was performed to investigate TGF-b and IL-10 production level in serum of patients. Results: Correlation analysis demonstrated simultaneously increasing of foxp3+ regulatory T cells and CD200 expression in AML patients (p = 0.003; r = 0.73). Our clinically poor prognosis patients have shown both higher expression of CD200 and frequency of foxp3+ regulatory T cells (p = 0.01 and p = 0.03, respectively). Moreover, the serum concentration of TGF-b (but not IL-10) was significantly correlated with the expression of CD200 on leukemic cells (p = 0.02). Conclusions: These data signify the CD200 roles in repression and suppression of anti tumor immune system response by stimulation of regulatory mechanisms in AML patients and can suggest that CD200 may have prognostic value in this malignancy.
[Show abstract][Hide abstract] ABSTRACT: Tumor infiltrating lymphocytes (TILs) and T regulatory cells (Tregs) have been associated with prognosis in ovarian cancer, but their prognostic significance in ascites has not been studied. We performed a prospective study of T lymphocytes isolated from ascites from patients with ovarian carcinoma and we compared them with the respective populations in blood and tumors.
Mononuclear cells from ascites (n=71) and blood were isolated by Ficoll, while tumor lymphocytes (n=20) were obtained upon mechanical dissociation. Phenotypic analysis was performed with flow cytometry. Ascites from 10 patients with cirrhosis was used as control.
Tregs containing CD4(+)CD25(+) cells, NK-T containing CD3(+)CD56(+) cells and CD69 and HLADR expression of CD4 and CD8 lymphocytes were significantly increased in tumor ascites compared to blood and control ascites. A selective accumulation of these populations in the ascites of cancer patients, was suggested by the significantly higher ascites/blood (A/B) ratios in cancer patients but not controls. Cancer cell content in ascites was correlated with CD4(+)CD25(+), CD4(+)CD69(+), CD4(+)HLADR(+) and CD8(+)CD69(+) cells. There was no correlation of lymphocyte populations between ascites and samples from peritoneal metastases. Higher tumor grade was associated with increased A/B CD4(+)CD25(+) ratio and reduced CD3(+)CD56(+) cells, while platinum resistance was associated with reduced A/B CD3(+)CD56(+) ratio.
There are significant differences of CD3(+)CD56(+) and CD25(+)CD4(+) lymphocytes and increase in lymphocyte activation between blood, ascites and peritoneal metastases from patients with ovarian cancer. The selective accumulation of CD3(+)CD56(+) population in ascites may be a predictive factor for platinum resistance.
[Show abstract][Hide abstract] ABSTRACT: We recently demonstrated that supernatants from cultures of peripheral blood mononuclear cells (PBMC) activated with anti-CD3-specific antibody (ACD3S) can induce, upon brief exposure, tumor-reactive lymphocytes in cancer patients. Here, we report that ACD3S can also induce rapid and stable maturation of dendritic cells (DC) which can be used as antigen presenting cells in in vitro protocols and for cancer immunotherapy in vivo.
A short (4-day) priming of CD14+ monocytes with granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) followed by only a 24 hour-incubation in ACD3S, is sufficient to generate fully mature and stable DC.
These DC (i) stimulated strong T cell proliferative responses in the mixed lymphocyte reaction, (ii) when pulsed with unfractionated peptides from autologous tumor membrane extracts activated CD4+ T cells which proliferated in response to the autologous tumor and CD8+ cytotoxic T cells (CTL) which specifically lyse autologous tumor targets and (iii) produced high levels of IL-12.
ACD3S-treated DC are functionally superior to monocyte-conditioned medium (MCM)-treated DC generated under the same short-term protocol and as efficient as DC induced by the standard 10-day protocol. Our data present an efficient and effective method for generating in a very short period of time, highly mature and functionally competent DC.
Anticancer research 01/2001; 21(2A):1199-206. · 1.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The response of lymphokine-activated killer (LAK) and natural killer (NK) cells from mice lacking tumor necrosis factor-alpha (TNF-alpha-/- mice) was impaired in cytotoxicity assays against various tumor cell targets. Furthermore, allogeneic cytotoxic T lymphocyte (CTL) responses were also impaired as compared to TNF-alpha+/+ littermates (control mice). Cytotoxicity was restored both upon in vitro incubation of TNF-alpha-/- lymphocytes with recombinant TNF-alpha (rTNF-alpha) or upon in vivo treatment of TNF-alpha-/- mice with rTNF-alpha. Using combinations of monoclonal antibodies we were able to show that TNF-alpha-/- effector lymphocytes exhibit both perforin- and Fas ligand-based cytotoxicity. Furthermore, upon in vivo administration of rTNF-alpha these effectors, in addition to perforin and Fas ligand, are also armed with TNF-alpha cytotoxic molecules, thus resembling to the cytotoxic effectors from control mice. In a tumor model, immunized TNF-alpha-/- mice failed to reject the syngeneic fibrosarcoma MC57X, but did so when injected with rTNF-alpha. In vivo administration of anti-TNF-alpha mAb neutralized the effect of rTNF-alpha supporting the growth of MC57X cells. Our data provide novel evidence for TNF-alpha as an essential factor in (i) controlling cytotoxicity in vitro and in vivo and (ii) promoting tumor rejection in vivo.
European Journal of Immunology 08/2000; 30(7):1957-66. DOI:10.1002/1521-4141(200007)30:7<1957::AID-IMMU1957>3.0.CO;2-7 · 4.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The present study investigated the ability of supernatants collected from cultures of healthy donor-derived peripheral blood mononuclear cells (HD-PBMCs) stimulated with anti-CD3 monoclonal antibody (MAb) (allogeneic CD3 supernatants; ACD3S) to induce, upon brief exposure, tumour-reactive cytotoxic lymphocytes in cancer patients' PBMCs. ACD3S enhanced natural killer (NK) and lymphokine-activated killer (LAK) cell-mediated cytotoxicity. ACD3S contained increased levels of interleukins (IL) 1, 2, 6, 7 and 12, as well as of granulocyte-macrophage colony-stimulating factor (GM-CSF), gamma-interferon (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha). MAbs against these cytokines significantly reduced the ACD3S-induced cytotoxicity. ACD3S-induced cytotoxicity was not inhibited by anti-CD4, CD8 and MHC class I MAbs, but was markedly reduced in the presence of MAb against CD18. In contrast to HD-PBMC, ACD3S derived from cancer patients' lymphocytes exhibited lower levels of the above-mentioned cytokines and exerted reduced biological activity. In conclusion, ACD3S are able to activate, upon short-term incubation, tumour-reactive lymphocytes from cancer patients' PBMCs that lyse a variety of tumour targets, including autologous tumours. ACD3S contain high levels of certain cytokines that positively influence the induction of autologous tumour-reactive lymphocytes. Such supernatants can be collected easily from healthy donors and stored until use in clinical trials for adoptive cellular therapy of cancer. They may also be indicated in the construction of cytokine cocktails that have the ability to induce anti-tumour cytotoxicity.
British Journal of Cancer 02/1997; 76(8):1072-80. DOI:10.1038/bjc.1997.510 · 4.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Interferon-alpha (IFN-alpha) has been found to exert multiple enhancing effects in the immune response in vitro, IFN-alpha has been also used in clinical trials with variable response rates. The aim of the present study was to assess the effectiveness of IFN-alpha in the treatment of 25 patients with malignant pleural or peritoneal effusions caused by lung, and metastatic breast and ovarian cancer. Clinical responses were correlated with a) the ratio of malignant effusion (ME)-associated tumor cells to ME-associated mononuclear cells (MEMNC), b) MEMNC-derived cytotoxic responses against autologous or allogeneic tumor targets, and c) major histocompatibility complex (MHC) antigen expression on tumor cells. After partial drainage of pleural or peritoneal fluid, the patients were allocated to receive 10 million units of IFN-alpha by intrapleural or intraperitoneal injection at weekly intervals. The treatment was terminated if the malignant effusion disappeared or the patients had received four to six consecutive procedures. None of the patients received concomitant systemic chemotherapy or radiation therapy. MEMNC and tumor cells were isolated by centrifugation on discontinous percoll density gradients. Cytotoxic and phenotypic profiles of MEMNG were analyzed before and after treatment with IFN-alpha. An improvement was observed in patients with increased ratios of tumor cells to malignant effusion-associated mononuclear cells (MEMNC) in the effusions. In the same patients MEMNC were overpopulated by CD8+ T lymphocytes. In this group of patients the administration of IFN-alpha was associated with 25% complete response and 75% partial response rates. In contrast only 17% partial responses were achieved in patients whose effusions had decreased tumor cell to MEMNC ratios. The immunomodulation induced by IFN-alpha in vivo was also tested. Thus in a group of 6 patients, treatment with IFN-alpha resulted in the induction of CD8+ cell-mediated lysis against autologous tumor cells which was associated with PR (two patients). Natural killer (NK)-cell activity, and MHC class I antigen expression on effusion-associated tumor cells were also enhanced during treatment, but were not correlated with the outcome of the therapy since similar findings were also observed in the 4 non-responders. Local infusions of IFN-alpha provide an effective alternative treatment for malignant effusion in patients with lung, breast, and ovarian cancer. Increased ratios of tumor cells to MEMNC and the presence of CD8+ T lymphocytes within the malignant effusions may play an important role in the outcome of such a treatment with IFN-alpha but more patients need to be studied for definite conclusions.
Anticancer research 01/1996; 16(6B):3855-60. · 1.83 Impact Factor