Culture Shock: An Investigation into the Tolerance of Pathogenic Biofilms to Antiseptics in Environments Resembling the Chronic Wound Milieu.

Credible assessment methods must be applied to evaluate antiseptics' in vitro activity reliably. Studies indicate that the medium for biofilm culturing should resemble the conditions present at the site of infection. We cultured S. aureus, S. epidermidis, P. aeruginosa, C. albicans, and E. coli biofilms in IVWM (In Vitro Wound Milieu)-the medium reflecting wound milieu-and were compared to the ones cultured in the laboratory microbiological Mueller-Hinton (MH) medium. We analyzed and compared crucial biofilm characteristics and treated microbes with polyhexamethylene biguanide hydrochloride (PHMB), povidone-iodine (PVP-I), and super-oxidized solution with hypochlorites (SOHs). Biofilm biomass of S. aureus and S. epidermidis was higher in IVWM than in MH medium. Microbes cultured in IVWM exhibited greater metabolic activity and thickness than in MH medium. Biofilm of the majority of microbial species was more resistant to PHMB and PVP-I in the IVWM than in the MH medium. P. aeruginosa displayed a two-fold lower MBEC value of PHMB in the IVWM than in the MH medium. PHMB was more effective in the IVWM than in the MH medium against S. aureus biofilm cultured on a biocellulose carrier (instead of polystyrene). The applied improvement of the standard in vitro methodology allows us to predict the effects of treatment of non-healing wounds with specific antiseptics.

The Role of Soy Isoflavones in the Prevention of Bone Loss in Postmenopausal Women: A Systematic Review with Meta-Analysis of Randomized Controlled Trials.

The aim of the report was to determine the effects of soy isoflavones on lumbar spine, femoral neck, and total hip bone mineral density (BMD) in menopausal women. MEDLINE (PubMed), EMBASE, and Cochrane Library databases were searched for articles published in English during 1995-2019. Studies were identified and reviewed for inclusion and exclusion eligibility. Weighted mean differences (WMD) were calculated for each study and were pooled by using the random effects model. Eighteen randomized controlled trials were selected for meta-analysis. Different types of soy phytoestrogens, i.e., genistein extracts, soy isoflavones extracts, soy protein isolate, and foods containing diverse amounts of isoflavones were used in the studies. The analysis showed that daily intake of 106 (range, 40-300) mg of isoflavones for 6-24 months moderately but statistically significantly positively affects BMD, compared with controls: lumbar spine WMD = 1.63 (95% CI: 0.51 to 2.75)%, p = 0004; femoral neck WMD = 1.87 (95% CI: 0.14 to 3.60)%, p = 0.034; and total hip WMD = 0.39 (95% CI: 0.08 to 0.69)%, p = 0.013. Subgroups analyses indicated that the varying effects of isoflavones on BMD across the trials might be associated with intervention duration, racial diversity (Caucasian, Asian), time after menopause, form of supplements (especially genistein), and dose of isoflavones. Our review and meta-analysis suggest that soy isoflavones are effective in slowing down bone loss after menopause.

Small extracellular vesicles containing arginase-1 suppress T-cell responses and promote tumor growth in ovarian carcinoma.

Tumor-driven immune suppression is a major barrier to successful immunotherapy in ovarian carcinomas (OvCa). Among various mechanisms responsible for immune suppression, arginase-1 (ARG1)-carrying small extracellular vesicles (EVs) emerge as important contributors to tumor growth and tumor escape from the host immune system. Here, we report that small EVs found in the ascites and plasma of OvCa patients contain ARG1. EVs suppress proliferation of CD4+ and CD8+ T-cells in vitro and in vivo in OvCa mouse models. In mice, ARG1-containing EVs are transported to draining lymph nodes, taken up by dendritic cells and inhibit antigen-specific T-cell proliferation. Increased expression of ARG1 in mouse OvCa cells is associated with accelerated tumor progression that can be blocked by an arginase inhibitor. Altogether, our studies show that tumor cells use EVs as vehicles to carry over long distances and deliver to immune cells a metabolic checkpoint molecule - ARG1, mitigating anti-tumor immune responses.

Biological markers of prognosis, response to therapy and outcome in ovarian carcinoma.

INTRODUCTION: Ovarian cancer (OvCa) is among the most common types of cancer and is the leading cause of death from gynecological malignancies in western countries. Cancer biomarkers have a potential for improving the management of OvCa patients at every point from screening and detection, diagnosis, prognosis, follow up, response to therapy and outcome. AREAS COVERED: The literature search has indicated a number of candidate biomarkers have recently emerged that could facilitate the molecular definition of OvCa, providing information about prognosis and predicting response to therapy. These potentially promising biomarkers include immune cells and their products, tumor-derived exosomes, nucleic acids and epigenetic biomarkers. Expert commentary: Although most of the biomarkers available today require prospective validation, the development of noninvasive liquid biopsy-based monitoring promises to improve their utility for evaluations of prognosis, response to therapy and outcome in OvCa.

Antagonism of tumoral prolactin receptor promotes autophagy-related cell death.

Therapeutic upregulation of macroautophagy in cancer cells provides an alternative mechanism for cell death. Prolactin (PRL) and its receptor (PRLR) are considered attractive therapeutic targets because of their roles as growth factors in tumor growth and progression. We utilized G129R, an antagonist peptide of PRL, to block activity of the tumoral PRL/PRLR axis, which resulted in inhibition of tumor growth in orthotopic models of human ovarian cancer. Prolonged treatment with G129R induced the accumulation of redundant autolysosomes in 3D cancer spheroids, leading to a type II programmed cell death. This inducible autophagy was a noncanonical beclin-1-independent pathway and was sustained by an astrocytic phosphoprotein (PEA-15) and protein kinase C zeta interactome. Lower levels of tumoral PRL/PRLR in clinical samples were associated with longer patient survival. Our findings provide an understanding of the mechanisms of tumor growth inhibition through targeting PRL/PRLR and may have clinical implications.

Exosomes in Plasma of Patients with Ovarian Carcinoma: Potential Biomarkers of Tumor Progression and Response to Therapy.

BACKGROUND: In patients with Ovarian Cancer (OvCa) exosomes released by tumor cells are present in the plasma and could be involved in tumor progression. This study examines the association between the exosome presence/protein content in plasma of OvCa patients and disease outcome, response to standard therapy and/or tumorresistance to therapies in patients studied at diagnosis and also serially during and after therapy. DESIGN AND METHODS: Exosomes were purified from OvCa patients' plasma (n=22), patients with benign tumors (n=10) or (n=10) healthy controls (NC) using ultracentrifugation. Exosomes were visualized by scanning electron microscopy. Their protein content was measured. The presence of MAGE 3/6 and TGF-ß1 in exosomes was evaluated in Western blots. RESULTS: The OvCa patients' plasma contained higher levels of exosomal proteins (p<0.05) compared to those isolated from plasma of patients with benign tumors or NC. Exosomes isolated from OvCa patients's plasma carried TGF-ß1 and MAGE3/6, which distinguished OvCa patients from those with benign tumors and NC. High protein levels of exosomes were seen in newly diagnosed patients; however in advanced stages of OvCa patients the protein content of isolated exosomes was significantly higher than that of early stages. The exosome levels variably changed during/after chemotherapy, and correlations between the changes in exosomal protein levels and clinical data suggested that the protein content of exosomes might be useful in predicting responses to therapy and prognosis in OvCa patients. CONCLUSION: Analysis of plasma exosomes levels offers a novel approach to diagnosis and monitoring response to therapies in OvCa patients.

17ß Hydroxysteroid dehydrogenase type 12 (HSD17B12) is a marker of poor prognosis in ovarian carcinoma.

OBJECTIVE: 17ß-hydroxysteroid dehydrogenase isoform 12 (HSD17B12) overexpression is associated with poor clinical outcome in invasive ductal carcinoma of the breast. Here, we evaluated HSD17B12 overexpression and its activity in ovarian carcinoma (OvCa) to determine its role in the growth and progression of this tumor. METHODS: Immunohistochemical analysis of HSD17B12 expression was performed in 100 tissue samples of untreated OvCa and was correlated with clinicopathologic characteristics and patient outcome. In A2780 OvCa cell line expressing HSD17B12, siRNA knockdown of the enzyme was performed, and its effects on tumor cell growth and Annexin V binding were determined. RESULTS: HSD17B12 expression was detected in all tumor samples, but the staining intensity was variable. Normal ovarian epithelium was negative. Patients with tumor showing weak/moderate expression of HSD17B12 had a better overall survival than those with strongly positive tumors (p<0.001). The time to first recurrence was longer for patients with tumors with heterogeneous staining relative to patients with tumors that were uniformly positive (p<0.001). Upon silencing of HSD17B12 in tumor cells, their growth was inhibited (p<0.005) and apoptosis was increased (p<0.05). Arachidonic acid but not estradiol reversed the growth inhibition mediated by HSD17B12 knockdown. CONCLUSION: HSD17B12 overexpression is shown to be a marker of poor survival in patients with OvCa. Expression in the tumor and function of this enzyme facilitates OvCa progression.

A gynecologic oncology group phase II trial of two p53 peptide vaccine approaches: subcutaneous injection and intravenous pulsed dendritic cells in high recurrence risk ovarian cancer patients.

PURPOSE: Peptide antigens have been administered by different approaches as cancer vaccine therapy, including direct injection or pulsed onto dendritic cells; however, the optimal delivery method is still debatable. In this study, we describe the immune response elicited by two vaccine approaches using the wild-type (wt) p53 vaccine. EXPERIMENTAL DESIGN: Twenty-one HLA-A2.1 patients with stage III, IV, or recurrent ovarian cancer overexpressing the p53 protein with no evidence of disease were treated in two cohorts. Arm A received SC wt p53:264-272 peptide admixed with Montanide and GM-CSF. Arm B received wt p53:264-272 peptide-pulsed dendritic cells IV. Interleukin-2 (IL-2) was administered to both cohorts in alternative cycles. RESULTS: Nine of 13 patients (69%) in arm A and 5 of 6 patients (83%) in arm B developed an immunologic response as determined by ELISPOT and tetramer assays. The vaccine caused no serious systemic side effects. IL-2 administration resulted in grade 3 and 4 toxicities in both arms and directly induced the expansion of T regulatory cells. The median overall survival was 40.8 and 29.6 months for arm A and B, respectively; the median progression-free survival was 4.2 and. 8.7 months, respectively. CONCLUSION: We found that using either vaccination approach generates comparable specific immune responses against the p53 peptide with minimal toxicity. Accordingly, our findings suggest that the use of less demanding SC approach may be as effective. Furthermore, the use of low-dose SC IL-2 as an adjuvant might have interfered with the immune response. Therefore, it may not be needed in future trials.

Blast-derived microvesicles in sera from patients with acute myeloid leukemia suppress natural killer cell function via membrane-associated transforming growth factor-beta1.

BACKGROUND: Natural killer cell cytotoxicity is decreased in patients with acute myeloid leukemia in comparison to that in normal controls. Tumor-derived microvesicles present in patients' sera exert detrimental effects on immune cells and may influence tumor progression. DESIGN AND METHODS: We investigated the microvesicle protein level, molecular profile and suppression of natural killer cell activity in patients with newly diagnosed acute myeloid leukemia. RESULTS: The patients' sera contained higher levels of microvesicles compared to the levels in controls (P<0.001). Isolated microvesicles had a distinct molecular profile: in addition to conventional microvesicle markers, they contained membrane-associated transforming growth factor-ß1, MICA/MICB and myeloid blasts markers, CD34, CD33 and CD117. These microvesicles decreased natural killer cell cytotoxicity (P<0.002) and down-regulated expression of NKG2D in normal natural killer cells (P<0.001). Sera from patients with acute myeloid leukemia contained elevated levels of transforming growth factor-ß, and urea-mediated dissociation of microvesicles further increased the levels of this protein. Neutralizing anti-transforming growth factor-ß1 antibodies inhibited microvesicle-mediated suppression of natural killer cell activity and NKG2D down-regulation. Interleukin-15 protected natural killer cells from adverse effects of tumor-derived microvesicles. CONCLUSIONS: We provide evidence for the existence in acute myeloid leukemia of a novel mechanism of natural killer cell suppression mediated by tumor-derived microvesicles and for the ability of interleukin-15 to counteract this suppression.

Mechanisms of tumor escape from the immune system: adenosine-producing Treg, exosomes and tumor-associated TLRs.

Human tumors utilize many different mechanisms of immunosuppression to prevent immune cells from exercising their antitumor activities. These mechanisms, which enable the tumor to escape from the host immune system and to progress, are being intensively investigated in hope of finding therapeutically safe and effective inhibitors able to counteract tumor-induced immunosuppression. Three of more recently discovered tumor-related suppression mechanisms, i.e. accumulations of adenosine-producing regulatory T-cells (Treg) in the tumor microenvironment, release by tumors of suppressive microvesicles (TMV) and expression of toll-like receptors (TLR) on the tumor cell surface, are described in this review. All contribute in a varying degree to creating a milieu favorable for the tumor and unfavorable for immune effector cells. Tumor escape has been a major problem in cancer immunotherapy and it has been held responsible for the failure of many immune interventions in cancer. For this reason, it is important to study and understand the various suppressive pathways human tumors utilize. Future antitumor immunotherapies are likely to include inhibitors of tumor-induced suppression with the goal of restoring antitumor immune responses in patients with cancer.

Mechanisms of T-cell protection from death by IRX-2: a new immunotherapeutic.

OBJECTIVES: IRX-2 is a novel immunotherapeutic containing physiologic quantities of several cytokines which protects human T lymphocytes from tumor-induced or drug-induced apoptosis. Here, we investigate the mechanisms responsible for IRX-2-mediated protection of T lymphocytes exposed to tumor-derived microvesicles (TMV). METHODS: Jurkat cells or primary human T cells ± IRX-2 were co-incubated with TMV and then examined by flow cytometry or Western blots for expression of molecules regulating cell survival (FLIP, Bcl-2, Bcl-xL, Mcl-1) or death (Fas, caspase 8, caspase 9, Bax, Bid). ANX V binding, caspase activation or cytochrome c release were also measured ± cycloheximide (CHX) or ± the Akt-specific inhibitor. Jurkat cells transfected with the cFLIP gene were used to evaluate the role of cFLIP in IRX-2-mediated protection. Effects of CHX on IRX-2-mediated protection and activation of NF-κB upon the TMV/IRX-2 treatment were also measured. RESULTS: IRX-2 protected T cells from apoptosis by preventing Fas overexpression induced by TMV and blocking caspase 8 activation by up-regulating cFLIP. Jurkat cells overexpressing cFLIP were more resistant to TMV-induced apoptosis than the mock-transfected cells (p < 0.02). Signaling via the PI3K/Akt pathway, IRX-2 corrected the imbalance of pro- versus anti-apoptotic proteins induced by TMV and promoted NF-κB translocation to the nucleus. CHX abolished IRX-2-mediated protection in T cells, suggesting that IRX-2 induces de novo synthesis of one or more proteins that are required for protection. CONCLUSIONS: This biologic may be therapeutically useful for protection of activated T cells from tumor-induced immune suppression and death.

[Expression of survivin, SDF-1 and CXCR4 on tumor cells in ovarian cancer]. / Ekspresja surwiwiny, SDF-1 i CXCR4 na komórkach nowotworowych raka jajnika.

BACKGROUND: epithelial ovarian cancer (EOC) has the highest mortality rate among patients with gynecologic malignancies. Lack of specific and early symptoms and of screening tests causes that most patients are diagnosed in advanced stage of disease. Radical surgery followed by chemotherapy does not bring satisfactory curative effects. OBJECTIVES: the urgent need exists to define the optimum biomarker for ovarian cancer to predict patients' response to curative therapy. Our current study aimed at correlation between the expression of survivin, SDF-1, CXCR-4 on tumor tissue and clinical outcome of patients with ovarian cancer. RESULTS: We showed that survivin expression correlates with histological grading of the tumor. No correlation was found in terms of SDF-1/ CXCR-4 expression and clinicopathologic data. CONCLUSIONS: further studies covering larger number of patients are needed to determine whether SDF-1 and CXCR-4 might be considered as biomarkers for ovarian cancer.

Tumor-derived microvesicles induce, expand and up-regulate biological activities of human regulatory T cells (Treg).

BACKGROUND: Tumor-derived microvesicles (TMV) or exosomes are present in body fluids of patients with cancer and might be involved in tumor progression. The frequency and suppressor functions of peripheral blood CD4(+)CD25(high)FOXP3(+) Treg are higher in patients with cancer than normal controls. The hypothesis is tested that TMV contribute to induction/expansion/and activation of human Treg. METHODOLOGY/PRINCIPAL FINDINGS: TMV isolated from supernatants of tumor cells but not normal cells induced the generation and enhanced expansion of human Treg. TMV also mediated conversion of CD4(+)CD25(neg) T cells into CD4(+)CD25(high)FOXP3(+) Treg. Upon co-incubation with TMV, Treg showed an increased FasL, IL-10, TGF-beta1, CTLA-4, granzyme B and perforin expression (p<0.05) and mediated stronger suppression of responder cell (RC) proliferation (p<0.01). Purified Treg were resistant to TMV-mediated apoptosis relative to other T cells. TMV also increased phospho-SMAD2/3 and phospho-STAT3 expression in Treg. Neutralizing Abs specific for TGF-beta1 and/or IL-10 significantly inhibited TMV ability to expand Treg. CONCLUSIONS/SIGNIFICANCE: This study suggests that TMV have immunoregulatory properties. They induce Treg, promote Treg expansion, up-regulate Treg suppressor function and enhance Treg resistance to apoptosis. Interactions of TMV with Treg represent a newly-defined mechanism that might be involved in regulating peripheral tolerance by tumors and in supporting immune evasion of human cancers.

Adenosine and prostaglandin E2 cooperate in the suppression of immune responses mediated by adaptive regulatory T cells.

Adaptive regulatory T cells (Tr1) are induced in the periphery upon encountering cognate antigens. In cancer, their frequency is increased; however, Tr1-mediated suppression mechanisms are not yet defined. Here, we evaluate the simultaneous involvement of ectonucleotidases (CD39/CD73) and cyclooxygenase 2 (COX-2) in Tr1-mediated suppression. Human Tr1 cells were generated from peripheral blood mononuclear cell-derived, sorted CD4(+)CD25(-) T cells and incubated with autologous immature dendritic cells, irradiated COX-2(+) or COX-2(-) tumor cells, and IL-2, IL-10, and IL-15 (each at 10-15 IU/ml) for 10 days as described (Bergmann, C., Strauss, L., Zeidler, R., Lang, S., and Whiteside, T. L. (2007) Cancer Immunol. Immunother. 56, 1429-1442). Tr1 were phenotyped by multicolor flow cytometry, and suppression of proliferating responder cells was assessed in carboxyfluorescein diacetate succinimidyl ester-based assays. ATP hydrolysis was measured using a luciferase detection assay, and levels of adenosine or prostaglandin E(2) (PGE(2)) in cell supernatants were analyzed by mass spectrometry or ELISA, respectively. Intracellular cAMP levels were measured by enzyme immunoassay. The COX-2(+) tumor induced a greater number of Tr1 than COX-2(-) tumor (p < 0.05). Tr1 induced by COX-2(+) tumor were more suppressive, hydrolyzed more exogenous ATP (p < 0.05), and produced higher levels of adenosine and PGE(2) (p < 0.05) than Tr1 induced by COX-2(-) tumor. Inhibitors of ectonucleotidase activity, A(2A) and EP(2) receptor antagonists, or an inhibitor of the PKA type I decreased Tr1-mediated suppression (p < 0.05), whereas rolipram, a PDE(4) inhibitor, increased the intracellular cAMP level in responder cells and their susceptibility to Tr1-mediated suppression. Tr1 present in tumors or the peripheral blood of head and neck squamous cell carcinoma patients co-expressed COX-2, CD39, and CD73. A concomitant inhibition of PGE(2) and adenosine via the common intracellular cAMP pathway might be a novel approach for improving results of immune therapies for cancer.

Reciprocal granzyme/perforin-mediated death of human regulatory and responder T cells is regulated by interleukin-2 (IL-2).

Human CD4(+)CD25(high)FOXP3(+) T regulatory cells (Treg) can suppress responder T cell (RC) functions by various mechanisms. In co-cultures of Treg and autologous activated RC, both cell subsets up-regulate the expression of granzymes and perforin, which might contribute to Treg-mediated suppression. Here, we investigate the sensitivity and resistance of Treg and RC to granzyme/perforin-mediated death. CD4(+)CD25(neg) RC were single cell-sorted from the peripheral blood of 25 cancer patients and 15 normal controls. These RC were carboxyfluorescein diacetate succinimidyl ester (CFSE) labeled and co-cultured with autologous CD4(+)CD25(high)FOXP3(+) Treg +/- 150 or +/-1,000 IU/mL of interleukin-2 (IL-2) to evaluate suppression of RC proliferation. In addition, survival of the cells co-cultured for 24 h and 5 days was measured using a flow-based cytotoxicity assay. Freshly isolated Treg and RC expressed granzyme A (GrA), granzyme B (GrB), and perforin. Percentages of positive cells were higher in cancer patients than controls (p < 0.01) and increased upon OKT3 and IL-2 stimulation. Treg, co-cultured with RC at 150 IU/mL of IL-2, no longer expressed cytotoxins and became susceptible to RC-mediated, granzyme/perforin-dependent death. However, in co-cultures with 1,000 IU/mL of IL-2, Treg became resistant to apoptosis and induced GrB-dependent, perforin-independent death of RC. When the GrB inhibitor I or GrB-specific and GrA-specific small inhibitory ribonucleic acids were used to block the granzyme pathway in Treg, RC death, and Treg-mediated suppression of RC, proliferation were significantly inhibited. Human CD4(+)CD25(high) Treg and CD4(+)CD25(neg) RC reciprocally regulate death/growth arrest by differentially utilizing the granzyme-perforin pathway depending on IL-2 concentrations.

Regulatory T cell suppression of Gag-specific CD8 T cell polyfunctional response after therapeutic vaccination of HIV-1-infected patients on ART.

We tested the hypothesis that therapeutic vaccination against HIV-1 can increase the frequency and suppressive function of regulatory, CD4(+) T cells (Treg), thereby masking enhancement of HIV-1-specific CD8(+) T cell response. HIV-1-infected subjects on antiretroviral therapy (N = 17) enrolled in a phase I therapeutic vaccine trial received 2 doses of autologous dendritic cells (DC) loaded with HIV-1 peptides. The frequency of CD4(+)CD25(hi)FOXP3(+) Treg in blood was determined prior to and after vaccination in subjects and normal controls. Polyfunctional CD8(+) T cell responses were determined pre- and post-vaccine (N = 7) for 5 immune mediators after in vitro stimulation with Gag peptide, staphylococcal enterotoxin B (SEB), or medium alone. Total vaccine response (post-vaccine-pre-vaccine) was compared in the Treg(+) and Treg-depleted (Treg-) sets. After vaccination, 12/17 subjects showed a trend of increased Treg frequency (P = 0.06) from 0.74% to 1.2%. The increased frequency did not correlate with CD8(+) T cell vaccine response by enzyme linked immunosorbent assay for interferon gamma production. Although there was no significant change in CD8(+) T cell polyfunctional response after vaccination, Treg depletion increased the polyfunctionality of the total vaccine response (P = 0.029), with a >2-fold increase in the percentage of CD8(+) T cells producing multiple immune mediators. In contrast, depletion of Treg did not enhance polyfunctional T cell response to SEB, implying specificity of suppression to HIV-1 Gag. Therapeutic immunization with a DC-based vaccine against HIV-1 caused a modest increase in Treg frequency and a significant increase in HIV-1-specific, Treg suppressive function. The Treg suppressive effect masked an increase in the vaccine-induced anti-HIV-1-specific polyfunctional response. The role of Treg should be considered in immunotherapeutic trials of HIV-1 infection.

Generation and accumulation of immunosuppressive adenosine by human CD4+CD25highFOXP3+ regulatory T cells.

Naturally occurring regulatory T cells (nTreg) are crucial for maintaining tolerance to self and thus preventing autoimmune diseases and allograft rejections. In cancer, Treg down-regulate antitumor responses by several distinct mechanisms. This study analyzes the role the adenosinergic pathway plays in suppressive activities of human nTreg. Human CD4(+)CD25(high)FOXP3(+) Treg overexpress CD39 and CD73, ectonucleotidases sequentially converting ATP into AMP and adenosine, which then binds to A(2a) receptors on effector T cells, suppressing their functions. CD4(+)CD39(+) and CD4(+)CD25(high) T cells express low levels of adenosine deaminase (ADA), the enzyme responsible for adenosine breakdown, and of CD26, a surface-bound glycoprotein associated with ADA. In contrast, T effector cells are enriched in CD26/ADA but express low levels of CD39 and CD73. Inhibitors of ectonucleotidase activity (e.g. ARL67156) and antagonists of the A(2a) receptor (e.g. ZM241385) blocked Treg-mediated immunosuppression. The inhibition of ADA activity on effector T cells enhanced Treg-mediated immunosuppression. Thus, human nTreg characterized by the presence of CD39 and the low expression of CD26/ADA are responsible for the generation of adenosine, which plays a major role in Treg-mediated immunosuppression. The data suggest that the adenosinergic pathway represents a potential therapeutic target for regulation of immunosuppression in a broad variety of human diseases.

Interleukin-15 enhances natural killer cell cytotoxicity in patients with acute myeloid leukemia by upregulating the activating NK cell receptors.

Interleukin-15 (IL-15) has a major role in NK-cell homeostasis. Modulation of the relative frequency and expression intensity of the NK-cell receptors by IL-15 may increase NK cell-mediated cytotoxicity in cancer patients. We investigated the receptor repertoire and measured NK-cell activity in newly diagnosed AML patients and evaluated the ex vivo effects of IL-15. The expression of the activating NK cell receptors was significantly decreased in the AML patients compared to that in NK cells of healthy donors. When NK cells obtained from AML patients were cultured with IL-15, expression of the activating receptors was significantly upregulated compared to pre-culture levels. Concomitantly, cytotoxic activity of NK cells against autologous leukemic blasts increased following IL-15 stimulation. This IL-15 induced increase in activity was blocked by neutralizing antibodies specific for the NK cell activating receptors. These pre-clinical data support the future use of IL-15 for NK cell- based therapies for AML patients.

Increased ectonucleotidase expression and activity in regulatory T cells of patients with head and neck cancer.

PURPOSE: Regulatory T cell (Treg) frequency and activity are increased in cancer patients and play a major role in tumor escape. Although disease progression is favored by the presence of Treg, mechanisms used by Treg to suppress antitumor immunity are unknown. The ectonucleotidases CD39 and CD73 are expressed in Treg and convert ATP into immunosuppressive adenosine. In this study, the involvement of the adenosinergic pathway in Treg-mediated suppression in head and neck squamous cell carcinoma (HNSCC) patients was evaluated. EXPERIMENTAL DESIGN: HNSCC patients with an active disease (n = 19) and patients with no evident disease after therapy (n = 14) were studied. Ectonucleotidase expression on CD4(+) T cells and CD4(+)CD25(high) Treg was evaluated by flow cytometry and compared with normal controls. Ectonucleotidase activity was also compared within these three groups. The data were analyzed for associations of ectonucleotidase expression/function with disease stage. RESULTS: The percentages and expression levels of CD39 and CD73 in CD4(+) T cells and Treg were greater in HNSCC than in normal controls and highest in patients with no evident disease. Patients' Treg hydrolyzed ATP at higher rates and produced higher levels of adenosine than normal controls' Treg. The increased frequency and enzymatic activity of CD4(+)CD39(+) cells corresponded to increased adenosine-mediated suppression of effector T cells, which was partly inhibited by ARL67156, an ectonucleotidase inhibitor, and by ZM241385, a selective A(2a)/A(2b) receptor antagonist. CONCLUSIONS: CD39(+) Treg frequency and adenosine-mediated suppression are significantly increased in HNSCC patients. The adenosinergic pathway is involved in Treg-mediated immunosuppression in cancer and its attenuation could be a promising immunotherapeutic strategy for patients with HNSCC.

Tumor-derived microvesicles promote regulatory T cell expansion and induce apoptosis in tumor-reactive activated CD8+ T lymphocytes.

Sera of patients with cancer contain membraneous microvesicles (MV) able to induce apoptosis of activated T cells by activating the Fas/Fas ligand pathway. However, the cellular origin of MV found in cancer patients' sera varies as do their molecular and cellular profiles. To distinguish tumor-derived MV in cancer patients' sera, we used MAGE 3/6(+) present in tumors and MV. Molecular profiles of MAGE 3/6(+) MV were compared in Western blots or by flow cytometry with those of MV secreted by dendritic cells or activated T cells. These profiles were found to be distinct for each cell type. Only tumor-derived MV were MAGE 3/6(+) and were variably enriched in 42-kDa Fas ligand and MHC class I but not class II molecules. Effects of MV on signaling via the TCR and IL-2R and proliferation or apoptosis of activated primary T cells and T cell subsets were also assessed. Functions of activated CD8(+) and CD4(+) T lymphocytes were differentially modulated by tumor-derived MV. These MV inhibited signaling and proliferation of activated CD8(+) but not CD4(+) T cells and induced apoptosis of CD8(+) T cells, including tumor-reactive, tetramer(+)CD8(+) T cells as detected by flow cytometry for caspase activation and annexin V binding or by DNA fragmentation. Tumor-derived but not dendritic cell-derived MV induced the in vitro expansion of CD4(+)CD25(+)FOXP3(+) T regulatory cells and enhanced their suppressor activity. The data suggest that tumor-derived MV induce immune suppression by promoting T regulatory cell expansion and the demise of antitumor CD8(+) effector T cells, thus contributing to tumor escape.