Comparative expression profiling of distinct T cell subsets undergoing oxidative stress.

The clinical outcome of adoptive T cell transfer-based immunotherapies is often limited due to different escape mechanisms established by tumors in order to evade the hosts' immune system. The establishment of an immunosuppressive micromilieu by tumor cells along with distinct subsets of tumor-infiltrating lymphocytes is often associated with oxidative stress that can affect antigen-specific memory/effector cytotoxic T cells thereby substantially reducing their frequency and functional activation. Therefore, protection of tumor-reactive cytotoxic T lymphocytes from oxidative stress may enhance the anti-tumor-directed immune response. In order to better define the key pathways/proteins involved in the response to oxidative stress a comparative 2-DE-based proteome analysis of naïve CD45RA(+) and their memory/effector CD45RO(+) T cell counterparts in the presence and absence of low dose hydrogen peroxide (H(2)O(2)) was performed in this pilot study. Based on the profiling data of these T cell subpopulations under the various conditions, a series of differentially expressed spots were defined, members thereof identified by mass spectrometry and subsequently classified according to their cellular function and localization. Representative targets responding to oxidative stress including proteins involved in signaling pathways, in regulating the cellular redox status as well as in shaping/maintaining the structural cell integrity were independently verified at the transcript and protein level under the same conditions in both T cell subsets. In conclusion the resulting profiling data describe complex, oxidative stress-induced, but not strictly concordant changes within the respective expression profiles of CD45RA(+) and CD45RO(+) T cells. Some of the differentially expressed genes/proteins might be further exploited as potential targets toward modulating the redox capacity of the distinct lymphocyte subsets thereby providing the basis for further studies aiming at rendering them more resistant to tumor micromilieu-induced oxidative stress.

High expression of GCLC is associated with malignant melanoma of low oxidative phenotype and predicts a better prognosis.

Reactive oxygen species (ROS) are strongly implicated in melanoma development, and treatment with antioxidants has shown efficacy in suppressing malignant transition and progression. Here, we investigated the significance of the glutamate-L: -cysteine ligase catalytic subunit (GCLC) expression, a key regulator of glutathione synthesis, for malignant melanoma. A large set of melanoma cell lines (n = 36) was analyzed, and higher GCLC levels were associated with lower presence of intracellular ROS and interestingly also lower rates of cell proliferation. Moreover, treatment with the antioxidant N-acetylcysteine efficiently reduced the growth speed of several investigated malignant cells. In addition GCLC expression was significantly linked to a prominent set of cellular antioxidants, accounting for the observed lower basal levels of oxidative stress and higher antioxidative capacity. Key attributes defining the malignant phenotype of melanoma cells including survival, invasiveness, and switch from E-cadherin to N-cadherin expression were more prominent in cells with lower GCLC expression. Our findings were further corroborated by observations in Rag2(-/-)γc(-/-)mice, in which melanoma cells with lower GCLC expression depicted a dramatically stronger tumor growth. Furthermore, prognostic significance of GCLC expression was investigated in patients (n = 28) with advanced malignant melanoma. High tumor immunoreactivity for GCLC was a significant determinant for better 5-year overall survival. Conclusively, we show for the first time that GCLC may serve a dual role, as a surrogate marker for cellular redox state as well as malignant potential of melanoma cells. These promising results regarding its prognostic significance as well as its potential as a pharmacological target require further in-depth investigations.

Nuclear HER3 is associated with favorable overall survival in uveal melanoma.

HER3 is a member of the epidermal growth factor receptor (EGFR) family and is expressed in several types of cancer. Both the cytoplasmic and nuclear appearances of the receptor have been reported. Here, we investigate the expression and subcellular distribution of HER3 in uveal melanoma (UM) cells and tissues and its potential impact on clinical outcome of patients. Paraffin-embedded samples from 128 consecutive UM patients, enucleated without alternative treatment on UM diagnosis, were evaluated for HER3 using immunohistochemistry. Immunoreactivity was scored for frequency, intensity of positive cells, and subcellular distribution. The results were correlated with the established clinicopathological parameters using univariate and multivariate statistical analyses. HER3 expression was shown in 70% of the cases (89/128). This contrasts with the other EGFR family receptors (EGFR, HER2 and HER4) that are infrequently expressed in UM. Surprisingly, HER3 was found to be localized solely in the cell nuclei in 56 cases. The remaining 33 HER3 positive cases showed diffuse distribution (cytoplasmic ± nuclear). Nuclear HER3 was independently correlated with a more favorable overall survival (p = 0.043 and hazard ratio = 0.618) compared to cases with diffuse and/or no HER3. Nuclear localization of HER3 was also confirmed in fresh UM material and in UM cell lines. In conclusion, HER3 is frequently localized solely in the cell nuclei in UM and as such it predicts a more favorable overall survival.

Multipotent mesenchymal stromal cells express FoxP3: a marker for the immunosuppressive capacity?

Multipotent mesenchymal stromal cells (MSCs) have immunosuppressive capacity but the exact mechanism by which they suppress proliferation of T lymphocytes is not fully understood. Recently, the characteristics and function of regulatory T lymphocytes (Tregs) have become better defined. Tregs and MSCs have immunosuppressive features in common. Here, we looked for a common basis for immunosuppression in these distinct cell types. Forkhead box P3 (FoxP3) and CD39 expression in MSCs was measured by flow cytometry and real-time quantitative polymerase chain reaction. The importance of FoxP3 in MSC-mediated immunosuppression was investigated by siRNA technology and mixed lymphocyte culture (MLC). The effect of 5-azacytidine and other immunosuppressive drugs on FoxP3 expression and immunosuppression by MSCs was explored by flow cytometry, MLC, and real-time quantitative polymerase chain reaction. MSCs express FoxP3 at variable levels, but they do not express CD39. FoxP3 MSCs suppress MLC to a greater extent than cells with lower FoxP3 expression. However, FoxP3-decreased MSCs were found to retain their immunosuppressive properties. 5-azacytitine had no effect on FoxP3 expression or MLC suppression by MSCs. However, immunosuppressive drugs led to increased FoxP3 levels and MLC inhibition in FoxP3 MSCs. This is the first demonstration of FoxP3 expression by MSCs. Although MSCs share several features with Tregs, and FoxP3 MSCs tend to be more immunosuppressive, MSCs do not require functional FoxP3 for their immunosuppressive activity. The increased MSC-mediated suppression of immune responses by immunosuppressive drugs deserves further investigation.

The impact of inflammatory licensing on heme oxygenase-1-mediated induction of regulatory T cells by human mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are characterized by their manifold immunomodulatory and regenerative properties. The stress-responsive, cytoprotective, and immunoregulatory molecule heme oxygenase-1 (HO-1) was recently identified as a key contributor for MSC-mediated suppression of alloactivated T cells. As HO-1 has also been implicated in the induction of regulatory T cells (Tregs), we sought to examine its impact on MSC-driven promotion of Tregs. Human MSCs were shown to induce, in a HO-1-dependent fashion, IL-10(+) Tr1 and transforming growth factor-ß(+) Th3 Treg-subsets in allo- and T-cell receptor-activated lymphocytes. Because inflammatory stimuli modulate ("license") human MSCs, we were interested in whether an in vitro alloreactive micro-milieu within mixed lymphocyte reactions (MLRs) alters the HO-1 expression. We observed a substantial down-regulation of HO-1 facilitated by yet unidentified soluble factor(s) produced in an MLR, and most probably occurring at the level of its major transcription-factor NF-E2-related factor 2. Interestingly, HO-1 lost its impact regarding suppressiveness, Treg induction, and promotion of IL-10 production for MSCs, which were prelicensed in an MLR environment. Taken together, we show that HO-1 produced by human MSCs beyond its direct suppressive function promotes formation of Tr1 and Th3 Tregs and IL-10 production, functions, which are taken over by other molecules, among them COX-2, after an alloreactive priming.

T cell recognition of HLA-A2 restricted tumor antigens is impaired by the oncogene HER2.

The HER2 oncogene is frequently over-expressed in human cancers and a promising target for immune therapy. Previous studies have shown that over-expression of mouse or rat HER2 leads to markedly reduced levels of major histocompatibility complex (MHC) class I and molecules of the antigen processing and presentation machinery (APM), thus resulting in a phenotype promoting tumor escape from the immune system. Our study focuses on analyzing the effect of HER2 on MHC class I antigen presentation and sensitivity to tumor-antigen specific cytotoxic T lymphocytes (CTLs) in HLA-A2.1(+) melanoma cell lines. We demonstrate significant inverse correlations both between the expression of HER2 and total MHC class I surface expression as well as between HER2 and HLA-A2. A significant reduction of HLA-A2 levels was found when melanoma and carcinoma cell lines were transfected with a human HER2 gene. A signaling-competent HER2 molecule was crucial for the observed HLA-A2 down-regulation, as transfectants expressing high levels of HER2 mutated in the tyrosine signaling domain did not show altered HLA-A2 expression. Importantly, the human melanoma cell line EST049 demonstrated reduced HER2 and melanoma antigen-specific recognition by CTLs upon HER2 transfection. In addition, high expression of HER2 prevented both IFN-γ mediated HLA-A2 up-regulation and improved recognition by HLA-A2-restricted CTLs in treated cells. Moreover, key APM molecules were down-regulated by HER2. These findings implicate that HER2 over-expressing tumors may be more prone to escape from HLA-A2 restricted CTLs suggesting that immunotherapy approaches inducing an integrated humoral, cellular and innate immune response would be most effective.

Increased thioredoxin-1 production in human naturally occurring regulatory T cells confers enhanced tolerance to oxidative stress.

Levels of regulatory T cells (Tregs) are increased in different cancer types as well as in inflammatory diseases, such as rheumatoid arthritis. Treg accumulation may result from aberrant proliferation and trafficking as well as greater resilience to oxidative stress compared with conventional T cells. This enhanced antioxidative capacity of Tregs possibly serves as feedback inhibition during inflammation and prevents uncontrolled immune reactions by favoring survival of suppressor rather than effector cells. In this study, we demonstrate that human Tregs express and secrete higher levels of thioredoxin-1, a major antioxidative molecule. Thioredoxin-1 has an essential role in maintaining their surface thiol density as the first line of antioxidative defense mechanisms and is sensitive to proinflammatory stimuli, mainly tumor necrosis factor-α, in a nuclear factor-κB-dependent fashion. The antiapoptotic and oncogenic potential of (secreted) Trx-1 suggests that it may exert effects in Tregs beyond redox regulation.

Regulatory T cells in cancer.

At the present time, regulatory T cells (Tregs) are an integral part of immunology but the route from discovery of "suppressive" lymphocytes in the 1980s to the current established concept of Tregs almost 20 years later has been a rollercoaster ride. Tregs are essential for maintaining self-tolerance as defects in their compartment lead to severe autoimmune diseases. This vitally important function exists alongside the detrimental effects on tumor immunosurveillance and antitumor immunity. Beginning with the identification of CD4(+)CD25(+) Tregs in 1995, the list of Treg subsets, suppressive mechanisms, and knowledge about their various origins is steadily growing. Increase in Tregs within tumors and circulation of cancer patients, observed in early studies, implied their involvement in pathogenesis and disease progression. Several mechanisms, ranging from proliferation to specific trafficking networks, have been identified to account for their systemic and/or local accumulation. Since various immunotherapeutic approaches are being utilized for cancer therapy, various strategies to overcome the antagonistic effects exerted by Tregs are being currently explored. An overview on the biology of Tregs present in cancer patients, their clinical impact, and methods for modulating them is given in this review. Despite the extensive studies on Tregs in cancer many questions still remain unanswered. Even the paradigm that Tregs generally are disadvantageous for the control of malignancies is now under scrutiny. Insight into the specific role of Tregs in different types of neoplasias is the key for targeting them in a way that is beneficial for the clinical outcome.

Intratumoral forkhead box P3-positive regulatory T cells predict poor survival in cyclooxygenase-2-positive uveal melanoma.

BACKGROUND: Forkhead box P3 (FOXP3)-positive regulatory T cells (Tregs) are key mediators of peripheral tolerance and suppress efficient antitumor responses. Prostaglandin E(2) (PGE(2)) produced by inducible cyclooxygenase-2 (COX-2) can lead to Treg induction. COX-2 expression has been linked to tumorigenesis and growth in various malignancies. The objective of the current study was to investigate whether Tregs infiltrate uveal melanomas (UMs) and whether their prevalence is linked to COX-2 expression and the prediction of overall survival (OS). METHODS: One hundred patients who underwent enucleation after they were diagnosed with UM were included in the study. Immunohistochemical staining with monoclonal anti-FOXP3, anti-CD4, and anti-COX-2 antibodies was performed, and immunoreactivity was assessed. Correlations of COX-2 expression with the presence of Tregs, established clinicopathologic parameters, and OS were evaluated in univariate and multivariate analyses. RESULTS: High expression of COX-2 was predictive of shortened OS. FOXP3-positive Tregs were detectable in 24% of UMs and were restricted to malignant tissue. The extent of COX-2 expression was associated significantly with Treg prevalence (P = .004) and Treg intratumoral localization (P = .005). Intratumoral Tregs (but not the prevalence of Tregs) were independent marker for worse OS with a hazard ratio of 5.36 in patients with COX-2-positive tumors. CONCLUSIONS: The current results demonstrated that high COX-2 expression is associated with OS and Treg prevalence in UM. These findings are in line with the observations that COX-2/PGE(2) induces Tregs and that Tregs may alter antitumor responses, resulting in a negative effect on the clinical disease course. Intratumoral Tregs are an independent prognostic marker for COX-2-positive UM, and these results put COX-2 inhibitors and Treg depletion into the spotlight of potential novel treatment modalities for patients with UM.

Expression and prognostic significance of iNOS in uveal melanoma.

Uveal melanoma (UM) is the most common primary intraocular tumor in adults. Disease metastasis occurs in half of the patients and is uniformly fatal despite systemic therapy. Inducible nitric oxide synthase (iNOS) is associated with disease progression in various malignancies including cutaneous melanoma. In this retrospective cohort, we examined the prognostic value of iNOS in UM by performing immunohistochemistry on paraffin-embedded sections of primary tumors (90 patients) and matched primary and metastatic hepatic tumors (19 patients) with complete histopathological and clinical data. We show that iNOS is expressed in UM (57% of the patients) and high iNOS levels significantly (p = 0.04; hazard ratio (HR) = 2.3) predict disease-specific survival (DSS) as assessed by Kaplan-Meier analysis and univariate Cox's proportional hazards regression model. Furthermore, high iNOS expression in the UM primary tissue was significantly associated with metastatic disease and vice versa. Expression of iNOS in hepatic metastases significantly (p = 0.02) predicted a shortened survival as assessed by Kaplan-Meier analysis. However, iNOS did not appear to be a significant (p = 0.16; HR = 1.9) factor in the multivariate Cox's regression analysis performed together with the clinical parameters tumor diameter, tumor cell type, and tumor location in which only tumor diameter predicted DSS. In conclusion, iNOS predicts DSS in UM and may play a role in disease progression but it is not an independent prognostic factor.

Oxidative stress and lymphocyte persistence: implications in immunotherapy.

CD8(+) T cells respond to antigen stimulation through a process of activation, division, and differentiation generating a large pool of activated effector cytolytic T lymphocytes (CTLs). Many cancer patients harbor the accordant precursor CTLs capable of responding to various tumor-associated antigens (TAA). In selected cases, vaccination with these TAA can elicit detectable antitumor responses. Presently, the clinical outcome of cancer vaccination remains inadequate. The lack of clinical efficacy may be attributed to various molecular and cellular mechanisms developed by tumors to successfully evade the host immune system. Some of these mechanisms have been identified. It is becoming increasingly apparent that immunotherapy with the sole objective of inducing immune activation is in itself not sufficient to fully overcome the mechanisms averting efficient antitumor responses. Strategies to neutralize tumor-induced immune suppression have to be developed in parallel to antigenic stimulation. Our data show that both oxidative stress- and antigen-mediated preferential cell death of antigen-experienced memory CTLs may be a major contributor to tumor-induced immune dysfunction. The persistence of functional CTLs is a key element for an efficient antitumor response and affects the outcome of any immunotherapy protocol. We therefore propose that protecting CTLs from premature death by identifying and targeting the responsible pathway can lead to substantial enhancement in antitumor response. In this review, we discuss some of the fundamental factors that may be involved in the modulation of the different lymphocyte subsets towards sensitization or resistance to tumor-induced stress.

Prognostic significance of tumor iNOS and COX-2 in stage III malignant cutaneous melanoma.

PURPOSE: New prognostic markers are needed for malignant melanoma. Inducible nitric oxide synthase (iNOS) and cyclooxygenase type 2 (COX-2) have been described to correlate with progression of melanoma. Moreover, activating mutations in BRAF/NRAS oncogenes are often detected in melanoma. The BRAF/NRAS mutation status and expression of COX-2 and iNOS were examined to compare their prognostic value for overall survival (OS) in stage III malignant cutaneous melanoma. EXPERIMENTAL DESIGN: The expression of iNOS and COX-2 in metastatic lymph nodes from 21 rapidly progressing (OS from date of diagnosis of stage III disease < or =14 months) and 17 slowly progressing (OS > or =60 months) stage III cutaneous melanoma patients was examined by immunohistochemistry. The presence of BRAF/NRAS mutations was analyzed using direct DNA sequencing. Chi2 exact trend test and logistic regression analysis were used for statistical analysis. RESULTS: Both iNOS (P = 0.002) and COX-2 (P = 0.048) alone significantly predicted OS. The BRAF/NRAS mutation status did not significantly differ between patient groups, although iNOS significantly (P = 0.013) correlated with BRAF mutation frequency. Furthermore, the odds ratio (OR) with respect to OS of iNOS (OR = 10.4) was higher than that of COX-2 (OR = 5.6) and was stable in the multivariate analysis of OS together with disease stage IIIB/C, ulceration, number of metastatic lymph nodes, and Breslow tumor thickness. CONCLUSION: Our data show that iNOS is an independent and stronger prognostic factor for OS in stage III malignant cutaneous melanoma than COX-2.

Naturally occurring regulatory T cells show reduced sensitivity toward oxidative stress-induced cell death.

Although the authors of several studies report elevated numbers of immunosuppressive regulatory T cells (Tregs) in hematologic and solid malignancies, the underlying mechanism is not fully clarified. Cancer is associated with oxidative stress mediated through reactive oxygen species produced by malignant cells, granulocytes, tumor-associated macrophages, and myeloid-derived suppressor cells. Oxidative stress is known to have detrimental effects on natural killer (NK) and T cells during chronic inflammatory conditions and cancer. Paradoxically, greater numbers of Tregs can be detected at tumor sites, indicating that Tregs can persist in this environment of increased oxidative stress. We demonstrate that Tregs, especially naive CD45RA(+), exhibit reduced sensitivity to oxidative stress-induced cell death and maintain their suppressive function, a phenomenon that may be attributed to their observed high antioxidative capacity. This newly described characteristic could explain their enrichment in malignancies associated with increased levels of oxidative stress.

Transduction with the antioxidant enzyme catalase protects human T cells against oxidative stress.

Patients with diseases characterized by chronic inflammation, caused by infection or cancer, have T cells and NK cells with impaired function. The underlying molecular mechanisms are diverse, but one of the major mediators in this immune suppression is oxidative stress caused by activated monocytes, granulocytes, or myeloid-derived suppressor cells. Reactive oxygen species can seriously hamper the efficacy of active immunotherapy and adoptive transfer of T and NK cells into patients. In this study, we have evaluated whether enhanced expression of the antioxidant enzyme catalase in human T cells can protect them against reactive oxygen species. Human CD4(+) and CD8(+) T cells retrovirally transduced with the catalase gene had increased intracellular expression and activity of catalase. Catalase transduction made CD4(+) T cells less sensitive to H(2)O(2)-induced loss-of-function, measured by their cytokine production and ability to expand in vitro following anti-CD3 stimulation. It also enhanced the resistance to oxidative stress-induced cell death after coculture with activated granulocytes, exposure to the oxidized lipid 4-hydroxynonenal, or H(2)O(2). Expression of catalase by CMV-specific CD8(+) T cells saved cells from cell death and improved their capacity to recognize CMV peptide-loaded target cells when exposed to H(2)O(2). These findings indicate that catalase-transduced T cells potentially are more efficacious for the immunotherapy of patients with advanced cancer or chronic viral infections.

The CD16- CD56(bright) NK cell subset is resistant to reactive oxygen species produced by activated granulocytes and has higher antioxidative capacity than the CD16+ CD56(dim) subset.

Human NK cells can be divided into CD56(dim) and CD56(bright) subsets. These two types of NK cells respond to different types of stimuli, with CD56(dim) NK cells having direct cytotoxic ability and CD56(bright) NK cells having mainly an immunoregulatory function. We show that the CD16+ CD56(dim) NK subset is characterized by sensitivity to cell death induced by activated granulocytes. We identified hydrogen peroxide (H2O2) as the major effector molecule responsible for the cytotoxic effect of granulocytes on CD56(dim) NK cells, because the ability of granulocytes to kill CD56(dim) NK cells was completely abrogated in the presence of the hydrogen peroxide scavenger catalase. When exposing NK cells to H2O2, CD56(dim) cells showed rapid mitochondrial depolarization and down-regulation of activating NKRs, eventually resulting in cell death, whereas CD56(bright) cells remained unaffected. The difference in sensitivity to H2O2 was mirrored by a difference in intracellular oxidation levels between CD56(dim) and CD56(bright) NK cells, and cell lysates from the latter subset possessed a greater ability to block H2O2-mediated oxidation. Our data may explain the preferential accumulation of CD56(bright) NK cells often seen in environments rich in reactive oxygen species, such as at sites of chronic inflammation and in tumors.

Functional gap junctions facilitate melanoma antigen transfer and cross-presentation between human dendritic cells.

Previously, we found that human dendritic cells (hDCs) pulsed with a melanoma cell lysate (MCL) and stimulated with TNF-alpha (MCL/TNF) acquire a mature phenotype in vitro and are able to trigger tumor-specific immune responses when they are used in melanoma immunotherapy in patients. In this study, we describe that MCL/TNF induces gap junction (GJ)-mediated intercellular communications and promotes melanoma Ag transfer between ex vivo produced hDCs from melanoma patients. hDCs also exhibit increased expression of the GJ-related protein connexin 43, which contributes to GJ plaque formation after MCL/TNF stimulation. The addition of GJ inhibitors suppresses intercellular tumor Ag transfer between hDCs, thus reducing melanoma-specific T cell activation. In summary, we demonstrate that MCL/TNF-stimulated hDCs can establish functional GJ channels that participate in melanoma Ag transfer, facilitating Ag cross-presentation and an effective dendritic cell-mediated melanoma-specific T cell response. These results suggest that GJs formed between hDCs used in cancer vaccination protocols could be essentials for the establishment of a more efficient antitumor response.

FOXP3+CD4+CD25+ adaptive regulatory T cells express cyclooxygenase-2 and suppress effector T cells by a prostaglandin E2-dependent mechanism.

CD4+CD25+ regulatory T (T(R)) cells suppress effector T cells by partly unknown mechanisms. In this study, we describe a population of human suppressive CD4+CD25+ adaptive T(R) (T(R)(adapt)) cells induced in vitro that express cyclooxygenase 2 (COX-2) and the transcription factor FOXP3. T(R)(adapt) cells produce PGE(2) and suppress effector T cell responses in a manner that is reversed by COX inhibitors and PGE(2) receptor-specific antagonists. In resting CD4+CD25- T cells, treatment with PGE(2) induced FOXP3 expression. Thus, autocrine and paracrine effects of PGE(2) produced by COX-2-positive T(R)(adapt) cells may be responsible for both the FOXP3+ phenotype and the mechanism used by these cells to suppress effector T cells.

The epidermal growth factor-like growth factor amphiregulin is strongly induced by the adenosine 3',5'-monophosphate pathway in various cell types.

We examined the cAMP-mediated regulation of the epidermal growth factor-like growth factor amphiregulin (AR) in T cells and observed a strong cAMP-induced up-regulation of AR mRNA in a time- and concentration-dependent manner independent of T cell activation. This regulation may be mediated in part through activation of a cAMP-responsive element in the AR promoter, because the cAMP-responsive element conferred cAMP responsiveness to a luciferase reporter in Jurkat TAg cells. Similar effects of AR mRNA induction were seen in T cells treated with cAMP-elevating agents such as prostaglandin E(2) and forskolin as well as with the phosphodiesterase inhibitors rolipram and isobutylmethylxanthine. Furthermore, the induction of AR mRNA by cAMP was strongly suppressed by a protein kinase A type I-selective inhibitor, whereas treatment with an exchange protein directly activated by cAMP-specific agonist did not increase AR levels. In addition, an increase in AR gene transcripts by cAMP was seen in MCF-7 mammary carcinoma cells and H295R adrenal cells. Moreover, the potent cAMP-mediated induction of AR mRNA resulted in increased secretion (5-fold) of AR from T cells. Furthermore, supernatants from cAMP-stimulated T cells containing secreted AR induced phosphorylated MAPK in OVCAR-3 carcinoma cells. In conclusion, our data suggest that AR is under strong regulation by the cAMP pathway in various cell types, and that prostaglandin E(2)- and cAMP-induced AR secretion from T cells may be highly relevant in a microenvironment consisting of tumor cells and infiltrated immune cells, because AR by activating the MAPK pathway through a paracrine route may contribute to proliferation of tumor cells and thus add to neoplastic processes.

Cytokine networks are pre-activated in T cells from HIV-infected patients on HAART and are under the control of cAMP.

OBJECTIVE: Cytokines seem to play a critical role in HIV infection. The cAMP/protein kinase A (PKA) type I pathway is shown to be hyper-activated and contributes to T-cell immune dysfunction in HIV infection. Here, we analysed firstly the levels of cytokine gene expression in unstimulated CD3+T cells from HIV-infected patients on HAART, and secondly the regulation of cytokine and cytokine-related genes by cAMP agonist and antagonist in anti-CD3 activated T cells in order to understand their effects on cytokine networks. METHODS: Cytokine Macro Array and real-time RT-PCR techniques were used to study cytokine gene expression in T cells of HIV-positive patients. RESULTS: Of the cytokine-related genes analysed 45% were expressed at twofold or higher levels in unstimulated T cells from HIV-infected patients as compared with healthy controls, and one-third of these genes were hypo-responsive upon activation as compared with controls. Furthermore, cAMP modulated levels of expression of a number of cytokine-related genes differently in patient and control T cells. CXCR4, CCR5 and amphiregulin were up-regulated by cAMP agonist, whereas other cytokine-related genes including macrophage inflammatory protein 1 beta, tumour necrosis factor-alpha and lymphotoxin-beta were markedly down-regulated by cAMP agonist in T cells from both HIV-infected patients and controls. Moreover, members of the chemokine/chemokine receptor family were over-represented among genes regulated by cAMP agonist/antagonist in patient T cells. CONCLUSIONS: Our data indicate that T cells from HIV-infected patients are in a pre-activated state and that a set of cytokine genes is hypo-responsive to activation and under tonic regulation by cAMP in these T cells.