Heme oxygenase-1 (HO-1) is an intracellular enzyme that degrades heme and inhibits immune responses and inflammation in vivo. In most cell types, HO-1 is inducible by inflammatory stimuli and oxidative stress. Here we demonstrate that human monocyte-derived immature dendritic cells (iDCs) and several but not all freshly isolated rat splenic DC subsets and rat bone marrow-derived iDCs, spontaneously express HO-1. HO-1 expression drastically decreases during human and rat DC maturation induced in vitro. In IntroductionHeme oxygenases (HOs) are the rate-limiting intracellular enzymes that degrade heme to biliverdin, free divalent iron, and CO (for a review, see Otterbein and Choi 1 ). Three distinct HO enzymes have been identified: HO-1, HO-2, and HO-3. 1 HO-1 is a stress responsive gene whose expression is induced by a variety of stimuli including heme, heavy metals, inflammatory cytokines, and nitric oxide. 1 HO-1 is known for its cytoprotective effect against oxidative injuries and inflammation. 1 Induction of HO-1 expression by pharmacologic activators or gene transfer has had therapeutic effects in a variety of conditions or disorders involving the immune system, including transplantation and inflammatory disorders. [2][3][4][5][6][7][8] Biliverdin and its metabolite, bilirubin, are known for their antioxidant 9 and immunosuppressive effects. 10 HO-1 and CO have been shown to inhibit lipopolysaccharide (LPS)-induced expression of proinflammatory cytokines and to increase LPS-induced expression of interleukin 10 (IL-10) in macrophages. 11,12 Moreover, IL-10 induces HO-1 expression in macrophages. [13][14][15] We previously reported that overexpression of HO-1, obtained with an HO-1-encoding adenovirus in rats having heart transplants, results in long-term allograft survival associated with an inhibition of cellular allogeneic immune responses, which could be mediated by adenoviral transduction of dendritic cells (DCs). 6 DCs play a central role in the induction of immunity and tolerance (for a review, see Steinman et al 16 ). In the absence of inflammation, immature DCs (iDCs) located in peripheral tissues specialize in taking up innocuous and cell-associated self antigens.They continuously capture antigens and migrate to draining lymph nodes where they can induce tolerance. 16 In the presence of danger signals, DCs undergo maturation, a process involving upregulation of surface major histocompatibility complex (MHC) class II and costimulatory molecules, secretion of proinflammatory and anti-inflammatory cytokines, and the acquired ability to stimulate differentiation of naive T cells into effector cells.Our working hypothesis was that DCs can express HO-1, which can regulate DC functions. In this study, we demonstrate that human and rat iDCs express HO-1 and that HO-1 expression is down-regulated by maturation stimuli. Our results also demonstrate that induction of HO-1 expression renders DCs refractory to LPS-induced maturation, but preserves IL-10 secretion, suggesting that HO-1 may be used to regulate DC f...

We have previously shown that human monocyte-derived dendritic cells (DC) express indoleamine 2,3-dioxygenase (IDO), as well as several other enzymes of the kynurenine pathway at the mRNA level upon maturation. The tolerogenic mechanisms of this pathway remain unclear. Here we show that LPS-treated DC metabolize tryptophan as far as quinolinate. We found that IDO contributes to LPS and TNF-a + poly(I:C)-induced DC maturation since IDO inhibition using two different inhibitors impairs DC maturation. IDO knock-down using short-hairpin RNA also led to diminished LPSinduced maturation. In line with these results, the tryptophan-derived catabolites 3-hydroxyanthranilic acid and 3-hydroxykynurenine increased maturation of LPStreated DC. Concerning the molecular mechanisms of this effect, IDO acts as an intermediate pathway in LPS-induced production of reactive oxygen species and NF-jB activation, two processes that lead to DC maturation. Finally, we show that mature DC expand CD4 + CD25 high regulatory T cells in an IDO-dependent manner. In conclusion, we show that IDO constitutes an intermediate pathway in DC maturation leading to expansion of CD4 + CD25 high regulatory T cells.

Dendritic cell (DC) maturation is the process by which immature DC in the periphery differentiate into fully competent antigen-presenting cells that initiate the T cell response. However, DC respond to many distinct maturation stimuli, and different types of mature DC induce qualitatively different T cell responses. As DC maturation involves the coordinated regulation of hundreds of genes, comprehensive assessment of DC maturation status would ideally involve monitoring the expression of all of these transcripts. However, whole-genome microarrays are not well-suited for routine phenotyping of DC, as the vast majority of genes represented on such chips are not relevant to DC biology, and their cost limits their use for most laboratories. We therefore developed a DC-dedicated microarray, or "DC Chip", incorporating probes for 121 genes up-regulated during DC maturation, 93 genes down-regulated during maturation, 14 DC-specific genes, and 90 other genes with known or probable immune functions. These microarrays were used to study the kinetics of DC maturation and the differences in maturation profiles among five healthy donors after stimulation with tumor necrosis factor-alpha + polyI:C. Results obtained with the DC Chip were consistent with flow cytometry, enzyme-linked immunosorbent assay, and real-time polymerase chain reaction, as well as previously published data. Furthermore, the coordinated regulation of a cluster of genes (indoleamine dioxygenase, kynureninase, kynurenine monoxygenase, tryptophanyl tRNA synthetase, and 3-hydroxyanthranilate 3,4-dioxygenase) involved in tryptophan metabolism was observed. These data demonstrate the use of the DC Chip for monitoring the molecular processes involved in the orientation of the immune response by DC.

Glioblastoma multiforme (GBM), WHO grade IV astrocytoma, is the most dramatic primary brain cancer with a very poor prognosis due to inevitable disease recurrence. Less than 10% of GBM patients are still alive 5 years after diagnosis despite a multimodal treatment with surgical resection of the tumor, radiation therapy and chemotherapy. Cellular immunotherapy in gliomas, one of the promising new therapies, has shown convincing results in some patients with induction of antitumor immune responses and prolonged survival. In particular, several patients treated with dendritic cell vaccinations have demonstrated systemic antigen-specific cytotoxicity and intratumor infiltration of cytotoxic T cells. However, this is not always correlated with clinical improvement because GBM cells have multiple mechanisms that lead to suppression of the patient's antitumor immune responses. This article will focus on some aspects of the systemic immunosuppression observed in GBM patients as well as the multiple mechanisms of local immunoresistance developed by GBM.

Dendritic cells (DC) are powerful antigen-presenting cells that have drawn many attentions due to the recent development of anti-cancer vaccines. Clinical grade production of monocyte-derived DC (Mo-DC) is extensively studied, and many efforts are made to develop and improve clinical standard operating procedures. Most of the parameters involved, such as the cytokines and maturation agents, have been widely assessed. However, very few are investigated about how culture medium and additional protein components affect DC yield, viability and maturation. Thus, our study aimed to compare the impact of standard culture medium on Mo-DC differentiation and maturation. Commercially available media for hematopoietic cell culture as well as different protein supplementations, that is foetal calf serum (FCS), autologous plasma (AP), human serum (HS) and human serum albumin (HSA) were tested. Culture yields, cell viability and DC maturation were investigated. Differentiation yields were similar between the conditions used. However, we evidenced significant differences in terms of cytotoxicity and DC maturation (phenotypic and functional). This underscores the importance of defining culture medium composition in clinical standard operating procedures to insure quality control, and also when preparing DC for experimental uses.

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Dendritic cells (DCs) are professional antigen-presenting cells involved in the control and initiation of immune responses. In vivo, DCs exposed at the periphery to maturation stimuli migrate to lymph nodes, where they receive secondary signals from CD4+ T helper cells. These DCs become able to initiate CD8+ cytotoxic T lymphocyte (CTL) responses. However, in vitro investigations concerning human monocyte-derived DCs have never focused on their functional properties after such sequential maturation. Here, we studied human DC phenotypes and functions according to this sequential exposure to maturation stimuli. As fi rst signals, we used TNF-α/polyI:C mimicking infl ammatory and pathogen stimuli and, as second signals, we compared activated CD4+ T helper cells to a combination of CD40-L/ IFN-γ. Our results show that a sequential activation with activated CD4+ T cells dramatically increased the maturation of DCs in terms of their phenotype and cytokine secretion compared to DCs activated with maturation stimuli delivered simultaneously. Furthermore, this sequential maturation led to the induction of CTL with a long-term effector and central memory phenotypes. Thus, sequential delivery of maturation stimuli, which includes CD4 + T cells, should be considered in the future to improve the induction of long-term CTL memory in DC-based immunotherapy.