Human dendritic cells sequentially matured with CD4(+) T cells as a secondary signal favor CTL and long-term T memory cell responses.

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 first signals, we used TNF-α/polyI:C mimicking inflammatory 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.

Efficient monocyte-derived dendritic cell generation in patients with acute myeloid leukemia after chemotherapy treatment: application to active immunotherapy.

OBJECTIVE: While complete remission in acute myeloid leukemia (AML) can be achieved after chemotherapy (CT), relapses occur for the majority of patients, underlying the need to eliminate residual disease. Based on dendritic cell (DC) vaccination, the triggering of an immune response against residual leukemia cells after CT could maintain patients in remission. The aim of our study was to assess, for vaccine preparation, generation of monocyte-derived DCs in AML patients after CT. MATERIALS AND METHODS: We evaluated efficiency of the production, yields, maturation, and functional properties of DCs from 22 AML patients at different CT stages compared to those from 15 healthy donors. RESULTS: We demonstrated that monocyte-derived DC production is successful later than 3 weeks after the last CT cycle, whatever the CT was. Immature DCs demonstrated functional phagocytic activity. Mature DCs displayed migratory, T-cell stimulatory and Th1-activation capacities. Our results also suggest a favorable period from 20 to 60 days after CT for potent monocyte-derived DC production and immune activation. CONCLUSION: In defining patient-sampling conditions, this preclinical study has direct implications for AML DC-based immunotherapy.

Profiling dendritic cell maturation with dedicated microarrays.

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.

Mechanisms of immunomodulation in human glioblastoma.

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.

Human dendritic cells sequentially matured with CD4+ T cells as a secondary signal favor CTL and long-term T memory cell responses

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 first signals, we used TNF-α/polyI:C mimicking inflammatory 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.

IDO expands human CD4+CD25high regulatory T cells by promoting maturation of LPS-treated dendritic cells.

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-alpha + 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 LPS-induced maturation. In line with these results, the tryptophan-derived catabolites 3-hydroxyanthranilic acid and 3-hydroxykynurenine increased maturation of LPS-treated DC. Concerning the molecular mechanisms of this effect, IDO acts as an intermediate pathway in LPS-induced production of reactive oxygen species and NF-kappaB 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.

Stress response in Cu2+ and Cd2+ exposed oysters (Crassostrea gigas): an immunohistochemical approach.

Localization of heat shock proteins (HSPs) and metallothioneins (MTs) was investigated in a marine bivalve (Crassostrea gigas) by immunohistochemical methods. Differential protein expression was demonstrated in digestive gland, gonad and gills, using a polyclonal antibody against C. gigas proteins. Application of this technique showed the cellular and tissue immunolabelling specificity of the two proteins. HSPs and MTs were localized in the epithelium of the digestive gland and gills in contact with the palleal compartment. For the first time, localization of MTs was observed in mature gametes of bivalve molluscs. Our results establish a basis for the use of immmunodetection techniques to study the tissue-specific localization of stress proteins in marine bivalves exposed to metal stress.

Heme oxygenase-1 expression inhibits dendritic cell maturation and proinflammatory function but conserves IL-10 expression.

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 human tissues, iDCs also express HO-1, whereas mature DCs do not. Induction of HO-1 expression with cobalt protoporphyrin (CoPP) in human and rat DCs inhibits lipopolysaccharide (LPS)-induced phenotypic maturation and secretion of proinflammatory cytokines, resulting in the inhibition of alloreactive T-cell proliferation. CoPP-treated DCs, however, retain the ability to produce the anti-inflammatory cytokine interleukin 10 (IL-10). Reactive oxygen species induced by LPS in DCs were inhibited by induction of HO-1. In conclusion, we identify, for the first time, the capacity of HO-1 to block maturation of DCs and to inhibit proinflammatory and allogeneic immune responses while preserving IL-10 production. This novel immune function for HO-1 may be of interest for the inhibition of immune responses in autoimmune diseases, transplantation, and other conditions involving activation of the immune system.