The immunosuppressive effect of vincristine on allostimulatory potential of human dendritic cells interferes with their function and survival.

Mature dendritic cells (mDC) are professional and potent antigen presenting cells required for initiation of primary immune responses. In the present study, we have investigated the effect of vincristine on the T cell allostimulatory potential of human monocyte-derived mDC in allogeneic mixed lymphocyte reaction. Using T lymphocytes as responding cells and mDC as stimulating cells, our data indicate that incubation of DC with vincristine decreased the accessory potency dose dependently and resulted in a subsequent inhibition of T cell proliferative responses. Treatment of mDC with vincristine also led to the alteration of their capacity to produce IL-12 but enhanced their production of IL-10. Using flow cytometry, we demonstrated that vincristine had no effect on mDC phenotype (CD83, CD40, CD86, CD58, CD54) but promoted apoptotic cell death of these cells as revealed by PI and annexin-V. These findings suggest that DC may be potential targets of cytotoxic drugs and point out the possible impairment of the immunocompetence of these cells following chemotherapy.

Dendritic cell KLH loading requirements for efficient CD4+ T-cell priming and help to peptide-specific cytotoxic T-cell response, in view of potential use in cancer vaccines.

This study focuses on a Keyhole Limpet Hemocyanin (KLH) adjuvant strategy to augment efficacy of dendritic cell-based vaccines that use class I-restricted peptides. Requirements for loading dendritic cells (DC) with KLH were first determined in order to optimally prime CD4(+) T cells. These KLH-loaded cells were pulsed with antigenic peptide and cultured with T cells to induce a cytotoxic T lymphocyte (CTL) response against the peptide. Such a concomitant presentation of KLH and peptide by the same DC strongly augmented the peptide-specific CTL response, as compared to the response induced by DC pulsed with the peptide alone. This adjuvant effect was more pronounced for poorly immunogenic antigens. The use of optimised peptide and KLH-loaded DC may improve the efficacy of therapeutic anti-tumour peptide vaccination.

Butyrate affects differentiation, maturation and function of human monocyte-derived dendritic cells and macrophages.

We studied the in vitro effects of butyric acid on differentiation, maturation and function of dendritic cells (DC) and macrophages (M(Phi)) generated from human monocytes. A non-toxic dose of butyrate was shown to alter the phenotypic differentiation process of DC as assessed by a persistence of CD14, and a decreased CD54, CD86 and HLA class II expression. The more immature differentiation stage of treated cells was confirmed further by their increased phagocytic capability, their altered capacity to produce IL-10 and IL-12, and their weak allostimulatory abilities. Butyrate also altered DC terminal maturation, regardless of the maturation inducer, as demonstrated by a strong down-regulation of CD83, a decreased expression of CD40, CD86 and HLA class II. Similarly, butyrate altered M(Phi) differentiation, down-regulating the expression of the restricted membrane antigens and reducing the phagocytic capacity of treated cells. To investigate further the mechanism by which butyrate hampers the monocyte dual differentiation pathway, we studied the effects of 1,25(OH)2D3 alone or in combination with butyrate on the phenotypic features of DC. Unlike 1,25(OH)2D3, butyrate inhibited DC -differentiation without redirecting it towards M(Phi). Combined treatment gave rise to a new cell subset (CD14(high), CD86 and HLA-DR(low)) phenotypically distinct from monocytes. These results reveal an alternative mechanism of inhibition of DC and M(Phi) differentiation. Altogether, our data demonstrate a novel immune suppression property of butyrate that may modulate both inflammatory and immune responses and support further the interest for butyrate and its derivatives as new immunotherapeutic agents.

Human monocyte-derived macrophages and dendritic cells as targets for biomaterial cytocompatibility studies using an improved in vitro culture system.

Since macrophage plays a key role in the biocompatibility process, neoplastic macrophage cell lines and human blood monocytes are commonly used as target cells for in vitro biomaterial tolerance evaluation. However, tumor cells profoundly differ from normal tissue cells and monocytes are only precursors of macrophages. It has become possible to generate recently, under adherent-free conditions, fully mature macrophages and dendritic cells from human blood monocytes in the presence of GM-CSF and GM-CSF + IL4 respectively. In the present work, we examined the effects of titanium-alloy on morphology, adhesion, cell phenotype and TNF-alpha release activity of such differentiated cells grown in hydrophobic teflon bags. Scanning electron microscopy showed that macrophages substantially adhered and spread on titanium-alloy surface throughout the culture period, whereas only a few dendritic cells were adherent. The phenotype of both cell types remained unchanged in the presence of the tested material. However, titanium-alloy stimulated the secretion of TNF-alpha by the macrophages of some donors. This model of culture may offer new insights into the biomaterial evaluation and may be useful for studying individual responses induced by biomaterials.

Adherent-free generation of functional dendritic cells from purified blood monocytes in view of potential clinical use.

There is increasing interest in dendritic cells (DC) that are capable of initiating antitumor immune responses. An in vitro cell differentiation method has recently been developed that uses GM-CSF and IL-4 to generate human DC from adherent blood mononuclear cells cultured on tissue culture plastic. These cells are competent for antigen uptake but express relatively low levels of co-stimulatory molecules and thus correspond to immature resident tissue DC. We have adapted this method to consider some variables that are pertinent to clinical use, including a large scale differentiation of functional DC in a culture system suitable for clinical use. We report here that sizable numbers of monocytes purified by elutriation from blood leukocytes and cultured in Teflon bags develop with high efficiency into typical DC, as defined by morphology and membrane phenotype. When compared with usual adherent DC, cells generated under our adherent-free conditions exhibited lower CD1a expression and antigen capture capacity, but maintained the ability to present soluble antigens to T cells. They neoexpressed a high level of the co-stimulator molecule B7-2 (CD86) and was potent accessory cells for T cell proliferation, but they lacked the CD83 marker of DC full maturation. This study may constitute a prerequisite step for clinical investigations in tumor immunotherapy.