Specific stabilization of the 4F7 molecule on dendritic cells by contact allergens.

Our laboratory has recently developed the monoclonal antibody 4F7 which recognizes a molecule on dendritic cells in the dermis of mice that is upregulated after application of contact allergens in vivo. Furthermore, this antibody detects an antigen on dendritic cells in spleen, lymph nodes and colon. In order to study the influence of contact allergens on the surface expression of the 4F7 molecules on dendritic cells, FACScan analysis of splenic dendritic cells was carried out after in vitro application of contact allergens. Freshly isolated splenic dendritic cells were found to be positive for 4F7, 33D1, N418 (CD11c) and MHC class II. After overnight culture the expression of the dendritic…

Therapeutic application of T cell receptor mimic peptides or cDNA in the treatment of T cell-mediated skin diseases

An 8-amino acid peptide encoding a sequence of the transmembrane region of the T cell receptor alpha chain (TCR-alpha) was shown to inhibit T cell function by preventing functional assembly of the T cell receptor (mimic peptide). To avoid systemic immunosuppression by peptide application in vivo, we used a topical application of the peptide. In the system of murine contact sensitivity, topical application of the peptide inhibited the elicitation of contact sensitivity following application of a contact allergen in sensitized animals. Alternatively, when naked DNA encoding the peptide sequence was injected into skin before application of a contact allergen to sensitized animals, local immuno…

Interleukin-10-treated dendritic cells modulate immune responses of naive and sensitized T cells in vivo.

Interleukin-10 is a pleiotropic cytokine known to have inhibitory effects on the accessory functions of dendritic cells. In vitro, interleukin-10 converts immature dendritic cells into tolerizing antigen- presenting cells. To assess whether interleukin-10-treated dendritic cells exert tolerizing effects in vivo, CD4+ T cells from DO11.10 ovalbumin-T cell receptor transgenic mice were transferred to syngeneic BALB/c recipients. Recipient animals were treated with ovalbumin-pulsed/unpulsed, interleukin-10-treated/untreated CD11c+ dendritic cells thereafter and ovalbumin-specific proliferation of lymph node cells was assessed by restimulation with the peptide in vitro. In prophylactic experime…

Fetal Calf Serum-Free Generation of Functionally Active Murine Dendritic Cells Suitable for In Vivo Therapeutic Approaches

Standard protocols to generate mouse dendritic cells (DC) generally use culture medium supplemented with fetal calf serum; however, reinjection in vivo of DC cultured in fetal calf serum results in priming to xenogeneic proteins that clearly limits the use of such DC. We therefore established a fetal calf serum-free culture system for the generation of murine DC from bone marrow precursors. DC can be generated fetal calf serum-free using RPMI supplemented with 1.5% syngeneic mouse serum. Although the yield of DC grown under fetal calf serum-free conditions was somewhat lower than that of the standard culture, large numbers of DC could be generated without the exposure to xenogeneic proteins…

Human Keratinocyte-Derived IL-12 Affects LC-Induced Allogeneic T-Cell Responses

Our laboratory has recently defined the cytokines involved in the early induction phase of contact sensitivity that help LC to become more potent APC and that help to direct this classical Thl immune response1,2.

Is cytokine expression responsible for differences between allergens and irritants?

Abstract Irritant and allergic contact dermatitis are two very similar diseases, and differentiating between these two can be difficult clinically. Recently, cytokines have been identified as useful tools for differentiation. Thus, our laboratory has identified an early cytokine pattern in the induction phase of contact sensitivity that is specific for allergens and is not found after epicutaneous application of irritants or tolerogens. The upregulation of the Langerhans' cell—derived signal interleukin (IL)-1β early after allergen application especially seems to be highly specific for contact allergens. This cytokine was also found to be essential for the development of epicutaneous sensit…

FCS-free generation of functionally active murine dendritic cells suitable for in vivo therapeutic approaches

Production of functional IL-18 by different subtypes of murine and human dendritic cells (DC): DC-derived IL-18 enhances IL-12-dependent Th1 development

IL-18 is a recently described cytokine that shares biological activities with IL-12 in driving the development of Th1-type T cells. As dendritic cells (DC) are very potent inducers of T cell proliferation and differentiation we wondered whether they utilize IL-18 as a factor driving Th1 development. We demonstrate by Northern blot and reverse transcription-PCR that various subtypes of human and murine DC as well as the DC-line XS contain IL-18 mRNA. When supernatants of either enriched Langerhans cells (LC) or bone marrow-derived DC were analyzed for production of IL-18 protein, IL-18 production was detected in an IL-18-specific ELISA. To assess whether the IL-18 protein released by DC is f…

Pro-inflammatory cytokines and prostaglandins induce maturation of potent immunostimulatory dendritic cells under fetal calf serum-free conditions.

Culture conditions for human dendritic cells (DC) have been developed by several laboratories. Most of these culture methods, however, have used conditions involving fetal calf serum (FCS) to generate DC in the presence of granulocyte-macrophage colony-stimulating factor and interleukin (IL)-4. Recently, alternative culture conditions have been described using an additional stimulation with monocyte-conditioned medium (MCM) and FCS-free media to generate DC. As MCM is a rather undefined cocktail, the yield and quality of DC generated by these cultures varies substantially. We report that a defined cocktail of tumor necrosis factor (TNF)-alpha, IL-1beta and IL-6 equals MCM in its potency to …

Interleukin-10–Treated Human Dendritic Cells Induce a Melanoma-Antigen–Specific Anergy in CD8+ T Cells Resulting in a Failure to Lyse Tumor Cells

Abstract Dendritic cells (DC) are critically involved in the initiation of primary immune processes, including tumor rejection. In our study, we investigated the effect of interleukin-10 (IL-10)–treated human DC on the properties of CD8+ T cells that are known to be essential for the destruction of tumor cells. We show that IL-10–pretreatment of DC not only reduces their allostimulatory capacity, but also induces a state of alloantigen-specific anergy in both primed and naive (CD45RA+) CD8+ T cells. To investigate the influence of IL-10–treated DC on melanoma-associated antigen-specific T cells, we generated a tyrosinase-specific CD8+ T-cell line by several rounds of stimulation with the sp…