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…

H2-M, a facilitator of MHC class II peptide loading, and its negative modulator H2-O are differentially expressed in response to proinflammatory cytokines

H2-M is a major histocompatibility complex (MHC) class II-like molecule that catalyzes peptide binding to MHC class II molecules. Recently, the H2-O heterodimer, encoded by H2-Oa and H2-Ob in the MHC class II region, has been shown to be physically associated with H2-M in B cells and to downregulate H2-M function. Examination of H2-O expression in freshly isolated mouse organs revealed that H2-Oa- and H2-Ob-specific transcripts are present in both lymphoid and nonlymphoid tissues. To evaluate the gene regulation and functional impact of H2-O on antigen presentation, we examined the effects on MHCII, invariant chain (Ii), H2-M, and H2-O gene expression of interleukin (IL)-4, IL-10, and inter…

Local administration of antisense phosphorothioate oligonucleotides to the c-kit ligand, stem cell factor, suppresses airway inflammation and IL-4 production in a murine model of asthma.

Abstract Background: The c-kit ligand, stem cell factor (SCF), is an important activating and chemotactic factor for both mast cells and eosinophils. These cells are known to play a fundamental role in the pathogenesis of asthma. Objective: Our goal was to analyze the functional role of SCF in the pathogenesis of asthma. Methods: The expression of SCF was targeted in fibroblasts, epithelial cells, and locally in a murine model of asthma in mice induced by ovalbumin sensitization with an antisense DNA strategy. Results: We could suppress SCF expression in NIH 3T3 fibroblasts and SP1 epithelial cells by a specific antisense phosphorothioate oligonucleotide overlapping the translation start si…

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

Interferon-regulatory factor 4 is essential for the developmental program of T helper 9 cells.

Summary Interferon-regulatory factor 4 (IRF4) is essential for the development of T helper 2 (Th2) and Th17 cells. Herein, we report that IRF4 is also crucial for the development and function of an interleukin-9 (IL-9)-producing CD4 + T cell subset designated Th9. IRF4-deficient CD4 + T cells failed to develop into IL-9-producing Th9 cells, and IRF4-specific siRNA inhibited IL-9 production in wild-type CD4 + T cells. Chromatin-immunoprecipitation (ChIP) analyses revealed direct IRF4 binding to the Il9 promoter in Th9 cells. In a Th9-dependent asthma model, neutralization of IL-9 substantially ameliorated asthma symptoms. The relevance of these findings is emphasized by the fact that the ind…