The mice were killed 1, 2, 4 or 6 weeks after injection and splenic CD4+ T cells were prepared and stimulated with 100 g of OVA for 4 days

The mice were killed 1, 2, 4 or 6 weeks after injection and splenic CD4+ T cells were prepared and stimulated with 100 g of OVA for 4 days. to injection with pAnti-CD3sFv DNA. In addition, pAnti-CD3sFv/IL-18 was more efficient than a mixture of pAnti-CD3sFv + pIL-18 in inducing OVA-specific, Th1 immune responses and also in inhibiting OVA-specific, IL-4 production. These studies indicate that vaccination with pAnti-CD3sFv/IL-18 fusion DNA efficiently induces the Th1 immune response in antigen-sensitized mice. Introduction The T-helper lymphocyte is responsible for orchestrating the appropriate immune response to a wide variety of pathogens. The recognition of the polarized T-helper (Th) cell subsets Th1 and Th2 has led to an understanding of the role of these cells in co-ordinating a variety of immune responses.1,2 Th1 cells selectively secrete interferon- (IFN-), interleukin (IL)-2 and tumour necrosis factor- (TNF-), and regulate cell-mediated immunity characterized by the production of complement-fixing and opsonizing antibodies such as immunoglobulin G2a (IgG2a). Th2 Bambuterol cells produce IL-4, IL-5, IL-6 and IL-10, which are involved in the development of humoral immune responses, including expression of immunoglobulin G1 (IgG1) and immunoglobulin E (IgE) isotypes. These two Th subsets regulate each others’ function through the antagonistic activity of their respective cytokines. Increasing evidence indicates that the outcome of many diseases is determined by the balance between Th1- and Th2-mediated immune responses.3,4 Polarized Th1-and Th2-type responses play different roles in protection, Th1 being effective in the defence against intracellular pathogens and Th2 against intestinal nematodes. Moreover, Th1 and Th2 subsets are responsible for different types of Bambuterol immunopathological reactions. Th1 responses predominate in organ-specific autoimmune disorders, acute allograft rejection, recurrent abortions and in some chronic inflammatory disorders. In contrast, Th2 responses predominate in Omenn’s syndrome, transplantation tolerance, chronic graft-versus-host disease and allergic diseases. A number of factors determine which Th subsets predominate in an immune response.5 These include the physical form of the antigen, as well as the density and affinity of the peptide ligand, costimulatory signals provided by antigen-presenting cells (APCs), hormones released into the microenvironment and genetic background of the T cells.6,7 Most importantly, the cytokine microenvironment in which the initial antigen priming occurs is responsible for influencing the induction of either Th1- or Th2-mediated immune responses. IFN- and IL-12 induce Th1-mediated responses, while IL-4 drives the response into Th2-mediated immune responses. IL-18 is an 18 300 molecular-weight cytokine that was first identified as an IFN–inducing factor (IGIF) by its ability to induce IFN- production in mice with endotoxin shock.8 IL-18 is initially produced Bambuterol in a biologically inactive precursor form and, after cleavage with IL-1-converting enzyme (ICE; caspase-1), a bioactive, mature IL-18 is secreted.9 In collaboration with IL-12, IL-18 is known to stimulate Bambuterol Th1-mediated immune responses, which play a critical role in host defences against several infectious micro-organisms (including intracellular bacteria, fungi and protozoa) through the induction of IFN-, and also in tumour immunotherapy by its potent capacity to augment the cytotoxic activity of natural killer (NK) and T cells and models.18 In this report, our results show that pAnti-CD3sFv/IL-18 DNA increases the production of both OVA-specific IFN- and anti-OVA IgG2a in OVA-sensitized mice, thereby leading to Bambuterol the efficient induction of Th1 immune responses in an antigen-specific manner. Materials and methods Materials, cells and mice Murine VHL recombinant IFN- and recombinant IL-4 were obtained from Genzyme Co. (Cambridge, MA). Ovalbumin (OVA) was obtained from ICN Biomedicals (Irvine, CA). Anti-OVA IgG1 and IgG2a monoclonal antibodies (mAbs) were used as standards for each isotype-specific enzyme-linked immunosorbent assay (ELISA). Rabbit anti-OVA serum was purchased from Cappel Co. (Durham, NC). Polyclonal mouse anti-OVA were prepared from the sera of immunized mice after repeated injections of OVA in complete Freunds adjuvant, followed by OVA in incomplete Freunds adjuvant. Horseradish peroxidase (HRP)-labelled goat anti-mouse IgG1 or IgG2a were purchased from Southern Biotechnology Associates Inc. (Birmingham, AL). HeLa cells and spleen cells were grown in Dulbecco’s modified Eagle’s minimal essential medium (DMEM) containing 10% fetal bovine serum and antibiotics. HeLa cells were purchased from the American Type Culture Collection (ATCC, Rockville, MD). Female, 6- to 8-week-old BALB/c (H-2d) mice were purchased from Daehan Animal Co., Ltd (Seoul, Korea). The mice were maintained and treated according to the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Construction of an expression plasmid carrying an anti-CD3sFv/IL-18 gene A mammalian expression plasmid (donated from Dr M. E. Reff),19 containing an SV40 origin of replication and designed for expression of immunoglobulin genes, was modified to eliminate most of the immunoglobulin-coding regions as well as the neomycin-resistance gene. The plasmid pAnti-CD3sFv/IL-18 was constructed by first inserting the anti-CD3sFv cDNA in-frame with the human immunoglobulin leader sequence, to permit secretion of the translated protein. Then, the anti-CD3sFv cDNA was cloned by polymerase chain.