Specific IFN-γ Production Research Articles

Interferon-gamma response of PBMC indicates productive pseudorabies virus (PRV) infection in swine

In Chinese Meishan/German Landrace cross-bred swine F2 generation interferon gamma (IFN-γ) production by peripheral blood mononuclear cells (PBMC) was determined directly ex vivo at different time points after survival of a virulent pseudorabies virus (PRV) infection. This reactivity was compared with the reactivity of naïve PBMC. Significant IFN-γ production was determined in ELISA and ELISPOT only after in vitro PBMC re-stimulation with PRV and not with the closely related bovine herpesvirus BHV-1.The PRV-specific IFN-γ secretion from re-stimulated PBMC showed high levels 6 days after infection, before the presence of serum antibodies, and it persisted at a high level over a 3 months period. The response of a group of eight piglets infected intranasally with PRV varied. Only two animals showed the expected typical fever response. PRV specific IFN-γ production by PBMC clearly indicated that infection had occurred. Early significant IFN-γ production by primed PBMC turned out to be a reliable and specific ex vivo marker for cellular response against productive PRV infection in swine before antibody formation.

Pertussis Toxin Enhances Th1 Responses by Stimulation of Dendritic Cells

Pertussis toxin (PTX) has been widely used as an adjuvant to induce Th1-mediated organ-specific autoimmune diseases in animal models. However, the cellular and molecular mechanisms remain to be defined. In this study, we showed that dendritic cells (DC) stimulated with PTX (PTX-DC) were able to substitute for PTX to promote experimental autoimmune uveitis (EAU). EAU induced by PTX-DC revealed a typical Th1 response, characterized by high uveitogenic retinal Ag interphotoreceptor retinoid-binding protein (IRBP)-specific IFN-gamma and IL-12 production in the draining lymph nodes, as well as increased levels of anti-IRBP IgG2a and decreased levels of anti-IRBP IgG1 in the serum of IRBP-immunized mice. Furthermore, PTX-DC preferentially induced T cells to produce the Th1 cytokine, IFN-gamma. After being stimulated with PTX, DC exhibited up-regulation of MHC class II, CD80, CD86, CD40, and DEC205. PTX-DC had also increased allostimulatory capacity and IL-12 and TNF-alpha production. Serum IL-12 was increased in naive mice that received PTX-DC i.p. In addition, PTX activated extracellular signal-regulated kinase in DC. Following the inhibition of extracellular signal-regulated kinase signaling, the maturation of PTX-DC was reduced. Subsequently, the ability of PTX-DC to promote IFN-gamma production by T cells in vitro and to induce EAU in vivo was blocked. The results suggest that PTX might exert an adjuvant effect on DC to promote their maturation and the production of proinflammatory cytokines, thereby eliciting a Th1 response.

Oxidised mannan-listeriolysin O conjugates induce Th1/Th2 cytokine responses after intranasal immunisation

Clearance of infectious organisms does not always require polarised Th1 or Th2 responses and it may be advantageous for both Th1 and Th2 responses to be elicited for effective protection against an invading pathogen. It was the aim of this study to investigate oxidised mannan as a possible Th1/Th2 adjuvant. Oxidised mannan was conjugated to two candidate antigens and delivered intranasally to mice. Immunisation with the oxidised conjugate resulted in significant antigen specific proliferative responses, IL-2, IFN-γ and IL-4 production when compared to unconjugated controls.

Immunogenicity of the Brucella melitensis recombinant ribosome recycling factor-homologous protein and its cDNA

A study was conducted to evaluate the immunogenicity of the Brucella melitensis ribosome recycling factor (RRF)-homologous protein (CP24). The CP24 gene was cloned, expressed in Escherichia coli and purified. The resulting purified recombinant protein (rCP24) produced delayed-type hypersensitivity (DTH) reactions in B. melitensis-infected mice but not in naive controls. Thus, we decided to characterise the immune responses generated with DNA vaccination (pcDNACP24) or immunisation with the rCP24 in adjuvant. Animals injected with pcDNACP24 exhibited a dominance of IgG2a to IgG1 while mice injected with rCP24 developed a higher response of IgG1 than IgG2a. Both immunisation protocols were capable of eliciting CP24-specific gamma interferon (IFN-γ) producing cells. Spleen cells from pcDNACP24-immunised mice did not produce interleukin (IL)-4, IL-10 or up-regulation of IL-2 mRNA. Cells from rCP24-immunised mice produced IL-10, up-regulated IL-2 mRNA but did not produce IL-4. Neither immunisation with purified CP24 nor injection of pcDNACP24 protected mice against challenge with live smooth B. melitensis. However, the potential of CP24 for a Brucella diagnostic test based on an in vitro antigen (Ag)-specific IFN-γ production or DTH test would be worth testing.

Targeting of tumor cells for human gammadelta T cells by nonpeptide antigens.

Human Vgamma2/Vdelta2(+) gammadelta T cells respond to low molecular-mass nonpeptide Ags in a gammadelta TCR-dependent manner. Although requirements of Ag presentation have remained controversial, we have indicated that specific responses of the primary gammadelta T cells to pamidronate were dependent on monocytic adherent cells for Ag presentation. Here, we show that human tumor cells can efficiently present aminobisphosphonate and pyrophosphomonoester compounds to gammadelta T cells, inducing specific proliferation and IFN-gamma production. gammadelta TCR dependency of the response to Ag-pulsed tumor cells was confirmed by using a Jurkat line transfected with a Vgamma2/Vdelta2 gammadelta TCR. Furthermore, gammadelta T cells exhibited markedly enhanced cytotoxicity against the Ag-pulsed tumor cells as compared with untreated tumor cells. Survey of a number of human tumor cell lines of different origins revealed that the majority of them became susceptible for gammadelta T cell-mediated cytotoxicity following the Ag pulsing except for breast cancer lines so far examined, while normal PHA blast cells remained resistant. The results not only imply a unique mode of nonpeptide Ag recognition by human gammadelta T cells but also may provide a novel strategic clue for immunotherapy of human malignancy.

Analysis of the antigen-specific IFN-γ producing T-cell subsets in cattle experimentally infected with Mycobacterium bovis

Three 10 months old cattle were infected by the intratracheal route with 10 6 cfu of a field strain of Mycobacterium bovis. Blood samples were regularly collected for in vitro IFN-γ production after antigenic stimulation. Peripheral blood cells of infected animals produced IFN-γ in response to crude M. bovis antigens (live and heat-inactivated BCG and protein-purified derivative (PPD)) 3–4 weeks after infection. The ratio of the response to bovine PPD versus avian PPD indicated a specific sensitisation for M. bovis antigens. Three months post-infection (PI), animals were culled and M. bovis was cultured from tubercle lesions. At different time points, the frequency of specific M. bovis IFN-γ producing CD4+, CD8+ and WC1+ T-cells in the peripheral blood was examined by flow cytometry. Two colour immunofluorescence staining of intracellular IFN-γ and bovine cell surface molecules showed that both CD4+ and CD8+, but not WC1+, T-cells produced IFN-γ following stimulation with PPD, live or killed BCG. In two animals analysed, the relative percentage of circulating IFN-γ producing CD8+ cells decreased between week 5 and week 9 PI. The same evolution was not observed for IFN-γ secreting CD4+ cells. Magnetic positive selection of T-cells from infected animals showed that CD4+ T-cells produced specific IFN-γ only in the presence of antigen presenting cells (APCs). Positively selected CD8+ T-cells secreted IFN-γ only in the presence of recombinant human IL-2 and APCs. In vitro depletion of the CD4+ T-cells, but not the depletion of CD8+ or WC1+ T-cells, resulted in abrogation of the specific IFN-γ production showing the key role of this cell population for the specific IFN-γ production.

Immunization with plasmid DNA encoding MHC class II binding peptide/CLIP-replaced invariant chain (Ii) induces specific helper T cells in vivo: the assessment of Ii p31 and p41 isoforms as vehicles for immunization

A single helper T cell (Th) epitope-specific T cell subset was successfully induced in vivo by immunization with plasmid DNA encoding MHC class II binding peptide/class II-associated invariant chain peptide (CLIP)-replaced murine Ii molecules. Spleen cells from mice immunized by gene gun bombardment with plasmid DNA for Ii p31 and p41 molecules, whose CLIP regions were replaced with an I-A d-restricted Th epitope, ovalbumin (OVA) 323–336, showed the specific proliferation and interferon-γ (IFN-γ) production. A20-2J B cell lines having these plasmids were capable of stimulating spleen cells from the immunized mice and naı̈ve DO10-transgenic mice bearing the epitope-specific T cell receptor (TCR) transgenes by examining the specific proliferative response and IFN-γ production. Some mice immunized with the Ii p41-OVA323, but not with the Ii p31-OVA323 plasmid, produced the peptide-specific antibodies, suggesting the functional difference between Ii isoforms.

Dendritic cell vaccination protects mice against lethality caused by genital herpes simplex virus type 2 infection

We have evaluated the ability of antigen pulsed bone-marrow derived dendritic cells (bmDC), to induce protective immunity against a genital tract infection with herpes simplex virus type 2 (HSV-2) in mice. Intravenous but not vaginal administrations of bmDC pulsed in vitro with UV-inactivated HSV-2, or with purified HSV-2 envelope glycoproteins gave rise to complete protection against disease, as well as death caused by genital herpes infection. Protection was dependent on the antigens being presented by the bmDC as neither the antigens alone, nor the mock-pulsed bmDC prevented disease. Immunity was associated with HSV-2 specific IFN-γ and antibody production, and was shown to be dependent on CD4 + cells secreting IFN-γ. Thus, ex vivo antigen-pulsed bmDC represents a powerful tool for the study of protective immunity to genital herpes infection, and for the identification of protective antigens. These findings might also have an impact on the design of vaccines against other sexually transmitted viral diseases.

Advantage of gene gun-mediated over intramuscular inoculation of plasmid DNA vaccine in reproducible induction of specific immune responses

Utilizing a plasmid DNA encoding a single cytotoxic T lymphocyte (CTL) epitope and that encoding ovalbumin (OVA), we compared the reproducibility in the induction of immune responses by gene gun and intramuscular immunization. As compared to intramuscular inoculation, gene gun DNA immunization appeared to bring about highly reproducible and reliable results in the induction of specific CTL and IFN-γ production to the CTL epitope and production of anti-OVA IgG. The results obtained by intramuscular inoculation vary significantly. Our data shown here strongly suggest that gene gun immunization of skin is a much more reliable method for DNA vaccination to induce effective immune responses in an animal model.

Anti-IL-12 antibody prevents the development and progression of multiple sclerosis-like relapsing–remitting demyelinating disease in NOD mice induced with myelin oligodendrocyte glycoprotein peptide

Treatment with monoclonal anti-IL-12 antibody injected on day 0, 7 and 10 after immunization with myelin oligodendrocyte glycoprotein (MOG) peptide 35–55 in NOD mice resulted in significant suppression of the development and the severity of the chronic relapsing–remitting experimental autoimmune encephalomyelitis (EAE) both clinically and histologically. The spleen cells from anti-IL-12 antibody treated mice displayed markedly inhibited MOG 35–55 specific proliferation and IFN-γ production. MOG 35–55 specific antibody production was enhanced by anti-IL-12 antibody treatment. These results suggest that IL-12 is critically involved in the pathogenesis of MOG-induced EAE and that antibody to IL-12 could be an effective therapeutic agent in the clinical treatment of autoimmune demyelinating diseases such as multiple sclerosis (MS).

Iscoms with different quillaja saponin components differ in their immunomodulating activities

In this study we demonstrate that iscoms made with different defined fractions of quillaja saponins (fractions A and C) exhibit strikingly different immunomodulatory activities. Fraction A has a potent immunomodulatory activity by enhancing antigen specific proliferation and production of IL-2 and IFN-γ, interestingly, this is not reflected by increased levels of IgG2a. The C-fraction on the other hand strongly enhance antibody production and has the ability to modulate the IgG subclass pattern towards IgG2a, in spite of the fact that C-iscoms induce only low levels of IFN-γ. Iscoms containing both fractions A and C (Iscoprep™703) induced higher serum antibody responses than iscoms made of either of the fractions alone and showed an enhanced ability to modulate the serum subclass pattern towards IgG2a compared to C-iscoms.

Trichuris muris: Host Intestinal Epithelial Cell Hyperproliferation during Chronic Infection Is Regulated by Interferon-γ

Artis, D., Potten, C. S., Else, K. J., Finkelman, F. D., and Grencis, R. K. 1999. Trichuris muris: Host intestinal epithelial cell hyperproliferation during chronic infection is regulated by interferon-γ. Experimental Parasitology92, 144–153. Chronic infection with the intestinal nematode Trichuris muris is associated with an inappropriate type 1 cytokine response (production of predominantly IFN-γ), whereas resistance to infection requires the induction of a protective type 2 response with the production of interleukin (IL)-4, IL-5, IL-9, and IL-13. T. muris inhabits an intracellular niche within murine intestinal epithelial cells of the caecum and in common with other intestinal helminth infections is associated with gross morphological changes in gut architecture. The purpose of this study was to characterise cytokine production during chronic infection in AKR and severe-combined-immunodeficient (SCID) mice and investigate what effect the anti-parasite response had on epithelial cell proliferation and so regulation of intestinal pathology. Pulse labeling with tritiated thymidine is employed to generate a sensitive cell position-linked proliferation index of the intestinal epithelium at various times postinfection. Infection in AKR mice is characterized by a marked elevation in antigen specific IFN-γ production from restimulated mesenteric lymph node cells and a significant increase in proliferation of pluripotent epithelial stem cells and transit cells within the crypts. Similarly, elevated IFN-γ production was observed in the mesenteric lymph nodes and intestinal mucosa of infected SCID mice, with epithelial cell hyperproliferation and the development of crypt hyperplasia in the caecum. Critically, in vivo depletion of IFN-γ during infection in SCID mice resulted in no significant increase in epithelial cell proliferation and effectively precluded the development of crypt hyperplasia without altering infection outcome. Taken together, the data provides the first detailed cell position linked analysis of epithelial dysregulation during chronic T. muris infection and identifies a critical role for IFN-γ, either directly or indirectly, in regulation of epithelial cell proliferation during the chronic intestinal inflammation associated with infection.

A new cell enzyme-linked immunosorbent assay demonstrates gamma interferon suppression by beta interferon in multiple sclerosis.

Multiple sclerosis (MS) is a demyelinating disorder of the central nervous system of unknown etiology. Immune mechanisms involving the proinflammatory cytokine gamma interferon (IFN-gamma) are believed to play an important role in the pathogenesis of MS. IFN-beta-1b has been introduced as a treatment for MS and was found to reduce the number and severity of clinical exacerbations. To examine the influence of IFN-beta-1b on myelin basic protein (MBP)-specific and phytohemagglutinin-induced IFN-gamma production, we developed a cell-released capturing enzyme-linked immunosorbent assay (CRC-ELISA), which rapidly measures spontaneous and antigen- or mitogen-induced cellular IFN-gamma production. CRC-ELISA documented a significant MBP-specific T-cell response in the blood of untreated MS patients, as assessed by IFN-gamma production. This response was suppressed in MS patients treated with IFN-beta-1b. The present work confirms in vivo the in vitro suppressive effects of IFN-beta-1b on IFN-gamma production in MS. Moreover, it provides a powerful new technique for detection of cytokines.

Improvement of heterologous protein productivity by controlling postinduction specific growth rate in recombinant Escherichia coli under control of the PL promoter.

To develop the optimal operational strategy for the PL promoter of interferon (IFN)-gamma-producing Escherichia coli, some rational medium feeding methods, carried out just after temperature induction to control the postinduction specific growth rate (mu i), were designed. Using experimental data from various batch cultures, we correlated mu i with specific IFN-gamma production rate (qpi) to find out how mu i just after induction affected qpi. It was revealed that sustaining mu i above a certain value after induction was the key factor for high-level production of IFN-gamma. Stepwise and constant-rate medium feeding, in which the temperature induction was done simultaneously, at the late-exponential growth phase of batch culture resulted in a dramatic extension of the production period as a result of sustaining mu i after temperature induction. These methods led to the overcoming of harsh conditions after temperature induction and the improvement of IFN-gamma productivity. Finally, by both a high-cell-density culture using an exponential fed-batch culture for cell growth and constant-rate medium feeding after temperature induction for IFN-gamma production, we accomplished an increase in IFN-gamma production by 23-fold, 7.43 g/L, as compared with that of batch culture.