Higher Interferon-gamma Production Research Articles

Genome-Wide Association Study Reveals Quantitative Trait Loci and Candidate Genes Associated with High Interferon-gamma Production in Holstein Cattle Naturally Infected with Mycobacterium Bovis.

Mycobacterium bovis (Mb) is the causative agent of bovine tuberculosis (bTb). Genetic selection aiming to identify less susceptible animals has been proposed as a complementary measure in ongoing programs toward controlling Mb infection. However, individual animal phenotypes for bTb based on interferon-gamma (IFNɣ) and its use in bovine selective breeding programs have not been explored. In the current study, IFNɣ production was measured using a specific IFNɣ ELISA kit in bovine purified protein derivative (bPPD)-stimulated blood samples collected from Holstein cattle. DNA isolated from the peripheral blood samples collected from the animals included in the study was genotyped with the EuroG Medium Density bead Chip, and the genotypes were imputed to whole-genome sequences. A genome-wide association analysis (GWAS) revealed that the IFNɣ in response to bPPD was associated with a specific genetic profile (heritability = 0.23) and allowed the identification of 163 SNPs, 72 quantitative trait loci (QTLs), 197 candidate genes, and 8 microRNAs (miRNAs) associated with this phenotype. No negative correlations between this phenotype and other phenotypes and traits included in the Spanish breeding program were observed. Taken together, our results define a heritable and distinct immunogenetic profile associated with strong production of IFNɣ in response to Mb.

Association between High Interferon-Gamma Production in Avian Tuberculin-Stimulated Blood from Mycobacterium avium subsp. paratuberculosis-Infected Cattle and Candidate Genes Implicated in Necroptosis.

The mechanisms underlying host resistance to Mycobacterium avium subsp. paratuberculosis (MAP) infection are largely unknown. In the current study, we hypothesize that cows with an ability to produce higher levels of interferon-gamma (IFNɣ) might control MAP infection more successfully. To test this hypothesis, IFNɣ production was measured using a specific IFNɣ ELISA kit in avian purified protein derivative (aPPD)-stimulated blood samples collected from 152 Holstein cattle. DNA isolated from peripheral blood samples of the animals included in the study was genotyped with the EuroG Medium-Density Bead Chip, and the genotypes were imputed to whole-genome sequencing. A genome-wide association analysis (GWAS) revealed that high levels of IFNɣ in response to the aPPD were associated with a specific genetic profile (heritability = 0.64) and allowed the identification of 71 SNPs, 40 quantitative trait loci (QTL), and 104 candidate genes. A functional analysis using the 104 candidate genes revealed a significant enrichment of genes involved in the innate immune response and, more specifically, in necroptosis. Taken together, our results define a heritable and distinct immunogenetic profile associated with the production of high IFNɣ levels and with the capacity of the host to lyse MAP-infected macrophages by necroptosis.

Induction of a Th1 immune response and suppression of IgE via immunotherapy with a recombinant hybrid molecule encapsulated in liposome-protamine-DNA nanoparticles in a model of experimental allergy.

Liposome-protamine-DNA nanoparticles (LPD) are safe, effective, and non-toxic adjuvants that induce Th1-like immune responses. We hypothesized that encapsulation of allergens into liposomes could be an appropriate option for immunotherapy. The present study evaluated the immunotherapeutic potential of a recombinant hybrid molecule (rHM) encapsulated in LPD nanoparticles in a murine model of Chenopodium album allergy. BALB/c mice were sensitized with the allergen in alum, and the immunotherapy procedure was performed by subcutaneous injections of LPD-rHM, rHM, or empty LPD at weekly intervals. Sensitized mice developed a Th2-biased immune response characterized by strong specific IgG1 and IgE production, IL-4, and the transcription factor GATA3 in spleen cell cultures. Treatment with the LPD-rHM resulted in a reduction in IgE and a marked increase in IgG2a. The LPD-rHM induced allergen-specific responses with relatively high interferon-gamma production, as well as expression of the transcription factor T-bet in stimulated splenocytes. In addition, lymphoproliferative responses were higher in the LPD-rHM-treated mice than in the other groups. Removal of the nanoparticles from the rHM resulted in a decrease in the allergen's immunogenicity. These results indicate that the rHM complexed with LPD nanoparticles has a marked suppressive effect on the allergic response and caused a shift toward a Th1 pathway.

Prime-boost BCG vaccination with DNA vaccines based in β-defensin-2 and mycobacterial antigens ESAT6 or Ag85B improve protection in a tuberculosis experimental model

The World Health Organization (WHO) has estimated that there are about 8 million new cases annually of active Tuberculosis (TB). Despite its irregular effectiveness (0–89%), the Bacillus Calmette-Guérin) BCG is the only vaccine available worldwide for prevention of TB; thus, the design is important of novel and more efficient vaccination strategies. Considering that β-defensin-2 is an antimicrobial peptide that induces dendritic cell maturation through the TLR-4 receptor and that both ESAT-6 and Ag85B are immunodominant mycobacterial antigens and efficient activators of the protective immune response, we constructed two DNA vaccines by the fusion of the gene encoding β-defensin-2 and antigens ESAT6 (pDE) and 85B (pDA). After confirming efficient local antigen expression that induced high and stable Interferon gamma (IFN-γ) production in intramuscular (i.m.) vaccinated Balb/c mice, groups of mice were vaccinated with DNA vaccines in a prime-boost regimen with BCG and with BCG alone, and 2 months later were challenged with the mild virulence reference strain H37Rv and the highly virulent clinical isolate LAM 5186. The level of protection was evaluated by survival, lung bacilli burdens, and extension of tissue damage (pneumonia). Vaccination with both DNA vaccines showed similar protection to that of BCG. After the challenge with the highly virulent Mycobacterium tuberculosis strain, animals that were prime-boosted with BCG and then boosted with both DNA vaccines showed significant higher survival and less tissue damage than mice vaccinated only with BCG. These results suggest that improvement of BCG vaccination, such as the prime-boost DNA vaccine, represents a more efficient vaccination scheme against TB.

Novel Th1-biased adjuvant, SPO1, enhances mucosal and systemic immunogenicity of vaccines administered intranasally in mice

Oil-in-water emulsions are potent human adjuvants commonly used in effective pandemic influenza vaccines; however, such emulsions that can induce both Th1-biased systemic immune responses and strong mucosal immune responses via an easy method of administration are lacking. To address this need for new adjuvants, we developed a novel oil/water emulsion, SPO1, which allows convenient mucosal immunization via an intranasal spray as well as by parenteral routes. Our report shows that SPO1 was able to boost up immunological resistance by inducing effective mucosal and serum antibodies, and the immune response was polarized to a Th1 pattern, as demonstrated by high IgG2α antibody levels and interferon-gamma production by splenocytes from intranasally (i.n.) immunized mice. Up-regulation of co-stimulatory and antigen-presenting molecules on dendritic cells was also observed in vivo after i.n. immunization, suggesting a possible mechanism for the adjuvant effects of SPO1. Another explanation may simply be a depot of antigen at the immunization site, as evidenced by in vivo imaging of i.n. immunized mice. In conclusion, our results demonstrate that a novel oil/water emulsion, SPO1, is a potent Th1 adjuvant for use in influenza and other vaccines, as it induces strong mucosal and systemic immune responses.

Coexpression of Flt3 ligand and GM-CSF genes modulates immune responses induced by HER2/neu DNA vaccine

DNA vaccine and dendritic cells (DCs)-based vaccine have emerged as promising strategies for cancer immunotherapy. Fms-like tyrosine kinase 3-ligand (Flt3L) and granulocyte-macrophage-colony-stimulating factor (GM-CSF) have been exploited for the expansion of DC. It was reported previously that combination of plasmid encoding GM-CSF with HER2/neu DNA vaccine induced predominantly CD4(+) T-cell-mediated antitumor immune response. In this study, we investigated the modulation of immune responses by murine Flt3L and GM-CSF, which acted as genetic adjuvants in the forms of bicistronic (pFLAG) and monocistronic (pFL and pGM) plasmids for HER2/neu DNA vaccine (pN-neu). Coexpression of Flt3L and GM-CSF significantly enhanced maturation and antigen-presentation abilities of splenic DC. Increased numbers of infiltrating DC at the immunization site, higher interferon-gamma production, and enhanced cytolytic activities by splenocytes were prominent in mice vaccinated with pN-neu in conjunction with pFLAG. Importantly, a potent CD8(+) T-cell-mediated antitumor immunity against bladder tumors naturally overexpressing HER2/neu was induced in the vaccinated mice. Collectively, our results indicate that murine Flt3L and GM-CSF genes coexpressed by a bicistronic plasmid modulate the class of immune responses and may be superior to those codelivered by two separate monocistronic plasmids as the genetic adjuvants for HER2/neu DNA vaccine.

Lamina Propria c-kit+ Immune Precursors Reside in Human Adult Intestine and Differentiate Into Natural Killer Cells

Recent studies have revealed that murine intestinal mucosa contains several kinds of lineage markers (lin)(-) c-kit(+) immune precursor cells. However, immune precursors in the human adult intestine have not been studied extensively. Lamina propria mononuclear cells and intraepithelial lymphocytes from surgically resected human adult intestine were examined for the surface antigen expression and cytokine profile by immunohistochemistry and flow cytometry. The transcriptional profile of these cells was analyzed by reverse-transcription polymerase chain reaction. The phenotypic and functional characterization of the in vitro differentiating cells from the precursors was examined by flow cytometry. We identified lin(-) c-kit(+) cells scattered throughout lamina propria of the human adult intestine. These intestinal immune precursors expressed CD34, CD38, CD33, interleukin-2R alpha, and interleukin-7R alpha, and they had much more abundant expression of Id2, PU.1, SpiB1, and lymphotoxin than thymocytes. The lin(-) c-kit(+) immune precursors mainly differentiated into CD56(+) c-kit(dim) cells during in vitro culture. These in vitro differentiating cells corresponded to intestinal natural killer (NK) cells, which had distinct characteristics from their peripheral counterparts, such as CD83 and integrin alpha(E) expression, less cytotoxic activity, and higher interferon-gamma production. Furthermore, both c-kit(dim) cells and NK cells were increased in lamina propria of Crohn's disease, although there was no change for peripheral blood NK cells. The human intestine may have the unique NK cell differentiation system, which may contribute to maintenance of immune homeostasis in the intestine.

CC chemokine receptor (CCR)-2 prevents arthritis development following infection by Mycobacterium avium

The host factors that influence autoimmune arthritides such as rheumatoid arthritis have not been fully elucidated. We previously found that genetic inactivation of CC chemokine receptor 2 (CCR2) in the arthritis-prone DBA/1j mouse strain significantly increases the susceptibility of this strain to autoimmune arthritis induced by immunization with collagen type II (CII) and complete Freund's adjuvant (CFA). Here, we show that following intradermal infection with Mycobacterium avium, a similar arthritis phenotype was detected in Ccr2-null mice in the DBA/1j, but not in the BALB/c background. The failure to develop arthritis in Ccr2-null BALB/c mice occurred in the face of high bacterial burdens and low interferon gamma (IFNgamma) production. By contrast, Ccr2-null DBA/1j mice had low bacterial burdens, produced normal amounts of IFNgamma, and had high titers of autoantibodies against CII. Thus, the Ccr2-null state in an arthritic-prone genetic background leads to increased arthritis susceptibility following infectious (M. avium) and noninfectious (CII/CFA) challenges. Because CCR2 serves as a negative regulator of murine arthritis, caution might need to be exercised while testing CCR2 blockers in human arthritis or other diseases. These findings also indicate that Ccr2-null DBA/1j mice might serve as a valuable model system to uncover the immunological determinants of arthritis and to test novel antiarthritic agents.

Natural killer T‐cell populations in C57BL/6 and NK1.1 congenic BALB.NK mice—a novel thymic subset defined in BALB.NK mice

Natural killer (NK) T lymphocytes are a subpopulation of T lymphocytes regarded as early regulators of immune responses. The majority of NKT cells are restricted by the CD1d molecule. NKT cells have mostly been studied in one single mouse strain, C57BL/6 (B6), because of the absence of NK1.1 in other common mouse strains, and the lack of other reliable surface markers for CD1d-restricted cells. To investigate NKT cell subsets in a mouse strain of a genetic background different from B6, we have back-crossed the NKT cell marker NK1.1 from the B6 mouse to the BALB/c mouse strain. We show that NKT cells in the congenic BALB.B6-NK1.1(b) mouse share many characteristics with their B6 counterparts, but seem to be deficient in the functional NKT cell subtype characterized by low interleukin-4 and high interferon-gamma production, and surface expression of CD49b but not CD69. Moreover, in the thymus but not the spleen of BALB.B6-NK1.1(b) mice we find a novel Valpha14-Jalpha18 invariant NKT cell subset which is devoid of a set of NK markers, suggesting that these cells represent a less differentiated NKT cell stage, and carries high levels of the T-cell receptor and uses a skewed T-cell receptor Vbeta-repertoire.

Fc-Gamma Receptor IIIa and Interferon-Gamma SNPs Do Not Predict Responsiveness of Follicular Lymphoma to Galiximab (Anti-CD80 Antibody).

CD80 is an immune costimulatory molecule expressed on the cell surface of follicular and other lymphomas. Galiximab, a macaque-human chimeric anti-CD80 monoclonal antibody, has been shown to have antitumor activity in patients with relapsed or refractory, follicular lymphoma. Preclinical work suggested antibody-dependent cellular cytotoxicity (ADCC) as a mechanism for galiximab-induced cell lysis. Single-nucleotide polymorphisms (SNPs) for high-affinity Fc gamma receptor IIIa and high interferon-gamma production have been associated with increased ADCC activity. For this report, genomic DNA from peripheral blood of clinical study patients was analyzed for Fc gamma receptor IIIa and interferon-gamma gene SNPs after PCR amplification. In addition, the frequency of interferon-gamma secreting cells in peripheral blood samples was evaluated by ELISPOT. SNP and ELISPOT results were then evaluated for correlation with clinical response (CR or PR) to galiximab and reduction in tumor burden (SPD of indicator lesions). Of 35 follicular lymphoma patients evaluable for response to galiximab, 2 CRs and 1 PR were observed; time to best response was delayed (Months 3, 9, and 12). Of 34 patients with Fc gamma receptor IIIa SNP results, 6 were homozygous for the high-affinity receptor. Of 33 pts with interferon-gamma SNP results, 4 were homozygous for the high-producing allele. None of the 3 responders were homozygous for high-affinity Fc gamma receptor IIIa or high interferon-gamma production, and the degree of tumor burden reduction was smaller in these homozygotes than in the others. In addition, pretreatment interferon-gamma ELISPOT data did not predict clinical response to galiximab or tumor burden reduction. In summary, high-affinity Fc gamma receptor IIIa and high interferon-gamma production do not predict clinical responsiveness of follicular NHL to galiximab, suggesting that ADCC may not be a primary mechanism of action for this antibody. Ongoing studies are evaluating other potential mechanisms including the immunomodulatory effects of blocking CD80.

Beta2-agonists prevent Th1 development by selective inhibition of interleukin 12.

Interleukin 12 (IL-12) plays a central role in the immune system by skewing the immune response towards T helper 1 (Th1) type responses which are characterized by high interferon-gamma and low IL-4 production. In this report we present evidence that beta2-agonists inhibit IL-12 production by both human monocytes in response to lipopolysaccharide (LPS) and dendritic cells stimulated via CD40. Inhibition of IL-12 production is selective, as other cytokines produced by monocytes are unaffected. IL-12 inhibition is dependent on beta2-adrenoceptor stimulation and correlates with increased levels of intracellular cAMP. In conjunction with their ability to suppress IL-12 production, when beta2-agonists are added at priming of neonatal T lymphocytes, they inhibit the development of Th1-type cells, while promoting T helper 2 (Th2) cell differentiation. Further, the in vivo administration of a therapeutic dose of salbutamol results in the selective inhibition of IL-12 production by whole blood lymphocytes stimulated in vitro with LPS. These findings provide new insight into the immunological consequences of the clinical use of beta2-agonists and may suggest new approaches for the treatment of Th1-mediated diseases.

Interleukin 12 induces stable priming for interferon gamma (IFN-gamma) production during differentiation of human T helper (Th) cells and transient IFN-gamma production in established Th2 cell clones.

Interleukin 12 (IL-12) facilitates the generation of a T helper type 1 (Th1) response, with high interferon gamma (IFN-gamma) production, while inhibiting the generation of IL-4-producing Th2 cells in polyclonal cultures of both human and murine T cells and in vivo in the mouse. In this study, we analyzed the effect of IL-12, present during cloning of human T cells, on the cytokine profile of the clones. The culture system used allows growth of clones from virtually every T cell, and thus excludes the possibility that selection of precommitted Th cell precursors plays a role in determining characteristics of the clones. IL-12 present during the cloning procedures endowed both CD4+ and CD8+ clones with the ability to produce IFN-gamma at levels severalfold higher than those observed in clones generated in the absence of IL-12. This priming was stable because the high levels of IFN-gamma production were maintained when the clones were cultured in the absence of IL-12 for 11 d. The CD4+ and some of the CD8+ clones produced variable amounts of IL-4. Unlike IFN-gamma, IL-4 production was not significantly different in clones generated in the presence or absence of IL-12. These data suggest that IL-12 primes the clone progenitors, inducing their differentiation to high IFN-gamma-producing clones. The suppression of IL-4-producing cells observed in polyclonally generated T cells in vivo and in vitro in the presence of IL-12 is not observed in this clonal model, suggesting that the suppression depends more on positive selection of non-IL-4-producing cells than on differentiation of individual clones. However, antigen-specific established Th2 clones that were unable to produce IFN-gamma with any other inducer did produce IFN-gamma at low but significant levels when stimulated with IL-12 in combination with specific antigen or insoluble anti-CD3 antibodies. This induction of IFN-gamma gene expression was transient, because culture of the established clones with IL-12 for up to 1 wk did not convert them into IFN-gamma producers when stimulated in the absence of IL-12. These results suggest that Th clones respond to IL-12 treatment either with a stable priming for IFN-gamma production or with only a transient low level expression of the IFN-gamma gene, depending on their stage of differentiation.

Peripheral Blood Lymphocytes Resistant to Epstein-Barr Virus Immortalization Manifest High Natural Killer (NK) Type Activity Against NK-Resistant Target Cells

Epstein-Barr virus (EBV) readily immortalizes human peripheral blood lymphocytes (PBL) in vitro. We found recently that PBL from two EBV-seropositive healthy adults were exceptionally resistant to immortalization by EBV. In contrast to PBL from other EBV-seropositive donors sensitive to immortalization by EBV (S-PBL), the "resistant" PBL (R-PBL) respond to EBV infection with an early interleukin-2 (IL-2) synthesis and high interferon gamma (IFN gamma) production. In order to determine whether these differences in cytokine responses between R-PBL and S-PBL could be associated with a detectable difference in lymphocyte cytotoxicity, we compared the natural killer (NK) activity of R-PBL and S-PBL effectors by using both NK-sensitive (i.e. K562) and NK-resistant (i.e. Raji) targets. We found that, while effectors from EBV-infected R-PBL and S-PBL cultures exhibited comparable NK activity against the K562 targets, they differed remarkably in their cytolytic activity against Raji cells. At days 3 and 5 of culture, effectors from EBV-infected R-PBL showed a significantly higher lytic activity against Raji targets, whereas S-PBL did not. Culture of EBV-infected R-PBL and S-PBL effectors in the presence of recombinant IL-2 (rIL-2) for 5 days resulted in increases of their lytic activity against Raji cells, whereas pretreatment of these effectors with recombinant IFN gamma (rIFN gamma) was found to increase only R-PBL cytotoxicity. These results suggest that the resistance of R-PBL to EBV immortalization could be associated with a lymphokine-mediated early cellular cytotoxic response of the NK/LAK (lymphokine-activated killer cell) type against EBV-infected cells.