Activation of bovine peripheral blood gammadelta T cells for cell division and IFN-gamma production

Bovine peripheral blood gammadelta T cells have been evaluated for effector function (IFN-gamma production) and clonal expansion in a variety of systems including following activation by mitogens, IL-12, and stimulation, through the T cell receptor (TCR) with anti-CD3 monoclonal antibody (mAb), a cell-bound molecule and a soluble antigenic extract. To evaluate cell division, carboxyfluorescein succinimidyl ester (CFSE) loading of cells and flow cytometric analysis were used, while IFN-gamma production was evaluated by intracytoplasmic staining. It was found that bovine gammadelta T cells produced IFN-gamma and clonally expanded when stimulated through the TCR/CD3 complex by a cell-associated autologous molecule on monocyte, by bacterial components following in vivo sensitization of gammadelta T cells with a leptospira vaccine or by anti-CD3 mAb. In addition, gammadelta T cells were activated efficiently for effector function but not clonal expansion by culturing with IL-12. In contrast, stimulation by Con A or PMA/ionomycin induced efficient replication but only low level IFN-gamma production which was not enhanced by the presence of IL-12. In several systems the amount of IFN-gamma produced per cell by gammadelta T cells was less than that produced by CD4 T cells in the same cultures.     

Equine herpesvirus-1 infection induces IFN-gamma production by equine T lymphocyte subsets

A commercial bovine IFN-gamma-specific monoclonal antibody was used to measure antigen-specific IFN-gamma production by equine lymphocytes. Paired PBMC samples were collected from six ponies prior to and 10 days after challenge infection with equine herpesvirus-1 (EHV-1). Each sample was stimulated in vitro with EHV-1, virus-free medium, or PMA and ionomycin, and labelled with monoclonal antibodies specific for various equine lymphocyte subset markers. Following fixation, intracellular IFN-gamma was detected using a FITC-conjugated bovine IFN-gamma-specific monoclonal antibody. In vitro restimulation of PBMC with EHV-1 induced IFN-gamma production by a significantly higher percentage of total (CD5(+)) T lymphocytes, and CD4(+) and CD8(+) T lymphocyte subsets among post-EHV-1 infection PBMC samples compared to pre-infection samples. This response was associated with an increase in virus-specific CTL activity, a critical immune effector for the control of EHV-1 infection and disease. No significant increase in IFN-gamma production by B lymphocytes was observed. These data demonstrate that EHV-1 challenge infection of ponies results in increased production of IFN-gamma by virus-specific T lymphocytes, and that this response can be quantitated using flow cytometry.     

Bovine interleukin-12 and modulation of IFNgamma production.

The effects of IL-12 on the responses of cattle peripheral blood mononuclear cells (PBMC) to bovine respiratory syncytial virus (BRSV) antigen and ovalbumin (OVA) were tested, in vitro. IL-12 did not affect the proliferative responses of PBMC to these antigens but markedly accelerated and augmented the level of IFNgamma secreted. When tested on lymphoblasts rather than resting T-cells IL-12 also enhanced proliferation. In contrast IL-4 and, to greater extent, IL-10 inhibited the response. The effect of IL-12 on IFNgamma synthesis was confirmed at the level of IFNgamma. mRNA expression using Taqman PCR. CD4 and CD8 T-cell populations produced IFNgamma, however, CD4 T-cells comprised the largest contributors to the IFNgamma production. Gamma/delta T-cells did not contribute markedly. A comparison of the species cross-reactivity showed bovine IL-12 was also active in the human system. This study shows that antigen-driven responses in cattle can be significantly influenced by exogenous cytokines and suggests the IL-12/IL-10 balance is crucial for regulation of IFNgamma.     

Induction of the 68 kDa major heat-shock protein in different Theileria annulata- and virus-transformed bovine lymphoblastoid cell lines.

Expression of the major inducible heat-shock protein of 68 kDa (hsp68) has been analyzed in peripheral blood mononuclear cells (PBMC) from cattle and in six Theileria annulata- and two bovine leukemia virus-transformed bovine lymphoblastoid cell lines (BoLCL). By metabolic labeling, hsp68 could be detected in PBMC and BoLCL only after heat-shock, but not under normal culture conditions. Immunoblot analysis with an hsp68 reactive monoclonal antibody similarly revealed a strong hsp68 response after heat-shock in BoLCL, and no hsp68 expression under normal culture conditions. Normally kept PBMC, however, were weakly positive with the antibody. The data are discussed with respect to the constitutive expression of hsp68 seen in several other cell lines.     

Porcine interleukin-12 fusion protein and interleukin-18 in combination induce interferon-gamma production in porcine natural killer and T cells

A pcDNA3 vector containing a gene encoding a porcine interleukin-12 (poIL-12) fusion protein was constructed, with the p40 chain and its signal peptide positioned first, followed by a linker and the p35 domain. When expressed in COS cells, secreted poIL-12 fusion protein showed high activity in terms of ability to induce interferon-gamma (IFN-gamma) production in porcine peripheral blood mononuclear cells (PBMCs) in vitro. The IFN-gamma production induced by poIL-12 fusion protein, as well as heterodimeric poIL-12 and human IL-12, was markedly dependent on the presence of human IL-18 (huIL-18). Furthermore, huIL-18 showed a dose-dependent induction of IFN-gamma production in PBMC in the presence of a constant concentration of huIL-12. A marked synergism between poIL-12 and IL-18 was consequently observed in poPBMC. The actual IFN-gamma producing cells were identified as probable NK cells (about 30%) and T lymphocytes (about 70%), using flow cytometry. Furthermore, a histidine-tagged poIL-12 fusion protein was expressed in Drosophila melanogaster Schneider 2 cells, using a modified pMT/V5-His vector lacking the V5 epitope. Such poIL-12 fusion protein was easily purified using Ni-NTA agarose and retained high biological activity.     

In vitro interactions of cytokines and bovine viral diarrhea virus in phytohemagglutinin-stimulated bovine mononuclear cells

Bovine viral diarrhea (BVD) virus inhibited phytohemagglutinin (PHA)-stimulated bovine peripheral blood mononuclear cell (PBMC) proliferation and bovine interleukin-2 (IL-2) production. In the controls, the heat-inactivated BVD virus was not capable of suppressing the PHA-stimulated PBMC proliferation. Presence of exogenous cytokines, such as purified human IL-2, recombinant bovine interleukin-1 (rbovIL-1), recombinant bovine IL-2, and recombinant human IL-6 failed to reverse the BVD virus-induced immunosuppression. Also, we found that the BVD virus inhibited PHA and IL-2 induced proliferation of bovine PBMC in the early and late stages of activation. In summary, our data suggest that BVD virus induced immunosuppression was not due to destruction of the PBMC but may be inhibiting one or more of the important intracellular enzymes that may regulate PBMC proliferation.     

Cloning of porcine interleukin (IL)-12 receptor beta2 (IL-12Rbeta2) gene and its application to a rapid biological assay for human/porcine IL-12

Porcine IL-12Rbeta2 gene was cloned from mRNA preparation of mitogen-activated peripheral blood mononuclear cells (PBMCs), and its complete nucleotide sequence was determined. To confirm the biological function, the entire open reading frame (ORF) was re-cloned into a mammalian expression vector, pcDNA3.1/Zeo(+), at the downstream of CMV promoter, and introduced to a Th1-like human lymphoma cell line, Jurkat E6-1. Antibiotic-resistant cells retaining the expression construct were selected then, isolated by the limiting dilution method. An established clone (10B10) constitutively expressed chimeric IL-12Rs composed of intrinsic (human) beta1 and extrinsic (porcine) beta2 subunits, and produced interferon (IFN)-gamma in response to IL-12 of both species with optimal PHA/PMA stimulation. The production of IFN-gamma was observed as early as 42 h after culture and appeared to be dose-dependent within the range between 20 and 2000 pg/ml. Thus, this clone not only reacts with IL-12 of both species but also provides a useful tool for quick and sensitive detection of IL-12 bioactivity.     

Molecular cloning, chromosomal location, and biological activity of porcine interleukin-21

A pig interleukin-21 (IL-21) cDNA was successfully cloned and sequenced from porcine peripheral blood lymphocytes (PBL) stimulated with 10 microg/ml concanavalin A (ConA), 10 microg/ml phytohemagglutinin P (PHA), 50 ng/ml phorbol 12-myristate 13-acetate (PMA), and 0.5 microg/ml anti-porcine CD3 antibody for 48 hr. The open reading frame of the porcine IL-21 cDNA is 459 base pairs in length and encodes 152 amino acids. The predicted amino acid sequence of the porcine IL-21 shows 86.2%, 77.7%, and 58.4% identity to the bovine, human, and murine IL-21, respectively. The porcine IL-21 gene was mapped to porcine chromosome 8 (8q22-->q23) by means of fluorescence in situ hybridization and radiation hybrid mapping, where the porcine IL-2 gene had been mapped nearby. The recombinant porcine mature IL-21 expressed by E. coli induced dose-dependent proliferation and IFN-gamma production from a human NK cell line, NK0. The porcine IL-21 identified in this study will be helpful for the enhancement of innate immune responses of pigs.     

Inhibition of phytohemagglutinin-stimulated bovine mononuclear cell proliferation, interleukin-2 production and protein kinase C activity by a protein kinase C inhibitor, H-7

1-(5-Isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), a potent and selective inhibitor of protein kinase C (PKC), inhibited PHA-stimulated bovine peripheral blood mononuclear cell (PBMC) proliferation, interleukin-2 (IL-2) production, and cytosolic PKC activity without affecting the cell viabilities. Presence of exogenous cytokines, such as purified human IL-2 or recombinant bovine IL-2 (rbovIL-2), reversed the H-7 inhibitory effects on PHA-stimulated PBMC proliferation. We conclude that the PKC enzyme plays an important role as a second messenger in bovine PBMC proliferation in the early stages of cell activation.     

Maternal and foetal immune responses of cattle following an experimental challenge with Neospora caninum at day 70 of gestation

The immune responses of pregnant cattle and their foetuses were examined following inoculation on day 70 of gestation either intravenously (iv) (group 1) or subcutaneously (sc) (group 2) with live NC1 strain tachyzoites or with Vero cells (control) (group 3). Peripheral blood mononuclear cell (PBMC) responses to Neospora antigen and foetal viability were assessed throughout the experiment. Two animals from each group were sacrificed at 14, 28, 42 and 56 days post inoculation (pi). At post mortem, maternal lymph nodes, spleen and PBMC and when possible foetal spleen, thymus and PBMC samples were collected for analysis. Inoculation with NC1 (iv and sc) lead to foetal deaths in all group 1 dams (6/6) and in 3/6 group 2 dams from day 28pi; statistically significant (p ≤ 0.05) increases in cell-mediated immune (CMI) responses including antigen-specific cell proliferation and IFN-γ production as well as increased levels of IL-4, IL-10 and IL-12 were observed in challenged dams compared to the group 3 animals. Lymph node samples from the group 2 animals carrying live foetuses showed greater levels of cellular proliferation as well as significantly (p ≤ 0.05) higher levels of IFN-γ compared to the dams in group 2 carrying dead foetuses. Foetal spleen, thymus and PBMC samples demonstrated cellular proliferation as well as IFN-γ, IL-4, IL-10 and IL-12 production following mitogenic stimulation with Con A from day 14pi (day 84 gestation) onwards. This study shows that the generation of robust peripheral and local maternal CMI responses (lymphoproliferation, IFN-γ) may inhibit the vertical transmission of the parasite.     

Immune production of interferon by cultured peripheral blood mononuclear cells from calves infected with BHV1 and PI3 viruses

Peripheral blood mononuclear cells (PBMC) from calves infected with bovine herpesvirus type 1 (BHV1) or parainfluenza 3 virus (PI3) were cultured in vitro in the presence of inactivated specific antigen presented on MDBK cells. In the presence of inactivated antigen, PBMC from both BHV1-infected and control calves produced interferon (IFN)-alpha in 24 hour cultures. Altering the culture conditions did not result in the detection of immune-specific IFN produced by mononuclear cells from BHV1-infected calves. However, spontaneous IFN was detected in the absence of antigen in 24 hour cultures from infected animals: this IFN was pH 2 labile and completely neutralised by antiserum to recombinant bovine IFN-gamma. Spontaneous IFN-gamma production was only seen in calves following a second BHV1 inoculation, given four to seven weeks after the primary dose. In contrast PBMC cultures from PI3 virus-infected calves did not produce IFN-gamma spontaneously, but did so in cultures which contained inactivated PI3 antigen. Mononuclear cells from control animals failed to produce either IFN-alpha or -gamma when cultured with inactivated PI3 virus. IFN-gamma was detected in PBMC cultures after the primary infection, with no increase in production occurring following subsequent PI3 virus inoculations. Immunospecific production of IFN-gamma provides a simple method for monitoring cell-mediated immunity in BHV1- and PI3 virus-infected calves and can be used for evaluating the efficacy of vaccines against these viruses.     

Altered surface antigen expression on peripheral blood mononuclear cells in cats infected with feline immunodeficiency virus.

Expression of CD4, CD8, IL-2 receptor alpha chain (IL-2R alpha), and MHC class II (MHC-II) on peripheral blood mononuclear cells were examined in cats infected with feline immunodeficiency virus (FIV). CD4/CD8 T cell ratio in FIV-infected cats was slightly decreased, as compared with that in specific-pathogen-free (SPF) cats. However, there was no statistical differences between them. The number of circulating IL-2R alpha+ cells in FIV-infected cats was higher than that in healthy cats, whereas induction of IL-2R alpha expression by concanavalin A (Con A) stimulation was depressed in FIV-infected cats. By using two-color cytofluorometry, Con A-induced enhancement of IL-2R alpha expression was found to be reduced in both CD4+ and CD8+ populations in PBMC from FIV-infected cats. The circulating MHC-II+ cells were also increased in FIV-infected cats. Furthermore, the induction of IL-2R alpha expression on PBMC after Con A-stimulation significantly depressed by FIV inoculation in vitro. These results suggest that FIV activates PBMC in vivo via direct and/or indirect mechanisms, leading to the unresponsive state of T cells to further stimuli in vitro.     

Interferon-gamma expression associated with suppression of bovine leukemia virus at the early phase of infection in sheep

Immunological control of bovine leukemia virus (BLV)-infection has been reported as dependent on the expression balance of types 1 and 2 cytokines. In this report, mRNA expression of interferon (IFN)-gamma and interleukin (IL)-2 (type 1 cytokines), and of IL-4 and IL-10 (type 2 cytokines) were evaluated in concanavalin A-stimulated peripheral blood mononuclear cells (PBMC) from BLV-infected sheep. Despite the same dose of BLV-infection, the extent of viral propagation was markedly different between eight individual sheep by 12 weeks post infection. The virus did not propagate well in three sheep, which showed augmented mRNA expression of IFN-gamma, a strong indicator of cell-mediated immunity, immediately after BLV-infection. Among the other five sheep having more than 2% of BLV-infected cells among PBMC at 12 weeks post infection, four sheep developed B-cell leukemia or lymphoma within 2 years after infection. These observations indicate IFN-gamma expression may play an important role in the protective mechanism against BLV propagation at the early phase of the infection.     

Antigen-specific IFN-gamma and IL-4 production in caprine herpesvirus infected goats

The analysis of cytokines secreted by antigen-specific lymphocytes is hampered in goats by the paucity of species-specific reagents yet it is crucial to study immune responses to infections. To overcome this limit, two commercial kits designed to measure soluble bovine IL-4 (by ELISA) and frequencies of bovine IFN-gamma secreting cells (by ELISPOT) were tested for cross-reactivity in goats. In addition, an ELISA specific to bovine/ovine IL-4 and employing two monoclonal antibodies, clones CC313 and CC314, was tested as well. Concanavalin A-stimulated caprine peripheral blood mononuclear cells (PBMCs) cultures were studied and they exhibited high levels of soluble IL-4 and high frequencies of IFN-gamma secreting cells. In addition, the two IL-4 ELISAs detected similar amounts of cytokine. To start defining the cytokine response triggered by caprine herpesvirus 1 (CpHV-1) infection, PBMC cultures were setup from goats naturally or experimentally infected with CpHV-1. High frequencies of IFN-gamma producing cells and low, when detectable, levels of soluble IL-4 were observed in CpHV-1-specific PBMC cultures from both groups of infected goats. Thus, the availability of cross-reactive research tools can expand cytokine studies in goats and can implement the research on immunity to other caprine infections.     

Genetic variation in Con A-induced production of interleukin 2 by porcine peripheral blood mononuclear cells

Genetic variation in concanavalin A (Con A)-induced proliferation and interleukin 2 (IL-2) production by peripheral blood mononuclear cells (PBMC) was studied in blood collected from 96 piglets, aged 7 weeks. The piglets were the offspring of seven sires and 24 dams. Pronounced differences between litters from various dams were observed in the immune parameters measured. Also, large individual differences in the magnitudes of Con A-induced proliferation and IL-2 production were seen for PBMC collected from individual pigs within each litter. Both the time course and magnitude of IL-2 activity showed genetic variation, as results from the offspring of the seven sires differed significantly. However, only the time course, not the magnitude, of proliferation differed among the offspring groups. It was possible to establish a rank order for the sires based on the IL-2 production of PBMC by their offspring. As IL-2 has a key role in regulating the immune response, mitogen-induced IL-2 activity seems to be a good candidate as a general marker for cell-mediated immunity in pigs.     

A monoclonal antibody specific for bovine T cell surface antigen associated with the E-rosette receptor

From mice immunized with T lymphocyte-enriched bovine peripheral blood mononuclear cells (PBMC), a monoclonal antibody termed BLMo-12 was obtained. BLMo-12 reacted with the antigen of Mr 56,000 in lysate of T lymphocytes. This mAb was found to inhibit spontaneous rosette formation by T-bovine lymphocytes with sheep red blood cells but it did not react with B lymphocytes, monocytes, neutrophils or eosinophils. In frozen section of the thymus, BLMo-12 showed a positive staining both the cortex and the medulla. In lymph nodes, the mAb stained the T-dependent paracortex. BLMo-12 reacted with 49.9% of PBMC and 82.5% of thymocytes. Recognition of the bovine homologue of CD2 on the T lymphocyte surface by this mAb was discussed.