Patterns of bovine T cell-mediated immune responses to bovine herpesvirus 1

Lymphocyte proliferation was used to evaluate T cell-mediated immune responses to different isolates of Bovine Herpesvirus 1 (BHV-1). Groups of high, moderate, and low responses were observed when lymphocytes from three breeds of dairy cattle were stimulated with each of the BHV-1 isolates. Proliferation of cells in the low responding group could be augmented by exogenous IL-2. The mechanism of unresponsiveness by cells from one individual whose response was not altered by IL-2 supplementation was further investigated. The patterns of response by limiting dilution frequency analysis eliminated the possibility that this individual lacked responsive cells but suggested the presence of regulatory cell interactions which resulted in the observed low proliferative response. These results show that animals exposed to the same environment can vary greatly in their ability to respond to BHV-1. At least two mechanisms may be responsible for low proliferative responses in vitro: inadequate levels of IL-2 and the presence of suppressor cells.     

Detection of impaired T cell-mediated immune responses to herpesvirus (BHV-1) in cattle

Interleukin 2 (IL-2) functions in the regulation of cell-mediated immune responses both in vivo and in vitro. Therefore, IL-2 production has been studied in patients with immunological disorders to determine the level of the immune defect. However, the cause(s) of low responses to selected antigens by cells from clinically normal individuals has not been examined. The ability of cells from clinically normal individual cattle to produce and respond to IL-2 was investigated as a measure of specific cell-mediated immune response. Cells from the majority of animals (6/7 in a representative experiment) could produce IL-2 in response to mitogen or a specific antigen, Bovine Herpesvirus 1 (BHV-1). Proliferation of lymphocytes from the majority of low responding animals (2/3 and 4/4 in separate experiments) could be restored to a level similar to high responders by the addition of exogenous IL-2 after endogenous IL-2 depletion. IL-2 receptor expression was indirectly assessed by the ability of activated cells to absorb IL-2. One individual's cells were incapable of absorbing exogenous IL-2 and failed to proliferate indicating a lack of activation and expression of receptors. In addition, exogenous IL-2 was able to enhance proliferation of both high and low responders in the presence of endogenous IL-2. These results suggest that proliferation of bovine peripheral blood mononuclear cells is dependent on the presence of IL-2. In addition, IL-2 production can be used to measure specific cell-mediated immune responses.     

Changes in cell-mediated immune responses after experimentally-induced anaphylaxis in dogs.

Experimentally-induced type 1 hypersensitivities were induced in normal dogs to either ovalbumin or Ascaris antigen. In vitro and in vivo cell-mediated immune responses were measured before sensitization and again at 1 and 6 days after induction of anaphylaxis by intravenous challenge with antigen. Histamine-modulated lymphocyte functions, such as histamine-induced suppression, histamine co-mitogen induced blastogenesis and the in vivo cutaneous responses to intradermally injected mitogens decreased post anaphylaxis. Spontaneous suppression of the autologous mixed-lymphocyte reaction increased post anaphylaxis. Lymphocyte blastogenic response to Concanavalin A (Con A) decreased at 6 (but not at 1) days post anaphylaxis probably due to a mediator other than histamine. Blastogenesis of 24 h preincubated cells by suboptimal concentration of Con A, declined post anaphylaxis, but Con A-induced suppression was not significantly altered. Dogs with atopic dermatitis have some altered cell-mediated immune responses. Altered histamine-induced and spontaneous suppression, histamine suppression of mitogenesis and decreased contact sensitivity observed in this experimental type 1 hypersensitivity mimicked that of atopic dogs. Increased cutaneous response to mitogens observed in atopic dogs was not reproduced in the type 1 hypersensitive dogs. These findings suggest some of the altered cell-mediated immune functions observed in dogs with atopic dermatitis result from type 1 hypersensitivity. The other abnormalities may be intrinsic to the atopic state.     

Characterization of porcine xenoreactive cytotoxic T lymphocytes.

Cytotoxic T lymphocytes (CTL) against mouse P815 cells were detected after stimulation of porcine peripheral blood mononuclear cells (PBMC) with irradiated Balb/c splenocytes. In vivo priming prior to in vitro stimulation slightly enhanced CTL activity, but lysis of targets was undetectable from lymphocytes from non-immune or immune animals that were not cultured with mouse splenocytes. After primary culture with Balb/c (H-2d) splenocytes, specific killing of P815 (H-2d) targets and not L929 (H-2k) targets indicated that recognition was specific for the H-2 locus. Similarly, CTL primed by mouse cells from either of two congenic strains recognized targets with alleles homologous to the stimulating cells. The anti-murine CTL was confirmed to be a CD8+ T cell based on studies using specific monoclonal antibodies to the porcine CD4 or CD8 cells. The cells responsible for the cytotoxicity of P815 targets lacked the characteristics of non-specific NK cells because (1) naive PBMC were unable to lyse NK targets (K562 cells) during the 4 h cytotoxic assay and (2) CTL killing of P815 targets increased with time after primary stimulation, whereas killing of K562 cells remained low at all times. These results suggest that porcine CTL can be readily generated against the xenogeneic mouse major histocompatibility complex.     

Evaluation of the cell-mediated immune response in cattle induced by 2,4-dinitrochlorobenzene

A technique has been evolved and standardized for evaluating the cell-mediated immune (CMI) response in cattle induced by 2,4-dinitrochlorobenzene (DNCB). Six healthy cross-bred Jersey cows were used. The dose and the number of applications for primary sensitization and the challenge dose after the 14th day have been standardized. The gross and histological pictures of the CMI response shown by these cattle are described in detail and their usefulness in assessing the CMI response in various disease conditions including tumour is indicated.     

Modulation of humoral and cell-mediated immune responses by dietary lutein in cats

The immuno-modulatory role of dietary lutein in domestic cats is unknown. Female Tabby cats (10-month old; n=56) were supplemented daily for 12 weeks with 0, 1, 5 or 10mg lutein. Blood was collected on Weeks 0, 2, 4, 8 and 12 to assess the following: (1) mitogen-induced peripheral blood mononuclear cells (PBMCs) proliferation, (2) changes in PBMC subpopulations, (3) interleukin-2 (IL-2) production and (4) plasma immunoglobulin (Ig)G production. In addition, delayed-type hypersensitivity (DTH) response to concanavalin A (Con A) or a polyvalent vaccine was performed on Weeks 0, 6 and 12. Dietary lutein increased plasma lutein concentrations in a dose-dependent manner (p<0.001) and concentrations had not reached steady state after 12 weeks of feeding in cats given 5 or 10mg lutein. Concentrations of plasma retinol and alpha-tocopherol were not influenced by diet. The DTH response to vaccine but not to Con A increased (p<0.05) in a dose-dependent manner on Week 6. Compared to control, cats fed lutein also showed enhanced Con A- and pokeweed mitogen-stimulated PBMCs proliferation. Dietary lutein also increased the percentages of CD4+ and CD21+ lymphocytes on Week 12 but had no significant effect on pan T, CD8 and MHC class II markers. Plasma IgG was higher (p<0.05) in cats fed 10mg lutein on Weeks 8 and 12. These results support the immuno-modulatory action of lutein in domestic cats.     

Humoral and T cell-mediated immune responses to bivalent killed bovine viral diarrhea virus vaccine in beef cattle

The objective of this research project was to evaluate the antibody and cell-mediated immune responses to a multivalent vaccine containing killed bovine viral diarrhea virus (BVDV) types 1 and 2. Twenty castrated male crossbred beef cattle (350-420kg body weight) seronegative to BVDV were randomly divided into two groups of 10 each. Group 1 served as negative mock-vaccinated control. Group 2 was vaccinated subcutaneously twice, 3 weeks apart, with modified live bovine herpesvirus 1, parainfluenza 3 virus and bovine respiratory syncytial virus diluted in diluent containing killed BVDV type 1 (strain 5960) and type 2 (strain 53637) in an adjuvant containing Quil A, Amphigen, and cholesterol. Serum samples were collected from all cattle at days -21, 0, and days 21, 28, 35, 56 and 70 post-vaccination. Standard serum virus neutralization tests were performed with BVDV type 1 (strain 5960) and type 2 (strain 125C). Anticoagulated blood samples were collected at day 0, and days 28, 35, 56 and 70 post-vaccination. Peripheral blood mononuclear cells (PBMCs) were isolated, stimulated with live BVDV type 1 (strain TGAN) and type 2 (strain 890) and cultured in vitro for 4 days. Supernatants of cultured cells were collected and saved for interferon gamma (IFNgamma) indirect enzyme-linked immunosorbent assay (ELISA). Four-color flow cytometry was performed to stain and identify cultured PBMC for three T cell surface markers (CD4, CD8, and gammadelta TCR) and to detect the activation marker CD25 (alpha chain of IL-2 receptor) expression. The net increase in %CD25+ cells (Delta%CD25+) of each T cell subset of individual cattle was calculated. The results of all post-vaccination weeks of each animal were plotted and the areas under the curve of each T cell subset were statistically analyzed and compared between groups. The mean area under the curve of the Delta%CD25+ data for days 0-70 of all subsets, except CD4-CD8+gammadelta TCR- (cytotoxic) T cell subset of both BVDV types 1 and 2 stimulated cells, of the vaccinated group were significantly higher than the control group (P<0.05). IFNgamma production by PBMC from the vaccinated group showed significantly higher results (P<0.05) than the control group in the BVDV types 1 and 2 stimulated cells for at least some time points after vaccination. The vaccinated group also had significantly (P<0.0001) higher neutralizing antibody titers than the control group from day 28 onward.     

Effect of aflatoxin on the humoral and cell-mediated immune systems of the chicken.

Chickens fed 2.5 microgram of aflatoxin/g of diet from 2 to 4 weeks of age or from hatching to 4 weeks were deficient in cell-mediated immunity, as measured at 4 weeks of age by the graft-versus-host reaction. Delayed-type hypersensitive skin reactions to tuberculin were also reduced in chickens given dietary aflatoxin from hatching to 7 weeks of age. Humoral immunity, as measured by the ability of 4-week-old chicks to produce natural agglutinins to rabbit red blood cells, was not significantly altered by dietary aflatoxin. A significant decrease in concentrations of serum immunoglobulins (Ig) IgG and IgA, but no IgM, however, did occur in chicks given dietary aflatoxin from hatching to 4 weeks or between 2 and 4 weeks of age. Aflatoxin consumption from 0 to 2 weeks of age produced no marked effect on either cell-mediated or humoral immunity in 4-week-old chicks.     

Temporal cell-mediated immune responses of cattle following experimental and natural exposure to living Brucella abortus.

A study on cell-mediated immune responses in cattle with different exposure experiences to Brucella abortus was conducted by an in vitro lymphocyte stimulation assay. The purpose of this study was to determine how soon the cell-mediated immune responses would be detected following experimental exposure to B. abortus and to study the cell-mediated immune trend following experimental and natural exposure of cattle to B. abortus. The first positive cell-mediated immune responses occurred one to two weeks after experimental inoculation with living B. abortus strain 2308. The cell-mediated immune responses in these animals appeared at least one week before the appearance of of B. abortus serum agglutinating antibodies. Animals which were naturally infected with B. abortus biotypes 1 and 2 demonstrated positive cell-mediated immune responses throughout the study.     

Humoral and cell-mediated immune response to crude antigens of Dermatophilus congolensis during experimental infection of rabbits.

Rabbits were infected with Dermatophilus congolensis and tested for humoral immune response by indirect haemagglutination and for cell-mediated immune response to crude antigens of D. congolensis. Lymphocyte transformation and macrophage migration inhibition assays were used as in vitro correlates of cell-mediated immune response while cutaneous delayed hypersensitivity was used in vivo. Endo-antigen and whole cell antigen were found to significantly induce cell-mediated immune response. In contrast, humoral responses were found to be more significantly induced by exo-antigen. A biphasic immune response was revealed by the lymphocyte transformation test.     

Production of murine cytotoxic T lymphocytes by bluetongue virus following various immunisation procedures

The induction of bluetongue virus specific cytotoxic T lymphocytes (CTLs) in C3H mice by various live and inactivated bluetongue virus preparations was studied. Live virus preparations were shown to induce good levels of CTLs; however, inactivation of virus preparations either by beta propriolactone or glutaraldehyde induced only a low level response. The use of Freund's adjuvants and double immunisation procedures failed to improve the response of the inactivated preparations. These findings are discussed in relationship to protection from bluetongue disease with various bluetongue virus vaccines.     

Potentiation of cell-mediated immune responses against recombinant HN protein of Newcastle disease virus by recombinant chicken IL-18

In this study, recombinant fowlpox viruses (rFPV/HN) expressing Newcastle disease virus (NDV) HN protein and rFPV/HN/chIL-18 co-expressing chicken IL-18 (chIL-18) and HN protein have been constructed and characterized. The co-expressed rHN/chIL-18 antigen or rchIL-18, expressed by our previous construct rFPV/chIL-18 and co-administered with NDV rHN, was assessed for its immunostimulatory activities and protection against NDV challenge in 2-week-old chickens. Chickens were vaccinated, intramuscularly, with various amounts of rHN or rHN/chIL-18 mixed with mineral oil. Production of hemagglutination-inhibition (HI) antibody depended on the concentration of the injected rHN or rHN/chIL-18. The lower HI antibody titers were obtained in chickens group rHN/chIL-18/6 and rHN/chIL-18/7, receiving 50 ng rHN/16.5 ng chIL-18 with mineral oil and 20 ng rHN/6.6 ng chIL-18 with mineral oil, respectively, compared to those in chickens rHN/6 and rHN/7, respectively receiving 50 ng and 20 ng rHN with mineral oil alone. However, the same protection rates were obtained from chickens in groups rHN/chIL-18/6 and rHN/6. Chicken groups rHN/chIL-18/7 and rHN/chIL-18/8 showed higher protective achievements than those in groups rHN/7 and rHN/8, respectively. When rchIL-18 was co-injected with 20ng rHN plus mineral oil, low level of HI antibody titer was produced; whereas, higher level of IFN-γ production and full protection rates were obtained. On the other hand, lower levels of IFN-γ production and lower protection rate (67%) were obtained in chickens injected with the same amount of rHN with mineral oil alone. Similar results were obtained when 10 ng rHN was used. Thus, when the concentration of rHN decreased to 50 ng or less, rchIL-18 reduced HI antibody production. The increase in IFN-γ production suggested that the enhancement of the cell-mediated immunity might confer the protection from NDV challenge, even accompanied with low HI antibody induction.     

Bovine resistance to Amblyomma americanum ticks: an acquired immune response characterized by cutaneous basophil infiltrates

Purebred Holstein calves acquired resistance to Amblyomma americanum adult ticks after 1 infestation and expressed resistance during a challenge infestation 26 days later. Tick yields from resistant animals were normal, but mean tick weight and egg-mass weight were reduced significantly (31 and 32%, respectively). Cutaneous cellular responses at tick feeding sites in primary (naive) hosts consisted almost exclusively of mononuclear cells, until 24-h post-infestation when granulocytes appeared. At 48 h, basophils were the most abundant granulocyte and comprised 19% of the total cellular response. Secondary and tertiary hosts expressed significant cutaneous basophil responses as early as 6 h post-tick infestation, indicative of an anamnestic response. Cutaneous basophil levels in tertiary hosts (16-34% of the infiltrate) were increased by 2-fold over secondary hosts (12-16% of the infiltrate). Neutrophils and eosinophils were fairly abundant in secondary hosts (22-33%, and 3-13%, respectively), but decreased significantly in tertiary hosts corresponding to increased basophil levels. Mast cells comprised less than 10% of the cells found in primary hosts, and less than 5% in secondary and tertiary hosts representing a consistent, but insignificant component of the total cells. Basophils were not observed in the blood of hosts during any tick infestation, but a peripheral blood basophilia (0.1-1.1%) marked the end of each tick feeding period. A neutropenia was observed in the blood of tertiary hosts corresponding to a decreased tissue neutrophil response. Blood eosinophil, lymphocyte and monocyte levels remained unchanged during each tick infestation compared to controls. In summary, calves acquired immunity to A. americanum ticks, resulting in significant decreases in feeding and ovipositional success associated with a local cutaneous basophil response and peripheral blood basophilia. These findings suggest that basophils are involved in bovine immunity to ticks as has been established in the guinea pig model.     

Quantitative analysis of the antigen-specific IFNgamma+ T cell-mediated immune response in conventional outbred pigs: kinetics and duration of the DNA-induced IFNgamma+ CD8+ T cell response

It is now well established that antigen-specific CD8⁺ T cells play a major role in vaccine-induced immunity against intracellular pathogens and tumor cells. The detection of these immune cells in outbred animals has been hampered mainly by the need to generate individual autologous antigen-presenting cells (APCs) due to the high degree of polymorphism of the major histocompatibility complex (MHC) Class I loci. We used individually derived immature porcine dendritic cells infected with a pox-based recombinant viral vector to ex vivo stimulate PBMCs from vaccinated conventional pigs. The frequencies of antigen-specific T cells was determined by the number of IFNγ-secreting cells in a quantitative enzyme-linked immune spot (ELISPOT) assay. Using this approach we were able to rank different pseudorabies virus (PRV) vaccines strategies for their ability to prime viral-specific IFNγ⁺ T cells. Plasmid DNA has recently emerged as a promising tool with multiple applications in the field of infectious diseases, allergy and cancer. We showed for the first time in this study that DNA immunization induced a long-lived antigen-specific IFNγ⁺ T cells response in conventional pigs. Additional studies allowed us to show that these virus-specific IFNγ⁺ responding cells detected in this ELISPOT assay were MHC-restricted and comprised in the CD8^bright pig T cell subset. These new data confirm the usefulness of DNA vaccines to control diseases requiring cellular immunity in pigs.     

Specific suppression of the bursa-dependent immune system of chicks with infectious bursal disease virus.

Chicks which had been inoculated with infectious bursal disease virus (IBDV) at 1 day of age had a severe depression of bursa-dependent humoral immune functions by day 42. Antibody responses against rabbit red blood cells or to immunization with bovine serum albumin were significantly suppressed. In contrast, chicks inoculated with IBDV at 21 days of age produced near normal antibody responses as compared with the responses in noninfected control chicks. The IBDV had no significant effect on the thymus-dependent cellular responses as measured by skin graft rejection or delayed type hypersensitivity reactions to tuberculin.     

Effect of age and pregnancy on the antibody and cell-mediated immune responses of horses to equine herpesvirus 1.

The cell-mediated immune response and antibody response of horses of varying ages and of pregnant horses to equine herpesvirus 1 antigen were examined. Six to eight month old horses showed either no increase or slight increases in anti-equine herpesvirus 1 serum neutralizing antibody following vaccination and revaccination with a modified live equine herpesvirus 1 vaccine. However, these same horses showed a marked increase in the cell-mediated immune response to equine herpesvirus 1 as measured by the lymphocyte transformation test. Eighteen to 21 month old horses showed four to 64-fold increases in anti-equine herpesvirus 1 serum neutralizing antibody titer following vaccination, but the cell-mediated immune response to equine herpesvirus 1 was low or absent. Only after revaccination did they show an increased cell-mediated immune response to equine herpesvirus 1. The cell-mediated immune response of mares in the latter stages of pregnancy to equine herpesivurs 1 was suppressed although antibody titers increased as much as 16-fold following exposure to virulent equine herpesvirus 1.     

Humoral immune response to feline immunodeficiency virus in cats with experimentally induced and naturally acquired infections.

Sera from cats with naturally acquired and experimentally induced feline immunodeficiency virus (FIV) infections were tested by immunoblot analysis, radioimmunoprecipitation assay (RIPA), and a complex trapping/blocking ELISA. In sequentially obtained samples from experimentally inoculated cats, antibodies against the envelope protein gp120 and the core protein p15 were the first to appear, as indicated by results of RIPA, using lysates of FIV-infected lymphocytes. Antibodies could be detected as early as 2 weeks after infection, followed by a response against p24, p43, and p50. By immunoblot analysis, p24 and p15 were the first proteins detectable between postinoculation weeks 3 and 5; an anti-envelope response was never found by use of this assay, but was found by RIPA. Using the latter test, most sera of naturally infected cats were found to recognize the major core protein p24 in addition to 1 or more minor core proteins. All 40 sera tested precipitated the envelope protein; 3 reacted exclusively with it. A complex trapping/blocking ELISA was developed to quantitate the anti-p24 response. Sera from healthy FIV-infected cats were shown to have higher anti-p24 titer than did those from diseased cats.     

Target recognition by bovine mononuclear, MHC-unrestricted cytotoxic cells.

A population of bovine non B/non T, cytotoxic lymphocytes with natural killer activity against virus-infected and non-infected embryonic kidney cells was functionally characterized. The data obtained in experiments of flow cytometry and immuno-peroxidase staining show that a CD2-, CD4-, CD8-, TcR gamma delta-, CD3+, CD45+, FcR+ lymphoid killer cell does exist within bovine peripheral blood leucocytes. This population can detect the down-regulation of class I MHC antigens or the expression of embryonic forms thereof, as shown by experiments of 17-hour 51Cr release and binding to target cells. This model was tested in vitro in experiments on virus-infected bovine kidney cells. The emerging picture was substantially in agreement with the "missing self" theory as a possible option for target cell recognition. In this respect, the profound alteration of MHC Class I expression could represent a major early event, recognized on virus-infected cells by the immune system.     

Evaluation of the recognition of Theileria parva vaccine candidate antigens by cytotoxic T lymphocytes from Zebu cattle

East Coast fever (ECF) is a highly fatal lymphoproliferative disease of cattle caused by Theileria parva, a tick-borne intracellular apicomplexan parasite. Parasite antigens that are targets of protective cytotoxic T lymphocyte (CTL) responses are required to formulate a sub-unit vaccine against ECF. A number of CTL target antigens have recently been identified and initial evaluation has shown their vaccine potential. This study aimed to evaluate whether these antigens were recognised by CTL obtained from six genetically diverse Zebu cattle immunized with a cocktail of T. parva stocks. T. parva Muguga specific polyclonal CD8(+) CTL lines were generated and confirmed to specifically lyse autologous infected cells. CTL recognition of autologous skin fibroblasts (iSF) transduced with recombinant modified vaccinia virus Ankara strain (MVA) expressing previously identified T. parva Muguga vaccine candidate antigens was evaluated using an IFN-gamma ELISpot assay. CTL lines from one of the four calves, BY120, responded specifically to cells infected with MVA expressing the antigen Tp2 and synthetic peptides were employed to map a new CTL epitope on this antigen. Immunoscreening of the T. parva genome with these CTL lines should identify novel antigens that will constitute valuable additions to the vaccine candidates currently being evaluated.