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ABSTRACT: Natural killer (NK) cells have innate antibacterial activity that could be targeted for clinical interventions for infectious disease caused by naturally occurring or weaponized bacterial pathogens. To determine a potential role for NK cells in immunity to Bacillus anthracis, we utilized primary human and murine NK cells, in vitro assays, and in vivo NK cell depletion in a murine model of inhalational anthrax. Our results demonstrate potent antibacterial activity by human NK cells against B. anthracis bacilli within infected autologous monocytes. Surprisingly, NK cells also mediate moderate antibacterial effects on extracellular vegetative bacilli but do not have activity against extracellular or intracellular spores. The immunosuppressive anthrax lethal toxin impairs NK gamma interferon (IFN-γ) expression, but neither lethal nor edema toxin significantly alters the viability or cytotoxic effector function of NK cells. Compared to human NK cells, murine NK cells have a similar, though less potent, activity against intracellular and extracellular B. anthracis. The in vivo depletion of murine NK cells does not alter animal survival following intranasal infection with B. anthracis spores in our studies but significantly increases the bacterial load in the blood of infected animals. Our studies demonstrate that NK cells participate in the innate immune response against B. anthracis and suggest that immune modulation to augment NK cell function in early stages of anthrax should be further explored in animal models as a clinical intervention strategy.
Infection and immunity 01/2012; 80(1):234-42. · 4.21 Impact Factor
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ABSTRACT: Prophylactic administration of CpG oligodeoxynucleotides (CpG ODNs) is known to confer protection against lethal sepsis caused by Burkholderia pseudomallei in the mouse model. The mechanisms whereby CpG regulates the innate immune response to provide protection against B. pseudomallei, however, are poorly characterized. In the present study, we demonstrate that intranasal treatment of mice with Class C CpG, results in recruitment of inflammatory monocytes and neutrophils to the lung at 48 h post-treatment. Mice infected with B. pseudomallei 48 h post-CpG treatment had reduced organ bacterial load and significantly altered cytokine and chemokine profiles concomitant with protection as compared to control animals. CpG administration reduced the robust production of chemokines and pro-inflammatory cytokines in blood, lung and spleen, observed following infection of non-treated animals. Death of control animals coincided with the time of peak cytokine production (day 1-3), while a moderate; sustained cytokine production in CpG-treated animals was associated with survival. In general, CpG treatment resulted in diminished expression of cytokines and chemokines post-infection, though IL-12p40 was released in larger quantities in CpG treated animals. In contrast to CpG-treated animals, the lungs of infected control animals were infiltrated with leukocytes, especially neutrophils, and large numbers of necrotic lesions were observed in lung sections. Therapeutic treatment of B. pseudomallei-infected animals with CpG at 24 h post-infection did not impact survival compared to control animals. In summary, protection of CpG-treated animals was associated with recruitment of inflammatory monocytes and neutrophils into the lungs prior to infection. These responses correspond with early control of bacterial growth, a dampened inflammatory cytokine/chemokine response, reduced lung pathology, and greatly increased survival. In contrast, a delay in recruitment of inflammatory cell populations, despite a robust production of pro-inflammatory cytokines, was associated with poorly controlled bacterial growth, severe lung pathology, and death of control animals.
PLoS ONE 01/2012; 7(3):e34176. · 4.09 Impact Factor
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Tuberculosis (Edinburgh, Scotland) 12/2011; 91 Suppl 1:S1-2. · 2.54 Impact Factor
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Jian Sha, Janice J Endsley,
Michelle L Kirtley,
Sheri M Foltz,
Matthew B Huante,
Tatiana E Erova,
Elena V Kozlova,
Vsevolod L Popov,
Linsey A Yeager,
Irina V Zudina,
Vladimir L Motin,
Johnny W Peterson,
Kristin L DeBord,
Ashok K Chopra
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ABSTRACT: We evaluated two commercial F1 antigen capture-based immunochromatographic dipsticks, Yersinia Pestis (F1) Smart II and Plague BioThreat Alert test strips, in detecting plague bacilli by using whole-blood samples from mice experimentally infected with Yersinia pestis CO92. To assess the specificities of these dipsticks, an in-frame F1-deficient mutant of CO92 (Δcaf) was generated by homologous recombination and used as a negative control. Based on genetic, antigenic/immunologic, and electron microscopic analyses, the Δcaf mutant was devoid of a capsule. The growth rate of the Δcaf mutant generally was similar to that of the wild-type (WT) bacterium at both 26 and 37 °C, although the mutant's growth dropped slightly during the late phase at 37 °C. The Δcaf mutant was as virulent as WT CO92 in the pneumonic plague mouse model; however, it was attenuated in developing bubonic plague. Both dipsticks had similar sensitivities, requiring a minimum of 0.5 μg/ml of purified F1 antigen or 1 × 10(5) to 5 × 10(5) CFU/ml of WT CO92 for positive results, while the blood samples were negative for up to 1 × 10(8) CFU/ml of the Δcaf mutant. Our studies demonstrated the diagnostic potential of two plague dipsticks in detecting capsular-positive strains of Y. pestis in bubonic and pneumonic plague.
Journal of clinical microbiology 03/2011; 49(5):1708-15. · 4.16 Impact Factor
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ABSTRACT: Leishmania (subgenus Viannia) braziliensis is the causative agent of mucocutaneous leishmaniasis (ML) in South America, and ML is characterized by excessive T- and B-cell responses to the parasite. We speculate that the unbalanced production of inflammatory mediators in response to L. braziliensis infection contributes to cell recruitment and disease severity. To test this hypothesis, we first examined the response of peripheral blood mononuclear cells (PBMCs) from healthy volunteers to L. braziliensis infection. We observed that while L. braziliensis infection induced the production of chemokine (C-X-C motif) ligand 10 (CXCL10) and interleukin-10 (IL-10) in human PBMCs and macrophages (MPhis), enhanced expression of CXCL10 and its receptor, chemokine CXC receptor (CXCR3), was predominantly detected in CD14(+) monocytes. The chemoattractant factors secreted by L. braziliensis-infected cells were highly efficient in recruiting uninfected PBMCs (predominantly CD14(+) cells) through Transwell membranes. Serum samples from American tegumentary leishmaniasis (ATL) patients (especially the ML cases) had significantly higher levels of CXCL10, CCL4, and soluble tumor necrosis factor (TNF) receptor II (sTNFRII) than did those of control subjects. Our results suggest that, following L. braziliensis infection, the production of multiple inflammatory mediators by the host may contribute to disease severity by increasing cellular recruitment.
Infection and immunity 11/2009; 78(1):301-8. · 4.21 Impact Factor
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ABSTRACT: The development of improved vaccines against tuberculosis (TB) is directly linked to the investigation of new and better correlates of protection after vaccination against TB. Cloning and characterization of bovine homologues of the antimicrobial protein granulysin (Bo-lysin) and perforin by our group could be used as potential biomarkers for TB vaccination efficacy. In the present study, we examined the kinetics of granulysin, perforin, IFNgamma and Fas-L responses to Mycobacterium bovis purified protein derivative (PPD) stimulation by peripheral blood mononuclear cells from M. bovisDeltaRD1-, BCG- and non-vaccinated cattle. Gene expression profiles following PPD stimulation showed significant increases in transcripts for granulysin and IFNgamma in both CD4(+) and CD8(+) T cells in BCG-vaccinated as compared with non-vaccinated animals. Perforin and IFNgamma examined by flow cytometry, showed a difference of 1-2% more PPD-specific cells in BCG-vaccinated than non-vaccinated animals. In the vaccine trial, granulysin and perforin were significantly increased in both vaccine groups as compared with control after vaccination and challenge. IFNgamma expression was increased only after vaccination and secretion was higher in the control, non-protected group as compared with both vaccine groups demonstrating no correlation with protection upon vaccination. In summary, results shown here provide evidence that granulysin and perforin are prospective candidates as biomarkers of protection after vaccination against TB.
Transboundary and Emerging Diseases 04/2009; 56(6-7):228-39. · 1.81 Impact Factor
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ABSTRACT: Granulysin is a cationic protein produced by human T cells and natural killer cells that can kill bacterial pathogens through disruption of microbial membrane integrity. Herein we demonstrate antimicrobial activity of the granulysin peptide derived from the active site against Bacillus anthracis, Yersinia pestis, Francisella tularensis, and Burkholderia mallei, and show pathogen-specific differences in granulysin peptide effects. The susceptibility of Y. pestis to granulysin is temperature dependent, being less susceptible when grown at the flea arthropod vector temperature (26 degrees C) than when grown at human body temperature. These studies suggest that augmentation of granulysin expression by cytotoxic lymphocytes, or therapeutic application of granulysin peptides, could constitute important strategies for protection against select agent bacterial pathogens. Investigations of the microbial surface molecules that determine susceptibility to granulysin may identify important mechanisms that contribute to pathogenesis.
The Open Microbiology Journal 02/2009; 3:92-6.
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ABSTRACT: Arenaviruses are important emerging pathogens and include a number of hemorrhagic fever viruses classified as NIAID category A priority pathogens and CDC potential biothreat agents. Infection of guinea pigs with the New World arenavirus Pichindé virus (PICV) has been used as a biosafety level 2 model for the Lassa virus. Despite continuing research, little is known about the molecular basis of pathogenesis, and this has hindered the design of novel antiviral therapeutics. Modulation of the host response is a potential strategy for the treatment of infectious diseases. We have previously investigated the global host response to attenuated and lethal arenavirus infections by using high-throughput immunoblotting and kinomics approaches. In this report, we describe the differential nuclear proteomes of a murine cell line induced by mock infection and infection with attenuated and lethal variants of PICV, investigated by using two-dimensional gel electrophoresis. Spot identification using tandem mass spectrometry revealed the involvement of a number of proteins that regulate inflammation via potential modulation of NF-kappaB activity and of several heterogeneous nuclear ribonuclear proteins. Pathway analysis revealed a potential role for transcription factor XBP-1, a transcription factor involved in major histocompatibility complex II (MHC-II) expression; differential DNA-binding activity was revealed by electrophoretic mobility shift assay, and differences in surface MHC-II expression were seen following PICV infection. These data are consistent with the results of several previous studies and highlight potential differences between transcriptional and translational regulation. This study provides a number of differentially expressed targets for further research and suggests that key events in pathogenesis may be established early in infection.
Journal of Virology 12/2008; 83(2):687-700. · 5.40 Impact Factor
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Janice J Endsley,
W Ray Waters,
Mitchell V Palmer,
Brian J Nonnecke,
Tyler C Thacker,
William R Jacobs,
Michelle H Larsen,
Alison Hogg,
Elisabeth Shell,
Martin McAlauy,
Charles F Capinosh Scherer,
Tracey Coffey,
Chris J Howard,
Bernardo Villareal-Ramos,
D Mark Estes
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ABSTRACT: Tuberculosis (TB) remains a major threat to public health. The identification of safe TB vaccine candidates beyond Mycobacterium bovis BCG, is an exciting prospect for control of human TB and necessary in the context of the human immunodeficiency virus (HIV) pandemic. Selection of vaccine candidates for human trials which are ultimately targeted for use in children less than 5 years of age or in newborns will require an animal model that closely approximates immune function and disease. We propose that the bovine neonate and adolescent is a robust animal model for preclinical safety and efficacy evaluation of TB candidate vaccines targeting this special human population. Parallel studies conducted in bovine neonates and non-human primates with a leading auxotrophic mutant with demonstrated efficacy/safety in a rodent TB model of TB demonstrated similar findings with respect to gross pathology scoring relative to BCG. The findings indicated more numerous and severe lesions in the lung in addition to higher levels of IFN-gamma producing cells. BCG vaccinates demonstrated higher levels of FoxP3 transcripts and lower levels of IL-4 mRNA.
Veterinary Immunology and Immunopathology 11/2008; 128(1-3):199-204. · 2.08 Impact Factor
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Janice J Endsley,
Alison Hogg,
Lis J Shell,
Martin McAulay,
Tracey Coffey,
Chris Howard,
Charles F Capinos Scherer,
W Ray Waters,
Brian Nonnecke,
D Mark Estes,
Bernardo Villarreal-Ramos
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ABSTRACT: The effector mechanisms used by CD4+ T cells to control mycobacteria differ between humans and rodent models of TB and should be investigated in additional animal models. In these studies, the bovine model was used to characterize the mycobactericidal CD4+ T cell response induced by vaccination with the attenuated Mycobacterium bovis bacillus Calmette-Guérin (BCG). Antigenic stimulation of peripheral blood CD4+ T cells from BCG-vaccinated cattle enhanced expression of perforin and IFNgamma in cells expressing a CD45RA-CD45RO+CD62L+ cell surface phenotype, enhanced transcription of granulysin, IFNgamma, perforin, IL-4, IL-13, and IL-21, and enhanced anti-mycobacterial activity of CD4+ T cells against BCG-infected macrophages.
Vaccine 01/2008; 25(50):8384-94. · 3.77 Impact Factor
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ABSTRACT: Bovine natural killer (NK) cells were recently identified by positive selection of a NK cell-activating receptor p46 (NKp46)+ CD3- lymphocyte population, which expresses CD25 and CD8 and lyses tumor cell lines following stimulation with recombinant interleukin-2. In the current work, we characterize the cytotoxic/effector potential of a CD3(-)CD8(-)CD11b- population isolated through negative selection of bovine peripheral blood leukocytes. This population is CD25(lo)CD62(hi) when isolated and becomes CD25hiCD62L(lo) following cytokine stimulation. Activated bovine NK cells increase expression of granulysin, interferon-gamma, and perforin and have cytotoxic activity against human tumor cells and Mycobacterium bovis bacillus Calmette-Guerin-infected alveolar and monocyte-derived macrophages. Expression of a bovine homologue of the CD56 neural adhesion molecule expressed by human NK cells was detected in mRNA from brain tissue but was not detected in peripheral blood mononuclear cells or purified NK cell mRNA. Analysis of mRNA from nonstimulated peripheral blood NK cells demonstrates the constitutive expression of homologues of human NK receptors NKp46, CD244, and CD94 and the granule proteins granulysin and perforin. Phorbol ester-stimulated CD8+ T cells also expressed CD244 and CD94, and CD4+ T cells expressed CD94. These NK cell receptors bearing T lymphocytes may represent memory subsets characterized in humans. The results of these studies demonstrate that bovine NK cells may play an important role in the innate immune responses of cattle.
Journal of Leukocyte Biology 02/2006; 79(1):71-9. · 4.99 Impact Factor
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ABSTRACT: Granulysin and NK-lysin are antimicrobial proteins found in the granules of human and swine cytotoxic lymphocytes. A murine counterpart to granulysin has not been identified to date, indicating the importance of additional models to fully characterize the role of granulysin-like molecules in the immune response to infectious disease. Two partial nucleotide sequences corresponding to the complete functional domain of granulysin and NK-lysin were amplified from bovine PBMC mRNA. Following stimulation with phorbol ester and calcium ionophore, expression of the bovine gene was detected in CD3(+) T cells, CD4(+) T cells, CD8(+) T cells, WC1(+) gammadelta T cells, and PBMC depleted of CD3(+) T cells, but was absent in CD21(+) cells and CD14(+) cells. Intracellular flow cytometry and immunoblotting confirmed the presence of protein corresponding to the bovine granulysin homologue in activated T lymphocytes and PBMC. Synthetic human, bovine, and swine peptides corresponding to the C terminus of helix 2 through helix 3 region of granulysin displayed potent antimicrobial activity against Escherichia coli, Salmonella enteritidis, Staphylococcus aureus, and Mycobacterium bovis bacillus Calmette-Guérin. Human and bovine peptides corresponding to helix 2 displayed antimycobacterial activity against M. bovis bacillus Calmette-Guérin. Expression of the bovine gene was detected in laser microscopy-dissected lymph node lesions from an M. bovis-infected animal. The identification of a biologically active bovine homologue to granulysin demonstrates the potential of the bovine model in characterizing the role of granulysin in the immune response to a variety of infectious agents.
The Journal of Immunology 09/2004; 173(4):2607-14. · 5.79 Impact Factor
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ABSTRACT: Passive antibody to bovine viral diarrhea virus (BVDV) acquired through colostrum intake may interfere with the development of a protective immune response by calves to this virus. The objective of this study was to determine if calves, with a high level of maternal antibody to bovine viral diarrhea virus (BVDV), develop CD4(+), CD8(+), or gammadelta T lymphocyte responses to BVDV in the absence of a measurable humoral immune response. Colostrum or milk replacer fed calves were challenged with virulent BVDV at 2-5 weeks of age and/or after maternal antibody had waned. Calves exposed to BVDV while passive antibody levels were high did not mount a measurable humoral immune response to BVDV. However, compared to nonexposed animals, these animals had CD4(+), CD8(+), and gammadelta T lymphocytes that were activated by BVDV after exposure to in vitro BVDV. The production of IFNgamma by lymphocytes after in vitro BVDV exposure was also much greater in lymphocytes from calves exposed to BVDV in the presence of maternal antibody compared to the nonexposed calves. These data indicate that calves exposed to BVDV while maternal antibody levels are high can develop antigen specific CD4(+), CD8(+), and gammadelta T lymphocytes in the absence of an active antibody response. A manuscript presented separately demonstrates that the calves with T lymphocytes specific for BVDV in this study were also protected from virulent BVDV genotype 2 challenge after maternal antibody became undetectable.
Viral Immunology 02/2004; 17(1):13-23. · 1.97 Impact Factor
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ABSTRACT: Bovine viral diarrhoea virus (BVDV) contributes significantly to health-related economic losses in the beef and dairy industry. Antibodies of maternal origin can be protective against BVDV infection, however, calves with low titres of maternal antibody or that do not receive colostrum may be at risk for acute BVDV infection. Interference by high titres of maternal antibodies prevents the development of an antibody response following vaccination with either a killed or attenuated BVDV vaccine. However, the T cell mediated immune response to BVDV may be generated in the absence of a detectable serum neutralizing antibody response. Two trials were conducted to evaluate the potential to elicit T cell mediated immune responses to BVDV in calves with circulating maternal antibody to BVDV. In the first trial, calves with high levels of circulating maternal antibody to BVDV 1 and BVDV 2 were experimentally infected with BVDV 2 (strain 1373) at two to five weeks of age. The T-cell mediated immune responses of the experimentally infected calves and non-infected calves were monitored monthly until circulating maternal antibody was no longer detectable in either treatment group. Calves experimentally infected with BVDV developed BVDV specific CD4(+), CD8(+), and delta T cell responses while high levels of maternal antibody were circulating. A second challenge with BVDV 2 (strain 1373) was performed in the experimentally infected and control calves once maternal antibody could no longer be detected. Previous exposure to BVDV in the presence of maternal antibody protected calves from clinical signs of acute BVDV infection compared to the control calves. In the second trial, three groups of calves with circulating maternal antibody to BVDV were given either a modified live vaccine (MLV) containing BVDV 1 and BVDV 2, a killed vaccine containing BVDV 1 and BVDV 2, or no vaccine, at seven weeks of age. Serum neutralizing antibody levels and antigen specific T cell responses were monitored for 14 weeks following vaccination. Calves vaccinated with MLV BVDV developed BVDV 1 and BVDV 2 specific CD4(+)T cell responses, and BVDV 2 specific gammadelta T cell responses, in the presence of maternal antibody. Vaccination with killed BVDV did not result in the generation of measurable antigen specific T cell immune responses. In this trial, a second vaccination was performed at 14 weeks to determine whether an anamnestic antibody response could be generated when calves were vaccinated in the presence of maternal antibody. Calves vaccinated with either a MLV or killed BVDV vaccine while they had maternal antibody developed an anamnestic antibody response to BVDV 2 upon subsequent vaccination. The results of these trials indicate that vaccinating young calves against BVD while maternal antibody is present may generate BVDV specific memory T and B cells. The data also demonstrated that seronegative calves with memory T and B cells specific for BVDV may be immune to challenge with virulent BVDV.
Biologicals 07/2003; 31(2):123-5. · 1.70 Impact Factor
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ABSTRACT: Expression of CD25 (interleukin-2 receptor alpha chain) was used to monitor antigen-specific activation of T lymphocyte subsets (CD4+, CD8+, and gamma delta T cells) from cattle immunized with modified-live virus (MLV) bovine viral diarrhea virus (BVDV) vaccines. Two groups of 15 animals each were vaccinated with one dose of either BVDV genotype 1 (BVDV-1) or BVDV-1 and BVDV genotype 2 (BVDV-1/2). Six animals negative for both BVDV antibody and BVDV virus were used as negative controls. Three animals vaccinated 7 and 5 weeks before the start of the experiment with MLV BVDV-1 vaccine served as positive controls. Blood samples were taken from the negative control group, the positive control group, and the BVDV-1/2 group 0, 21, 35, 60, and 90 days after vaccination. Blood samples were taken from the BVDV-1 group 0, 21, and 90 days after vaccination. Isolated peripheral blood lymphocytes from immunized and control animals were incubated for 5 days with and without BVDV-1 or BVDV-2. Compared with nonvaccinated animals, a significant (P <.05) increase in expression of CD25 by CD4+ (60 days), CD8+, and gammadelta T (35 to 90 days) lymphocytes from the group given BVDV-1/2 was detected following in vitro exposure to BVDV-1 or BVDV-2 after vaccination. The CD8+ and gammadelta T cells from the group vaccinated with BVDV-1 had significantly (P <.05) increased expression of CD25 compared with nonvaccinates following postvaccination exposure to in vitro BVDV-1 but not to BVDV-2. There was no significant difference between the two vaccinated groups in CD25 expression on any of the T cell subsets in response to BVDV-1 or BVDV-2 exposure. A single administration of MLV BVDV vaccine may be more effective at stimulating CD8+ and gammadelta T cell-specific immune responses to the homologous genotype than to the heterologous genotype.
Veterinary therapeutics: research in applied veterinary medicine 01/2002; 3(4):364-72. · 1.69 Impact Factor
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ABSTRACT: Expression of the high-affinity interleukin 2 receptor alpha chain (CD25) was used to monitor antigen-specific activation of T lymphocyte subsets (CD4+, CD8+, and gammadelta T cells) from cattle immunized with modified live bovine herpesvirus-1 (BHV-1). Calves seronegative for BHV-1 were either vaccinated with one dose of modified live vaccine containing BHV-1 or not vaccinated to serve as negative controls. Two animals vaccinated 7 and 5 weeks before the start of the experiment with two doses of modified live vaccine containing BHV-1 served as positive controls. Blood samples were taken from the nonvaccinate group, the positive control group, and the vaccinate group at 0, 21, 35, 60, and 90 days postinoculation (PI). Isolated peripheral blood mononuclear cells from immunized and control animals were incubated for 5 days with and without live BHV-1 ISU99. Compared to the nonvaccinates, a significant (p < 0.05) increase in expression of CD25 by CD4+, CD8+, and gammadelta T lymphocytes from the vaccinate group was detected following in vitro exposure to live BHV-1 after vaccination. This is apparently the first report using live BHV-1 to stimulate lymphocytes in vitro and showing CD8+ T cell activation. Peripheral blood from the positive control animals was depleted of CD4+, CD8+, or gammadelta T lymphocytes prior to incubation with BHV-1 to assess bystander activation in the CD25 expression assay. When incubated with live BHV-1, depletion of CD4+ T cells depressed the expression of CD25 by CD8+ T cells, but not gammadelta T cells. Depleting CD8+ or gammadelta T cells prior to in vitro culture with BHV-1 did not affect the expression of CD25 by the remaining T lymphocyte subsets. Vaccinates were protected from challenge with virulent BHV-1 at 110 days postvaccination compared to nonvaccinates. Expression of CD25 appears to be a useful marker for evaluating induction of antigen-specific T lymphocyte subset responses following vaccination.
Viral Immunology 01/2002; 15(2):385-93. · 1.97 Impact Factor
