Antigen-independent priming: a transitional response of bovine gammadelta T-cells to infection

Analysis of global gene expression in immune cells has provided unique insights into immune system function and response to infection. Recently, we applied microarray and serial analysis of gene expression (SAGE) techniques to the study of gammadelta T-cell function in humans and cattle. The intent of this review is to summarize the knowledge gained since our original comprehensive studies of bovine gammadelta T-cell subsets. More recently, we have characterized the effects of mucosal infection or treatment with microbial products or mitogens on gene expression patterns in sorted gammadelta and alphabeta T-cells. These studies provided new insights into the function of bovine gammadelta T-cells and led to a model in which response to pathogen-associated molecular patterns (PAMPs) induces 'priming' of gammadelta T-cells, resulting in more robust responses to downstream cytokine and/or antigen signals. PAMP primed gammadelta T-cells are defined by up-regulation of a select number of cytokines, including MIP1alpha and MIP1beta, and by antigens such as surface IL2 receptor alpha (IL-2Ralpha) and CD69, in the absence of a prototypic marker for an activated gammadelta T-cell, IFN-gamma. Furthermore, PAMP primed gammadelta T-cells are more capable of proliferation in response to IL-2 or IL-15 in the absence of antigen. PAMPs such as endotoxin, peptidoglycan and beta-glucan are effective gammadelta T-cell priming agents, but the most potent antigen-independent priming agonists defined to date are condensed oligomeric tannins produced by some plants.     

Changes in local IFN-gamma and TGF-beta4 mRNA expression and intraepithelial lymphocytes following Eimeria acervulina infection

Inbred chickens SC (B2B2) and TK (B15B21) display different levels of susceptibility to Eimeria acervulina infection. Following primary and secondary infections, SC chickens showed significantly lower oocyst production compared to TK chickens. Both strains produce significantly fewer oocysts during secondary infection (si) indicating that a protective host immune response had developed subsequent to primary infection (pi). To elucidate the immunologic differences between SC and TK chickens that may account for their different levels of disease susceptibility, cellular and molecular parameters of intestinal immunity were compared. CD4 T-lymphocytes increased significantly and more rapidly post-pi and si in SC relative to TK chickens during the later stages of infections. However, later during the infections, CD4 cells were higher in TK compared to SC chickens. Although the percentage of CD8 lymphocytes increased in both strains after pi, following si the percentage of these cells continued to increase in SC chickens but showed a marked decrease in TK chickens. Contrary to the effects on CD4 cells, the percentage of TCR1 cells was higher in TK chickens early after pi while the same cell subset was higher in SC chickens later following infection. The percentages of TCR2 cells were significantly higher in both strains following pi. At the molecular level, IFN-gamma mRNA expression in caecal tonsils and splenic lymphocytes was generally higher in SC compared to TK chickens following E. acervulina infection, while intraepithelial lymphocytes from the duodenum demonstrated reduced levels of this cytokine in both the strains, particularly following pi. TGF-beta4 mRNA levels generally increased in lymphocytes from the caecal tonsils, spleen and duodenum from both the strains. These differences in lymphocyte subpopulations and cytokine mRNA expression between SC and TK chickens following E. acervulina infection indicate a complex genetic control of the native immune response to coccidiosis.     

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.     

Effects of live attenuated and killed Salmonella vaccine on T-lymphocyte mediated immunity in laying hens

The impact of live and killed Salmonella vaccines on cell-mediated immunity (CMI) was investigated in 18- and 32-week-old White Leghorn chickens, by assessing splenic lymphocyte proliferation, expression of IL-2 mRNA in concanavalin A (Con A) stimulated cells and flow cytometric analysis of cell subpopulations. Con A and Salmonella enteritidis (SE) flagella induced proliferation of splenocytes were enhanced in the 18- and 32-week-old chickens treated with live vaccine, compared to the corresponding control chickens. Among the killed vaccine treated birds, Con A-mediated response was higher in the 18-week-old chickens compared to the corresponding control birds. Increased proliferation was accompanied by increased CD4 and reduced CD8 and gammadelta T-lymphocytes in the 18-week-old live vaccine treated chickens. Relative expression of IL-2 mRNA in Con A-stimulated splenocytes from 18-week-old birds was not affected by vaccine treatment. Overall, live vaccine was more effective in increasing the lymphocyte proliferation to Con A as well as SE antigen. This enhanced CMI may prove beneficial in protecting chickens against SE infection.     

Resistance to intestinal coccidiosis following DNA immunization with the cloned 3-1E Eimeria gene plus IL-2, IL-15, and IFN-gamma

A cloned Eimeria acervulina gene (3-1E) was used to vaccinate chickens in ovo against coccidiosis, both alone and in combination with genes encoding interleukin (IL)-1, IL-2, IL-6, IL-8, IL-15, IL-16, IL-17, IL-18, or interferon (IFN)-gamma. Vaccination efficacy was assessed by increased serum anti-3-1E antibody titers, reduced fecal oocyst shedding, and enhanced body weight gain following experimental infection with E. acervulina. When used alone, anti-3-1E antibody titers were transiently, but reproducibly, increased at 2 wk and 3 wk posthatching in a dose-dependent manner. Similarly, significantly reduced oocyst shedding and increased weight gain were observed at relatively high-dose 3-1E vaccinations (> or =25 microg/egg). Combined immunization with the 3-1E and IL-1, IL-2, IL-15, or IFN-gamma genes induced higher serum antibody responses compared with immunization with 3-1E alone. Following parasite infection, chickens hatched from embryos given the 3-1E gene plus the IL-2 or IL-15 genes displayed significantly reduced oocyst shedding compared with those given 3-1E alone, while 3-1E plus IL-15 or IFN-gamma significantly increased weight gain compared with administration of 3-1E alone. Taken together, these results indicate that in ovo immunization with a recombinant Eimeria gene in conjunction with cytokine adjuvants stimulates protective intestinal immunity against coccidiosis.     

Immune responses to an Eimeria acervulina infection in different broilers lines

The (T-cell) immune responses of two different broiler lines to a primary Eimeria acervulina infection were investigated. The lines used were a commercial fast-growing broiler line and a slow-growing type of broiler as used in organic farming. Seven-day-old broilers of both lines were infected with 5 x 10(4) oocysts of E. acervulina. The animals were weighed and a species-specific real-time PCR was used to quantify the total amount of parasites in the duodenum. In the fast-growing line, a lower parasite load was seen from day 4 onwards compared to the slow-growing line. In both lines the intestinal peak of Eimeria DNA was observed at day 5 post infection (p.i.). In the duodenum no increase in CD4(+) T-cells was found in both infected lines, but a fast increase in CD8(+) T-cells was observed in the fast-growing line. At day 3 p.i. in the slow-growing broilers an IL-18 mRNA response was observed. At day 4 p.i. strong IFN-gamma and IL-8 mRNA responses were found in both lines. No IL-4 mRNA responses were found in the duodenum. In conclusion, both lines have different growth rates and control and infected conditions. Based on the kinetics of observed phenomena a primary infection with E. acervulina in 7-day-old broilers seems to generate an early CD8alpha(+) response in fast-growing broilers compared to the slow-growing broilers. This difference in immune reaction after an E. acervulina infection could result in a different Eimeria load in the duodenum.     

Analysis of monoclonal antibodies reacting with molecules expressed on gammadelta T-cells.

Twenty-six monoclonal antibodies (mAbs) selected after the first round of analysis in the Third International Swine Workshop were grouped with additional mAbs from the first and second workshops and mAbs under study for further evaluation. Preparations of peripheral blood leukocytes were used in single and multicolor flow cytometric (FC) analyses. Six mAbs did not react with gammadelta T-cells. Two were negative for all tested specificities. Seven mAbs recognized molecules expressed on gammadelta T-cells that were not lineage restricted. One of these from the first workshop (2B11) yielded a pattern of labeling identical to a mAb under study (PGB73A). Ten mAbs were characterized in previous workshops and known to react with the gammadelta TCR or molecules expressed on subsets of gammadelta T-cells. One belonged to SWC4, two to SWC5, and one to SWC6. Two mAbs from the second workshop recognized a molecule or molecules expressed on subsets of gammadelta T-cells. A new mAb (PPT16) added late to the workshop following a request by the workshop chairs appeared to recognize a determinant expressed on the gammadelta TCR/CD3 molecular complex.     

Development and characterization of mouse monoclonal antibodies reactive with chicken interleukin-2 receptor αlpha chain (CD25)

This study was carried out to develop and characterize mouse monoclonal antibodies (mAbs) against chicken CD25 (chCD25), the alpha chain of the interleukin-2 (IL-2) receptor. A recombinant chimeric chCD25/IgG4 fusion protein was expressed in Chinese hamster ovary (CHO) cells and isolated from spent cell culture medium by protein G affinity chromatography. Purified chCD25 protein was used to immunize mice, from which 54 stable hybridomas secreting chCD25 mAbs were produced. Two mAbs, chCD25-32 and chCD25-54, with high binding affinity for chCD25-expressing CHO cells were selected for further characterization. By flow cytometry, both mAbs detected cells in the spleen, bursa of Fabricius, intestinal duodenum, and immunostained established chicken T cell, B cell, and macrophage cell lines. Both mAbs reacted with a 55 kDa protein on Western blots of lysates from concanavalin A (Con A)-stimulated spleen mononuclear cells. Intraperitoneal injection of chickens with bacterial lipopolysaccharide increased the percentage of chCD25(+) spleen cells by approximately 4-fold compared with untreated animals. In vitro stimulation of spleen cells with Con A increased the percentage of chCD25(+) cells by up to 50-fold compared with cells treated with medium alone. Finally, the chCD25-32 mAb suppressed IL-2-driven spleen cell proliferation and reduced IL-2-induced nitric oxide production. These mAbs may be useful for future investigation of chicken regulatory T cells.     

Immune responses in Eimeria acervulina infected one-day-old broilers compared to amount of Eimeria in the duodenum, measured by real-time PCR

T-cell responses are supposed to be the major immune reactions in broilers infected with Eimeria. The nature of such T-cell responses is influenced by the species of Eimeria involved, age of the host, amount of parasites and the preceding infection history. In young chicks the intestine is still developing in length while the lymphocyte populations in the gut develop and differentiate. In chicks infected at young age the immune response may be different in quality as compared to responses in adults. We investigated the (T-cell) immune responses of young broilers to a primary Eimeria acervulina infection in relation to the number of parasites used for infection. In our experiment we infected one-day-old broilers with a low (5 x 10(2) oocysts) and a high (5 x 10(4) oocysts) dose of E. acervulina. We used a newly developed species specific real-time PCR to quantify total amount of parasites in the duodenum as the number of oocysts in faeces may not be representative for the exposure of the gut immune system. We characterized T-cell subsets in the duodenum by means of FACS-analyses, lymphocyte proliferation assays with spleen lymphocytes and the mRNA profiles of different cytokines (TGF-beta2, -4, IFN-gamma, IL-2, -6, -8 and -18) in the duodenum by means of real-time PCR. From day 5 p.i. broilers with a high dose of E. acervulina had a significantly lower body weight than the control group. No increase in CD4(+) cells, but a strong increase in CD8(+) cells was observed at days 7 and 9 p.i. in the duodenum of broilers infected with a high dose E. acervulina. IL-8 mRNA responses were observed after infection with low and with high infection doses, but no IFN-gamma and TGF-beta mRNA responses were found in the duodenum. The specific proliferative T-cell responses to a low infectious dose were not significantly different as compared to the control group. In conclusion, based on the kinetics of observed responses a primary infection with a high dose of E. acervulina in one-day-old broilers seems to generate an immune response that shows a peak at the time of oocyst excretion, whereas the immune response to a low dose is less explicit.     

The WC1(+) gammadelta T-cell population in cattle: a possible role in resistance to intracellular infection

Intracellular infections are important in veterinary medicine and detailed understanding of the associated immune responses is needed for optimal development of strategies based on diagnosis and vaccination. It is generally accepted that cell-mediated immune responses are of greatest importance in intracellular infections and recent studies from several bovine models of infection indicate that WC1(+) gammadelta T-cells have a number of possible levels of involvement, which remain incompletely defined. Investigations of experimental infection with Mycobacterium bovis in cattle have indicated that WC1(+) gammadelta T-cells are among the first cells to accumulate at initial sites of infection, an observation which has been linked with decreased numbers of these cells in the circulation within days of infection. These WC1(+) gammadelta T-cells have been shown to respond in vitro, both to protein antigens and to non-protein, phosphate containing antigens of M. bovis and to be capable of producing IFN-gamma. Studies of M. bovis infection in calves depleted of WC1(+) gammadelta T-cells by monoclonal antibody have suggested that the presence of these cells is associated with development of a Th1-biased acquired immune response. In combination, these observations allow speculation regarding a possible role for WC1(+) gammadelta T-cells as a link between the innate and acquired immune systems which is instrumental in establishing an appropriate response.     

Cell-mediated immune responses to a killed Salmonella enteritidis vaccine: lymphocyte proliferation, T-cell changes and interleukin-6 (IL-6), IL-1, IL-2, and IFN-gamma production

Two experimental approaches were used to investigate the immunological responses of chickens to a commercial killed Salmonella enteritidis (SE) vaccine. In the first, the effects of host age on antigen-specific proliferative responses and cytokine production were examined. Compared with non-vaccinated controls, 4-wk-old vaccinated chickens showed higher proliferation to SE LPS and flagella. The lymphoproliferation responses to these antigens of 8-mo-old vaccinated chickens were not different compared to the non-vaccinated controls. Increased production of interferon-gamma (IFN-gamma) and interleukin-2 (IL-2) by antigen-stimulated splenocytes following vaccination were, in general, more often observed in 4-wk-old compared with 8-mo-old chickens, whereas serum levels of these cytokines were consistently higher in the vaccinated birds compared with controls regardless of age. The second set of experiments were designed to determine the effects of SE vaccination on mitogen- or antigen-induced splenocyte proliferation and serum nitric oxide (NO) and cytokine levels. Splenocytes from vaccinated chickens stimulated with SE flagella showed significantly increased numbers of TCRgammadelta+ cells at 7 days post-vaccination compared with non-vaccinated birds. In contrast, no differences were noted with CD4+, CD8+, or TCRalphabeta+ cells at any time points examined. Higher levels of NO production were observed following stimulation with SE flagella at 4, 7, 11, and 14 days after SE vaccination while serum levels of IFN-gamma, IL-1, IL-6, and IL-8 were elevated only at day 7 post-vaccination. In conclusion, younger chickens mounted a more robust antigen-specific immune response to the SE vaccine compared with older birds and vaccination induced not only T-cell-mediated responses but also host innate and pro-inflammatory responses.     

Failure of antigen-stimulated gammadelta T cells and CD4+ T cells from sensitized cattle to upregulate nitric oxide and mycobactericidal activity of autologous Mycobacterium avium subsp. paratuberculosis-infected macrophages

The function of gammadelta T cells during ruminant paratuberculosis (Johne's disease) is presently unknown. An ex vivo system was used to test the hypothesis that gammadelta T cells are capable of activating Mycobacterium avium subsp. paratuberculosis-(M. paratuberculosis)-infected macrophages. Peripheral blood-derived macrophages were infected in vitro with live M. paratuberculosis, and autologous LN-derived gammadelta T cells or CD4+ T cells were co-cultured with infected macrophages for 48h, at which time bacterial survival as well as production of nitrites and IFN-gamma was evaluated. Incubation of M. paratuberculosis-infected macrophages with autologous gammadelta T cells did not result in reduced intracellular bacterial viability compared to infected macrophage cultures without added T cells. IFN-gamma production by-infected cultures containing added gammadelta T cells was not enhanced compared to that of infected macrophages alone. Although infection of macrophage cultures caused increased production of nitrites at both post-infection day (PID) 0 and PID 60, the addition of gammadelta T cells did not further increase nitrite production. In contrast, addition of PPD-stimulated CD4+ T cells obtained at PID 60 to M. paratuberculosis-infected macrophages resulted in significantly increased IFN-gamma production compared to cultures without added T cells or cultures containing unstimulated CD4+ T cells or unstimulated or antigen-stimulated gammadelta T cells. However, the increased production of IFN-gamma by co-cultures containing PPD-stimulated CD4+ T cells did not result in increased bacterial killing or increased production of nitrites compared to cultures without added T cells. In additional in vitro experiments, M. paratuberculosis-infected macrophages, but not uninfected macrophages, were unable to increase nitrite production when stimulated with recombinant IFN-gamma. Taken together, the data suggest that (1) gammadelta T cells do not produce significant IFN-gamma and do not significantly increase NO production from M. paratuberculosis-infected macrophages in vitro, (2) the production of significant IFN-gamma by antigen-stimulated CD4+ T cells from infected calves is insufficient to enhance mycobacterial killing or nitrite production by infected macrophages, and (3) macrophages may have an impaired NO response following intracellular M. paratuberculosis infection, even in the presence of significant concentrations of IFN-gamma.     

Effect of L‐carnitine on proliferative response and mRNA expression of some of its associated factors in splenic mononuclear cells of male broiler chicks

The effect of L‐carnitine supplementation on mitogen (concanavalin A, Con A) induced proliferation of mononuclear cells (MNC) in the spleen was investigated in broiler chickens at different ages. Day‐old chickens were fed a diet supplemented with or without L‐carnitine (100 ppm) for 24 days. The carnitine‐supplemented group showed greater proliferation of MNC in the spleen in response to Con A than that of the control group at 24 days of age. In addition, at 24 days of age the carnitine‐supplemented group showed higher expression of interleukin (IL)‐2 and interferon (IFN)‐γ mRNA, but lower expression of inducible nitric oxide synthase (iNOS) in the Con A‐stimulated splenic MNC than the control group. The enhancement effect of L‐carnitine on MNC proliferation and IL‐2 mRNA expression was not found in chicks at 14 days of age. Addition of L‐carnitine (50 nmol/mL) to the culture medium enhanced proliferation and IL‐2 mRNA expression of splenic MNC obtained from 24‐day‐old but not from 14‐day‐old broiler chickens. The results suggest that L‐carnitine is capable of enhancing MNC proliferation in broiler chickens at 24 days of age partly through increasing IL‐2 and IFN‐γ production and decreasing NO production.     

The presence of CD25 on bovine WC1+ gammadelta T cells is positively correlated with their production of IL-10 and TGF-beta, but not IFN-gamma

WC1+ cells in cattle exhibit both regulatory and effector activities. However, it has not been elucidated whether they are so plastic that both activities co-exist in one cell or there are separate subpopulations of effector and regulatory cells. Since the production of IFN-gamma and IL-10 seems to be related to WC1+ cells' effector and regulatory function, respectively, the main aim of this study was to determine whether those cytokines are produced by separate subpopulations of WC1+, or are co-produced by the same cells. Due to increasingly frequent emphasised role of consumption of IL-2 in the mechanism of suppressor action of mouse CD25+CD4+ T regulatory cells, expression of the receptor's alpha chain for interleukin 2 (CD25) on WC1+ lymphocytes has been evaluated. An average of 5.21% of WC1+ cells obtained from PBMCs of 12-month-old heifers show constitutive expression of the CD25 molecule, with CD25(high)WC1+ and CD25(low)WC1+ cells accounting for 1.05% and 4.10% of WC1+ lymphocytes, respectively. For detection of intracellular cytokine production, PBMCs were stimulated with concanavalin A. Both IFN-gamma(-) and IL-10-producing cells within the CD25(-)WC1+ and CD25+WC1+ subpopulations were mainly separate subpopulations. The average percentage of IFN-gamma(+)IL-10(-), IFN-gamma(-)IL-10+ and IFN-gamma(+)IL-10+ cells among CD25(-)WC1+ lymphocytes was 4.03%, 2.67% and 0.51%, respectively. A positive correlation was observed between the presence of the CD25 molecule on WC1+ lymphocytes and production of IL-10 and TGF-beta, because the average percentage of IFN-gamma(-)IL-10+ and IFN-gamma(+)IL-10+ among CD25+WC1+ lymphocytes was 3 and 4.5 times higher as compared to the corresponding cells in the CD25(-)WC1+ subpopulation, whereas the percentage of IFN-gamma(+)IL-10(-) cells in both the subpopulations was not significantly different. The percentage of TGF-beta+ cells within the CD25+WC1+ subpopulation was 2.72 times as high as that of CD25(-)WC1+ lymphocytes. Therefore, with respect to the production of IFN-gamma, IL-10 and TGF-beta, CD25+WC1+ lymphocytes turn out to have a more suppressor profile than CD25(-)WC1+.