Philippe Totté

Recombinant bovine interferon gamma inhibits the growth of Cowdria ruminantium but fails to induce major histocompatibility complex class II following infection of endothelial cells

Recombinant bovine IFN gamma is a potent inhibitor of Cowdria ruminantium growth in vitro irrespective of the rickettsial stock, or the origin of the endothelial cells. These results suggest an important role for IFN gamma in protective immune responses against C. ruminantium infections. Here we also show that IFN gamma can induce the expression of MHC class II molecules on the surface of endothelial cells. However, treatment of endothelial cells with IFN gamma following infection with Cowdria fails to induce MHC class II expression. The implications of this pathogen-specific effect on class II expression by endothelial cells with regard to its recognition by the host immune system are discussed.

Immune Responses to Cowdria ruminantium Infections

Understanding the basis of protective immunity to Cowdria ruminantium will facilitate the development of an effective subunit vaccine against heartwater in ruminants and contribute to a better definition of protective immune mechanisms to obligate intracellular pathogens in general. Until recently, immunological studies of heartwater in ruminants concentrated solely on antibody responses. Since 1995, the mechanisms underlying cell-mediated immunity of heartwater have been analysed. Progress achieved in these areas is discussed here by Philippe Totté and colleagues, with special emphasis on ruminants, the natural hosts of C. ruminantium.

CD62L defines a subset of pathogen-specific bovine CD4 with central memory cell characteristics

Central memory T cells (Tcm) have not previously been characterized in cattle and any other ruminant species. Here we described two phenotypically and functionally different subsets of pathogen-specific memory CD4(+) T cells in cattle that survived infection with Mycoplasma mycoides subsp. mycoides small colony (MmmSC). The first subset is CD45RO(+)CD45R(-)CD62L(-) and comprises two thirds of IFN-gamma producing CD4(+) T cells after MmmSC recall stimulation. The second is CD45RO(+)CD45R(-)CD62L(+) and represents the majority of proliferating CD4(+) T cells after 7 days of stimulation. Cell sorting experiments confirmed that both CD4(+)CD62L(+) and CD4(+)CD62L(-) subsets are present in vivo and proliferate independently in recall responses to MmmSC. In addition, MmmSC stimulation strongly decreased CCR7 and increased CCR5 transcripts levels in CD4(+)CD62L(-) cells whereas CD4(+)CD62L(+) were only slightly affected. High levels of recall proliferation but low IFN-gamma production, together with the capacity to preferentially migrate through the lymph nodes (i.e., expression of CD62L and CCR7), are characteristics of Tcm, in humans and mice. Tcm are associated with long-term protective immunity and a privileged target for vaccine development. Our results demonstrate the existence of Tcm in cattle and suggest that CD62L may serve as a marker to monitor Tcm in infections and vaccine development studies in ruminant.

Analysis of Ehrlichia ruminantium-specific T1/T2 responses during vaccination with a protective killed vaccine and challenge of goats

Ehrlichia ruminantium is an obligate intracellular bacterium that causes heartwater in ruminants and for which T‐cell‐mediated immunity is believed to play an important role in protection. To better characterize protective cellular immunity, E. ruminantium‐specific IFN‐γ and IL‐4 recall responses in major T‐cell subsets were analysed by flow cytometry during immunization of goats with a killed vaccine and following a virulent challenge. The killed vaccine elicited both CD8+ and CD4+ subsets to produce cytoplasmic IFN‐γ in the absence of IL‐4, thus indicating a biased T1 response. The relative capacity of CD8+ T‐cells to produce IFN‐γ was significantly higher than CD4+ T‐cells but the final contribution of both subsets was comparable. Circulating ER‐specific CD4 and CD8 effectors substantially decreased in numbers after the booster injection and could not be detected in most animals during challenge, which warrants further investigation in immune compartments other than blood. Since IFN‐γ inhibits the growth of the pathogen in target cells, the information provided in this study on E. ruminantium‐specific T1 responses will be valuable to develop cellular tools for the identification of potential protective antigens.

Effect of isolation techniques, in vitro culture and IFNγ treatment on the constitutive expression of MHC Class I and Class II molecules on goat neutrophils

Previous studies on the ability of human neutrophils to synthesize cytokines and express MHC Class I and inducible Class II molecules have suggested a possible role of these cells as accessory or antigen presenting cells (APC). There is no information available to date concerning this aspect in ruminants. Therefore, as the first step of these investigations, we have studied the cell surface expression of MHC molecules on goat neutrophils. We show that goat neutrophils can be distinguished from eosinophils with monoclonal antibody (MoAb) ILA-24 which recognizes cattle monocytes and neutrophils. Goat neutrophils constitutively express MHC Class II molecules. However, cell surface expression of MHC Class I and Class II molecules is dramatically reduced on neutrophils purified by density gradient centrifugation in comparison to neutrophils obtained from whole blood after lysis of erythrocytes. Also, the level of expression of MHC Class I antigens is seasonal and donor-dependent and rapidly decreases after in vitro culture despite negligible necrosis and apoptosis of neutrophils. Although treatment with IFNgamma partially prevents the loss of MHC Class I molecules on neutrophils, it fails to induce MHC Class II antigens. Implications of these results for further studies on the potential role of neutrophils as APC are discussed.

Comparative analysis of four lipoproteins from Mycoplasma mycoides subsp. mycoides Small Colony identifies LppA as a major T-cell antigen.

Control of contagious bovine pleuropneumonia (CBPP), caused by Mycoplasma mycoides subsp. mycoides Small Colony (MmmSC), remains an important goal in Africa. Subunit vaccines triggering B and T-cell responses could represent a promising approach. To this aim, the T-cell immunogenicity of four MmmSC lipoproteins (LppA, LppB, LppC and LppQ), present in African strains and able to elicit humoral response, was evaluated. In vitro assays revealed that only LppA was recognized by lymph node lymphocytes taken from three cattle, 3 weeks after MmmSC exposure. Maintenance of the LppA-specific response, relying on CD4 T-cells and IFN gamma production, was then demonstrated 1 year after infection. LppA is thus an important target for the CD4 T-cells generated early after MmmSC infection and persisting in the lymph nodes of recovered cattle. Its role as a protective antigen and ability to in vivo trigger both arms of the host immune response remain to be evaluated.

A highly sensitive, non-radioactive assay for T cell activation in cattle: applications in screening for antigens recognised by CD4(+) and CD8(+) T cells.

We describe a highly sensitive, non-radioactive assay for T cell activation, based on the rapid induction of class II MHC expression by constitutively negative bovine endothelial cells, when cultured in the presence of supernatants derived from activated bovine T cells. We demonstrate the effectiveness of this assay in detecting rBoIFNgamma and activation of immune CD4(+) and CD8(+) T cell lines and clones in response to specific antigen and transfected COS-7 cells, respectively. We also demonstrate its utility in identifying purified pathogen fractions that activate immune CD4(+) T cell clones.

Protection against #Cowdria ruminantium# infection in mice with gamma interferon produced in animal cells

We report here that y interferon produced in animal cell culture injected intraperitoneally, efficiently protects mice against Cowdria ruminantium infection. None of the other cytokines tested exerted any effect. Neutralizing antibodies against the cytokines (anti IL6, anti TNF, anti γIFN), did not modify the course of the disease, indicating that these cytokines do not play any crucial role in the pathology. The results concerning the protective effect of interferon pave the way towards the establishment of a rational selection method for protective antigens.

Characterization of Anamnestic T-cell Responses Induced by Conventional Vaccines against Contagious Bovine Pleuropneumonia

A better understanding of how T1 vaccination confers immunity would facilitate the rational design of improved vaccines against contagious bovine pleuropneumonia (CBPP). We show here that mycoplasmas-induced recall proliferation and IFN-γ responses are detected in cattle that received multiple shots of T1 vaccines. These anamnestic responses were under the strict control of CD4(+) T lymphocytes. Moreover, CD62L expression indicated that both CD4(+) effector memory (Tem) and central memory (Tcm) T lymphocytes are elicited in these animals. Comparative analysis with data from cattle that completely recovered from CBPP infection revealed similar anamnestic T-cell responses albeit at a lower magnitude for T1-vaccinated animals, particularly in the Tcm compartment. In conclusion, we discuss how our current understanding of T-cell responses will contribute to ongoing efforts for the improvement of future CBPP vaccines.

Bovine CD4+ T-cell lines reactive with soluble and membrane antigens of Cowdria ruminantium

Cowdria-specific CD4+ T-cell lines generated from immunised cattle respond to both soluble and membrane proteins of the agent. Furthermore, the lines produced the Cowdria-inhibitory cytokine IFN-gamma in response to soluble antigens fractionated by gel filtration and FPLC. Activity eluted as a single peak around fraction 15 for all T-cell lines tested. This fraction induced the highest production of IFN-gamma by the lines and was shown by SDS-polyacrylamide gel electrophoresis and silver staining analysis to contain less than 10 different bands ranging from 22 to 32 kDa. Given their high sensitivity and specificity, these short-term CD4+ T-cell lines will be valuable tools for the identification of Cowdria antigens for incorporation in a subunit vaccine.