Responses of bovine WC1(+) gammadelta T cells to protein and nonprotein antigens of Mycobacterium bovis

WC1(+) gammadelta T cells of Mycobacterium bovis-infected cattle are highly responsive to M. bovis sonic extract (MBSE). In mycobacterial infections of other species, gammadelta T cells have been shown to respond to protein and nonprotein antigens, but the bovine WC1(+) gammadelta T-cell antigenic targets within MBSE require further definition in terms of the dominance of protein versus nonprotein components. The present study sought to characterize the WC1(+) gammadelta T-cell antigenic targets, together with the role of interleukin-2 (IL-2), in the context of M. bovis infection. This was achieved by testing crude and defined antigens to assess protein versus nonprotein recognition by WC1(+) gammadelta T cells in comparison with CD4(+) alphabeta T cells. Both cell types proliferated strongly in response to MBSE, with CD4(+) T cells being the major producers of gamma interferon (IFN-gamma). However, enzymatic digestion of the protein in MBSE removed its ability to stimulate CD4(+) T-cell responses, whereas some WC1(+) gammadelta T-cell proliferation remained. The most antigenic protein inducing proliferation and IFN-gamma secretion in WC1(+) gammadelta T-cell cultures was found to be ESAT-6, which is a potential novel diagnostic reagent and vaccine candidate. In addition, WC1(+) gammadelta T-cell proliferation was observed in response to stimulation with prenyl pyrophosphate antigens (isopentenyl pyrophosphate and monomethyl phosphate). High levels of cellular activation (CD25 expression) resulted from MBSE stimulation of WC1(+) gammadelta T cells from infected animals. A similar degree of activation was induced by IL-2 alone, but for WC1(+) gammadelta T-cell division IL-2 was found to act only as a costimulatory signal, enhancing antigen-driven responses. Overall, the data indicate that protein antigens are important stimulators of WC1(+) gammadelta T-cell proliferation and IFN-gamma secretion in M. bovis infection, with nonprotein antigens inducing significant proliferation. These findings have important implications for diagnostic and vaccine development.     

In vitro responsiveness of gammadelta T cells from Mycobacterium bovis-infected cattle to mycobacterial antigens: predominant involvement of WC1(+) cells

It is generally accepted that protective immunity against tuberculosis is generated through the cell-mediated immune (CMI) system, and a greater understanding of such responses is required if better vaccines and diagnostic tests are to be developed. gammadelta T cells form a major proportion of the peripheral blood mononuclear cells (PBMC) in the ruminant system and, considering data from other species, may have a significant role in CMI responses in bovine tuberculosis. This study compared the in vitro responses of alphabeta and gammadelta T cells from Mycobacterium bovis-infected and uninfected cattle. The results showed that, following 24 h of culture of PBMC with M. bovis-derived antigens, the majority of gammadelta T cells from infected animals became highly activated (upregulation of interleukin-2R), while a lower proportion of the alphabeta T-cell population showed activation. Similar responses were evident to a lesser degree in uninfected animals. Study of the kinetics of this response showed that gammadelta T cells remained significantly activated for at least 7 days in culture, while activation of alphabeta T cells declined during that period. Subsequent analysis revealed that the majority of activated gammadelta T cells expressed WC1, a 215-kDa surface molecule which is not expressed on human or murine gammadelta T cells. Furthermore, in comparison with what was found for CD4(+) T cells, M. bovis antigen was found to induce strong cellular proliferation but relatively little gamma interferon release by purified WC1(+) gammadelta T cells. Overall, while the role of these cells in protective immunity remains unclear, their highly activated status in response to M. bovis suggests an important role in antimycobacterial immunity, and the ability of gammadelta T cells to influence other immune cell functions remains to be elucidated, particularly in relation to CMI-based diagnostic tests.     

Modulation of WC1, a lineage-specific cell surface molecule of gamma/delta T cells augments cellular proliferation.

WC1, also known as T19, is the only unique gamma/delta T-cell differentiation antigen described to date other than the gamma/delta T-cell receptor. We present evidence that modulation of WC1 results in augmented proliferation of gamma/delta T cells. Immobilized IL-A29, a monoclonal antibody (mAb) specific for WC1, augmented proliferation of gamma/delta T cells in the autologous mixed leucocyte reaction (AMLR) as well as proliferation induced by either anti-CD3 or anti-CD5 mAb. In contrast, anti-CD5 mAb did not increase proliferation in the AMLR even though both CD5 and WC1 are members of the scavenger receptor cysteine-rich family of proteins and are expressed by bovine peripheral blood gamma/delta T cells. IL-A29 did not induce proliferation when assessed alone or in the presence of either phorbol myristate acetate (PMA) or interleukin-2. IL-A29 also did not induce detectable calcium mobilization when evaluated in the presence of monocytes, PMA, or following cross-linking of IL-A29 with anti-immunoglobulin antibody. We conclude that WC1 is a gamma/delta T-cell lineage-specific cell-surface differentiation antigen which is involved in activation of gamma/delta T cells using an as yet unidentified pathway.     

Early antibody responses to experimental Mycobacterium bovis infection of cattle

Bovine tuberculosis persists as a costly zoonotic disease in numerous countries despite extensive eradication and control efforts. Sequential serum samples obtained from Mycobacterium bovis-infected cattle were evaluated for seroreactivity to mycobacterial antigens. Animals received M. bovis by aerosol, intratonsil, intranasal, or intratracheal inoculation. Assays included the multiantigen print immunoassay for determination of antigen recognition patterns, immunoblot analysis for sensitive kinetic studies, and the VetTB STAT-PAK test, a novel, rapid test based on lateral-flow technology. Responses to MPB83 were detected for all M. bovis-infected animals regardless of the route or strain of M. bovis used for inoculation. Other less commonly recognized antigens included ESAT-6, CFP-10, and MPB70. Responses to MPB83 were detectable as early as 4 weeks after inoculation, were boosted upon injection of purified protein derivatives for skin testing, and persisted throughout the course of each of the four challenge studies. MPB83-specific immunoglobulin M (IgM) was detected prior to MPB83-specific IgG detection; however, early IgM responses rapidly waned, suggesting a benefit of tests that detect both IgM- and IgG-specific antibodies. The VetTB STAT-PAK test detected responses in sera from 60% (15/25) of the animals by 7 weeks after challenge and detected responses in 96% (24/25) of the animals by 18 weeks. These findings demonstrate the potential for new-generation antibody-based tests for the early detection of M. bovis infection in cattle.     

Immune responses to defined antigens of Mycobacterium bovis in cattle experimentally infected with Mycobacterium kansasii

Cross-reactive responses elicited by exposure to nontuberculous mycobacteria often confound the interpretation of antemortem tests for Mycobacterium bovis infection of cattle. The use of specific proteins (e.g., ESAT-6, CFP-10, and MPB83), however, generally enhances the specificity of bovine tuberculosis tests. While genes for these proteins are absent from many nontuberculous mycobacteria, they are present in M. kansasii. Instillation of M. kansasii into the tonsillar crypts of calves elicited delayed-type hypersensitivity and in vitro gamma interferon and nitrite concentration responses of leukocytes to M. avium and M. bovis purified protein derivatives (PPDs). While the responses of M. kansasii-inoculated calves to M. avium and M. bovis PPDs were approximately equivalent, the responses of M. bovis-inoculated calves to M. bovis PPD exceeded their respective responses to M. avium PPD. The gamma interferon and nitrite responses of M. kansasii-inoculated calves to recombinant ESAT-6-CFP-10 (rESAT-6-CFP-10) exceeded corresponding responses of noninoculated calves as early as 15 and 30 days after inoculation, respectively, and persisted throughout the study. The gamma interferon and nitrite responses of M. bovis-inoculated calves to rESAT-6-CFP-10 exceeded the corresponding responses of M. kansasii-inoculated calves beginning 30 days after inoculation. By using a lipoarabinomannan-based enzyme-linked immunosorbent assay, specific serum antibodies were detected as early as 50 days after challenge with M. kansasii. By a multiantigen print immunoassay and immunoblotting, serum antibodies to MPB83, but not ESAT-6 or CFP-10, were detected in M. kansasii-inoculated calves; however, responses to MPB83 were notably weaker than those elicited by M. bovis infection. These findings indicate that M. kansasii infection of calves elicits specific responses that may confound the interpretation of bovine tuberculosis tests.     

Prominence of gamma delta T cells in the ruminant immune system.

The lymphoid systems of sheep and cattle contain a large number of gamma delta T cells, in striking contrast to the lymphoid systems of humans and mice. In neonatal animals particularly, these cells comprise the predominant fraction of T cells in the blood. Here Wayne Hein and Charles Mackay discuss what is currently known about the ontogeny, phenotype, tissue distribution and function of gamma delta T cells in ruminants. There are a number of interesting molecular features that characterize ruminant gamma delta T cells, but these do not entirely explain the high frequency of use of the gamma delta T-cell receptor in these animals. Studies on sheep, cattle or other animals that preferentially use gamma delta T cells should provide insights into the biological significance of the existence of two distinct forms of the T-cell receptor.     

CD8(+) T cells participate in the memory immune response to Mycobacterium tuberculosis

The contribution of CD8(+) T cells to the control of tuberculosis has been studied primarily during acute infection in mouse models. Memory or recall responses in tuberculosis are less well characterized, particularly with respect to the CD8 T-cell subset. In fact, there are published reports that CD8(+) T cells do not participate in the memory immune response to Mycobacterium tuberculosis. We examined the CD8(+) T-cell memory and local recall response to M. tuberculosis. To establish a memory immunity model, C57BL/6 mice were infected with M. tuberculosis, followed by treatment with anti-mycobacterial drugs and prolonged rest. The lungs of memory immune mice contained CD4(+) and CD8(+) T cells with the cell surface phenotype characteristic of memory cells (CD69(low) CD25(low) CD44(high)). At 1 week postchallenge with M. tuberculosis via aerosol, > or =30% of both CD4(+) and CD8(+) T cells in the lungs of immune mice expressed the activation marker CD69 and could be restimulated to produce gamma interferon (IFN-gamma). In contrast, <6% of T cells in the lungs of naive challenged mice were CD69(+) at 1 week postchallenge, and IFN-gamma production was not observed at this time point. CD8(+) T cells from the lungs of both naive and memory mice after challenge were cytotoxic toward M. tuberculosis-infected macrophages. Our data indicate that memory and recall immunity to M. tuberculosis is comprised of both CD4(+) and CD8(+) T lymphocytes and that there is a rapid response of both subsets in the lungs following challenge.     

gamma delta T cells in autoimmunity

Conclusions  γδ T cells likely play a critical role in the host defenses, and recent evidence has indicated they are important in regulation of the immune response after pathogen challenge. Data are also accumulating that they play a part in autoimmunity and immune regulation. Evidence indicates that γδ T cells mediate these effects through cytokine production, via direct effects on cells of the adaptive immune response, or through interactions with innate immune components or barriers. Nevertheless, the biological function of γδ T cells in immune responses, especially in autoimmunity, largely remains unresolved. Further studies are needed to gain more knowledge about the immunobiology of these cells, asking why precisely they bear an activated phenotype in vivo, how their cytokine production is regulated, and how they interact with other cells of the innate and adaptive immune responses.     

Perturbation and proinflammatory type activation of V delta 1(+) gamma delta T cells in African children with Plasmodium falciparum malaria

gamma delta T cells have variously been implicated in the protection against, and the pathogenesis of, malaria, but few studies have examined the gamma delta T-cell response to malaria in African children, who suffer the large majority of malaria-associated morbidity and mortality. This is unfortunate, since available data suggest that simple extrapolation of conclusions drawn from studies of nonimmune adults ex vivo and in vitro is not always possible. Here we show that both the frequencies and the absolute numbers of gamma delta T cells are transiently increased following treatment of Plasmodium falciparum malaria in Ghanaian children and they can constitute 30 to 50% of all T cells shortly after initiation of antimalarial chemotherapy. The bulk of the gamma delta T cells involved in this perturbation expressed V delta 1 and had a highly activated phenotype. Analysis of the T-cell receptors (TCR) of the V delta 1(+) cell population at the peak of their increase showed that all expressed V gamma chains were used, and CDR3 length polymorphism indicated that the expanded V delta 1 population was highly polyclonal. A very high proportion of the V delta 1(+) T cells produced gamma interferon, while fewer V delta 1(+) cells than the average proportion of all CD3(+) cells produced tumor necrosis factor alpha. No interleukin 10 production was detected among TCR-gamma delta(+) cells in general or V delta 1(+) cells in particular. Taken together, our data point to an immunoregulatory role of the expanded V delta 1(+) T-cell population in this group of semi-immune P. falciparum malaria patients.     

The antituberculous Mycobacterium bovis BCG vaccine is an attenuated mycobacterial producer of phosphorylated nonpeptidic antigens for human gamma delta T cells.

The mycobacterial antigens stimulating human gamma delta T lymphocytes (R. L. Modlin, C. Permitz, F. M. Hofman, V. Torigian, K. Uemura, T. H. Rea, B. R. Bloom, and M. B. Brenner, Nature (London) 339:544-548, 1989; D. H. Raulet, Annu. Rev. Immunol. 7:175-207, 1989) have been characterized recently in Mycobacterium tuberculosis H37Rv as a group of four structurally related nucleotidic or phosphorylated molecules, termed TUBag1 to -4 (tuberculous antigens 1 to 4) (P. Constant, F. Davodeau, M. A. Peyrat, Y. Poquet, G. Puzo, M. Bonneville, and J. J. Fournie, Science 264:267-270, 1994). Here, we analyzed their distribution in different mycobacterial species of the M. tuberculosis group, with special emphasis on the human vaccine Mycobacterium bovis BCG. We show that the same four TUBag1 to -4 molecules are shared by these mycobacteria. Quantitative comparison reveals, however, that while the pathogen M. bovis and M. tuberculosis species produce rather high amounts of TUBag, all of the BCG strains have a surprisingly reduced production of TUBag. These observations suggest that among tuberculous mycobacteria, the bacterial TUBag load could, to some extent, constitute an immunological determinant of mycobacterial virulence for humans.     

Effects of different tuberculin skin-testing regimens on gamma interferon and antibody responses in cattle experimentally infected with Mycobacterium bovis

Although tuberculin skin testing has been a hallmark of bovine tuberculosis eradication campaigns, it lacks sensitivity, can be confounded by exposure to nontuberculous mycobacteria, and cannot be repeated for 60 days due to desensitization. To overcome these difficulties, an effective whole-blood cellular immunoassay for bovine gamma interferon (IFN-gamma) has been developed. The IFN-gamma test is commonly used in conjunction with tuberculin skin testing as a confirmatory test following a positive response to the caudal fold test (CFT). The present study was conducted to determine the effect of different tuberculin skin-testing regimens on IFN-gamma and antibody production by using calves that were experimentally infected with Mycobacterium bovis. Holstein calves were CFT tested 60 days after inoculation and the comparative cervical test (CCT) was conducted 7 (7-day CCT) or 55 (55-day CCT) days after the CFT. In both the 7-day CCT and 55-day CCT groups, IFN-gamma responses increased 3 days after the CFT; this was immediately followed by a decrease to pre-skin test levels 7 days after the CFT. In both groups, the application of the CCT at 7 or 55 days after the CFT resulted in no significant increase in IFN-gamma production. The administration of the CFT and the CCT to M. bovis-inoculated cattle boosted antibody responses to M. bovis PPD, rMPB83, ESAT-6, and the fusion protein Acr1-MPB83. The boosting effect was more pronounced in the 55-day CCT group. Increases in either IFN-gamma or antibody production were not seen in noninoculated cattle. Measurement of both IFN-gamma and antibody responses after skin testing may be useful in identifying M. bovis-infected cattle; however, the timing of collection of such samples may influence interpretation.     

Positive selection of gamma delta T cells

The issue of T-cell repertoire selection has been addressed recently by several laboratories. While evidence has been provided for both negative and positive selection of CD4+ and CD8+ alpha beta T cells, the molecular basis of positive selection remains unclear. In this article Juan Lafaille and colleagues describe molecular features of gamma delta T-cell selection in the fetal thymus. These features were deduced from extensive junctional sequence data of gamma delta T-cell receptor genes in fetal thymocytes. Their data suggest the active participation of a self peptide in the positive selection of gamma delta T cells.     

Involvement of gamma delta T cells in immunity to trypanosomiasis.

In this study the involvement of peripheral gamma delta T cells, prepared by flow cytometry, in the immune response of cattle to primary infection with Trypanosoma congolense was assessed. Negligible in vitro proliferative responses were observed in gamma delta T cells isolated from trypanosusceptible Boran (Bos indicus) cattle at all stages examined post-infection when stimulated in vitro with parasite antigens. In contrast, both CD8+ T cells and gamma delta T cells from trypanotolerant N'Dama (Bos taurus) cattle proliferated markedly when stimulated in vitro with a complex of invariant trypanosome antigens with MW between 100,000 and 140,000 (100,000 MW complex). Neither species of cattle exhibited significant T-cell recognition of trypanosome variable surface glycoprotein (VSG). To study further the functional and phenotypic characteristics of the gamma delta T-cell response, four T-cell lines were established from infected N'Dama cattle. These cell lines were comprised of up to 96% gamma delta (WC1+) T cells, the remainder being CD8+ T cells. Two of these gamma delta T-cell lines exhibited 100,000 MW complex antigen specificity which was not major histocompatibility complex (MHC) restricted in one line.     

Cytotoxic T-cell responses to Mycobacterium bovis during experimental infection of cattle with bovine tuberculosis

Cytotoxic T-cell responses are thought to play a significant role in the host defence against mycobacterial infections. Little is understood about such responses of cattle to Mycobacterium bovis, the causative agent of bovine tuberculosis. The work described in this report demonstrates the activity of cytotoxic cells during experimental infection of cattle with M. bovis. The cytotoxic cells were found to have the ability to specifically lyse macrophages infected with M. bovis and were detected in peripheral blood lymphocytes after in vitro re-exposure to M. bovis. Cytotoxic activity was detected 4 weeks after experimental infection with M. bovis; a similar level of activity was maintained during the infection and it was mediated by both WC1+gammadelta and CD8+ T cells. In addition, inhibition of the growth of M. bovis within infected macrophages was detected when they were exposed to cultures containing M. bovis-specific cytotoxic cells. The ability to detect cytotoxic cells after infection of cattle with M. bovis will allow their activity to be measured during vaccination trials. Correlation of cytotoxic activity with disease outcome may aid in the design of new vaccines and vaccination strategies.     

Immunoblot analysis of humoral immune responses to Mycobacterium bovis in experimentally infected cattle: early recognition of a 26-kilodalton antigen.

Development of a serodiagnostic test for bovine tuberculosis necessitates an understanding of the humoral immune responses of animals following infection with Mycobacterium bovis. The antibody responses in groups of calves challenged intranasally with different doses of M. bovis (approximately 10(2), 10(4), and 10(6) CFU) or placed in contact with the infected animals were analyzed by immunoelectrophoretic blotting in which a whole-cell sonicate of M. bovis was utilized as an antigen. Antibody responses were evident early in infections in which calves were exposed to high doses of M. bovis, while in groups exposed to lower doses, the time until antibody was detected increased as the challenge dose decreased. In cattle exposed to M. bovis, immunoblot analysis showed antibody responses to three main antigens of 26, 22, and 16 kDa. It was further demonstrated that antibody responses to the 26-kDa antigen appeared earliest in the course of infection. Preliminary investigations in this study have identified a 26-kDa antigen for potential use in improved serodiagnosis by enzyme-linked immunosorbent assays.     

The immunobiology of gamma delta+ T cells

T cells expressing the gd receptor are the major lymphocyte type represented in intraepithelial layers of various organs in the mouse. The gd cells found in distinct anatomical locations express distinct restricted subsets of Vg and Vd genes. Cells in each site are probably derived from progressive intrathymic waves of gd cells expressing these restricted V gene subsets. These features of the cells suggest a role in monitoring epithelial layers for distinctive ligands, and recent studies implicate mycobacterial "stress proteins' as antigens for g/d T cells. Thus gd T cells may represent an arm of the immune system devoted to elimination of infected, transformed or otherwise stressed autologous epithelial cells, based on recognition of induced stress proteins. They may also be devoted to mycobacterial immunity by virtue of specificity for mycobacterial stress proteins and other antigens.