Enhanced antigen-presenting activity and tumour necrosis factor-α-independent activation of dendritic cells following treatment with Mycobacterium bovis bacillus Calmette–Guérin

Dendritic cells (DCs) are most potent among the antigen-presenting cells and are believed to be crucial for the initiation of a primary T-cell response to foreign antigens. Mycobacterial infection within macrophages is controlled by cell-mediated immunity. To elucidate the stimulation of immune response by Mycobacterium bovis bacillus Calmette-Guérin (BCG), we purified DCs from precursor cells in human peripheral blood mononuclear cells (PBMC) by culturing them with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) and characterized their surface antigen expression. The interaction of cultured DCs with BCG resulted in increased surface expression of several DC-related marker antigens. BCG also induced reduction of endocytosis, enhancement of CD83 expression as well as B7 costimulatory molecules and IL-12 production, suggesting that BCG treatment directly induces DCs to mature. BCG-treated DCs were much more potent antigen-presenting cells in allogeneic immune response than untreated DCs. Moreover, while the neutralization of tumour necrosis factor-alpha (TNF-alpha) significantly blocked the DC maturation induced by lipopolysaccharide (LPS), it could not inhibit the induction of DC maturation by the BCG treatment, indicating that TNF-alpha production plays a minor role in the BCG-induced DC maturation. However, the neutralization of TNF-alpha resulted in decreased IL-12 production by activated DCs. These results suggest that infection with BCG might evoke direct activation and maturation of DC and the general immune stimulant effect of BCG might be related with the activation of DCs.     

Dendritic cells efficiently immunoselect mycobacterial-reactive T cells in human blood, including clonable antigen-reactive precursors.

Given the persistence of tuberculosis throughout the world, the delineation of mechanisms that lead to protective immunity to Mycobacterium tuberculosis is important. We have evaluated the presenting function of human dendritic cells for mycobacterial antigens, since these antigen-presenting cells (APC) are particularly effective in initiating antigen-specific T-cell responses. Dendritic cells from blood prove to be active APC for mycobacteria-specific proliferative responses by CD4+ T cells from bacillus Calmette-Guérin (BCG)-vaccinated individuals. In the first 24-48 hr of the response, dendritic cells that have been pulsed with mycobacterial antigens, including live BCG, effectively bind T cells forming discrete cell clusters. The clusters represent about 1% of the applied T cells. Clusters are highly enriched in mycobacterial reactivity while the non-clusters are depleted. Clustered T cells can be used as a starting point to expand antigen-specific cell lines. Mitogen and allogeneic feeder cells were used as APC to expand the mycobacterial-reactive lines, because the antigen-specific T cells had been preselected by virtue of their binding to antigen-pulsed dendritic cells. We discuss the advantages of obtaining antigen-reactive T cells by using dendritic cells as immunoadsorbents. These lines should help delineate the range of mycobacterial antigens and T-cell responses that participate in host responses to mycobacteria.     

Differential effects of control and antigen-specific T cells on intracellular mycobacterial growth

We investigated the effects of peripheral blood mononuclear cells expanded with irrelevant control and mycobacterial antigens on the intracellular growth of Mycobacterium bovis bacillus Calmette-Guérin (BCG) in human macrophages. More than 90% of the cells present after 1 week of in vitro expansion were CD3(+). T cells were expanded from purified protein derivative-negative controls, persons with latent tuberculosis, and BCG-vaccinated individuals. T cells expanded with nonmycobacterial antigens enhanced the intracellular growth of BCG in suboptimal cultures of macrophages. T cells expanded with live BCG or lysates of Mycobacterium tuberculosis directly inhibited intracellular BCG. Recent intradermal BCG vaccination significantly enhanced the inhibitory activity of T cells expanded with mycobacterial antigens (P < 0.02), consistent with the induction of memory-immune inhibitory T-cell responses. Selected mycobacterial antigens (Mtb41 > lipoarabinomannan > 38kd > Ag85B > Mtb39) expanded inhibitory T cells, demonstrating the involvement of antigen-specific T cells in intracellular BCG inhibition. We studied the T-cell subsets and molecular mechanisms involved in the memory-immune inhibition of intracellular BCG. Mycobacteria-specific gammadelta T cells were the most potent inhibitors of intracellular BCG growth. Direct contact between T cells and macrophages was necessary for the BCG growth-enhancing and inhibitory activities mediated by control and mycobacteria-specific T cells, respectively. Increases in tumor necrosis factor alpha, interleukin-6, transforming growth factor beta, and vascular endothelial growth factor mRNA expression were associated with the enhancement of intracellular BCG growth. Increases in gamma interferon, FAS, FAS ligand, perforin, granzyme, and granulysin mRNA expression were associated with intracellular BCG inhibition. These culture systems provide in vitro models for studying the opposing T-cell mechanisms involved in mycobacterial survival and protective host immunity.     

Protection against aerosol Mycobacterium tuberculosis infection using Mycobacterium bovis Bacillus Calmette Guérin-infected dendritic cells.

In the lung, dendritic cells (DC) are key antigen-presenting cells capable of triggering specific cellular responses to inhaled pathogens, and thus, they may be important in the initiation of an early response to mycobacterial infections. The ability of DC to enhance antigen presentation to naive T cells within the lungs was characterized with respect to Mycobacterium bovis Bacillus Calmette Guérin (BCG) vaccination against M. tuberculosis infection. In vitro derived DC were infected with BCG, which induced their maturation, as shown by the increased expression of MHC class II antigens, CD80 and CD86 co-stimulatory molecules. The synthesis of mRNA for IL-1, IL-6, IL-12, IL-10 and IL-1 receptor antagonist was also enhanced. When administered intratracheally in mice, infected DC induced a potent T cell response and the production of IFN-gamma to mycobacterial antigens in the mediastinal lymph nodes, leading to a significant protection against aerosol M. tuberculosis infection. Intriguingly, although the vaccination schedule for BCG-infected DC was much shorter than subcutaneous BCG vaccination (7 days as compared to 100 days), both types of vaccination showed similar levels of protection. These data confirm that DC can be potent inducers of a cellular immune response against mycobacteria and support the concept of combining DC strategies with mycobacterial vaccines for protective immunity against tuberculosis.     

Relative efficacy of uptake and presentation of Mycobacterium bovis BCG antigens by type I mouse lung epithelial cells and peritoneal macrophages

Flow cytometric studies indicated that both peritoneal macrophages (PMs) and primary lung epithelial (PLE) cells isolated from mouse lungs could take up fluorescence-tagged Mycobacterium bovis BCG. BCG uptake in both cases was significantly inhibited by cytochalasin D, indicating active internalization of BCG by these cells. Confocal microscopy data further confirmed that BCG was internalized by PLE cells. BCG sonicate antigen (sBCG) had marked toxicity toward PMs but was relatively nontoxic to PLE cells. Accordingly, BCG sonicate antigen induced a significantly higher apoptotic and necrotic response in PMs compared to that in PLE cells. Both PMs and PLE cells exposed to BCG antigens and fixed thereafter could efficiently present antigens to purified BCG-sensitized T helper cells, as assessed by the release of interleukin-2 (IL-2) and gamma interferon (IFN-γ). If, however, PLE cells were fixed before exposure to BCG, antigen presentation was abrogated, indicating that the PLE cells may in some way process the BCG antigen. A comparison of efficacies of BCG-pulsed PLE cells and PMs to present antigen at various antigen-presenting cell (APC)/T cell ratios indicated that PMs had only marginally greater APC function than that of PLE cells. Staining with specific monoclonal antibodies indicated that the cultured PLE cells used for antigen presentation essentially comprised type I epithelial cells. Our results suggest that type I lung epithelial cells may present BCG antigens to sensitized T helper cells and that their performance as APCs is comparable with that of PMs.     

Mycobacterial infection primes T cells and macrophages for enhanced recruitment of neutrophils.

C57BL/6 mice intraperitoneally infected with Mycobacterium bovis BCG (substrain Pasteur) recruited significantly higher numbers of neutrophils after an intraperitoneal inoculation of either BCG protein antigen or of endotoxin than uninfected control mice. Antigen-induced neutrophil recruitment was mediated by T cells of both CD4+ and CD8+ phenotype and was also observed in the C5-deficient DBA/2 mouse strain. The adoptive transfer of immune serum did not prime mice for enhanced antigen-mediated recruitment of neutrophils. The endotoxin-mediated recruitment of neutrophils was also enhanced in infected as compared to uninfected DBA/2 mice. Finally, endotoxin-pulsed purified macrophages from infected C57BL/6 mice recruited higher numbers of neutrophils than endotoxin-pulsed macrophages from normal mice in an adoptive transfer to peritoneal cavities of naive recipient mice. These data show that during mycobacterial infection, T cells and macrophages are primed for recruitment of neutrophils after being triggered in specific or nonspecific ways. This may represent a means to cope with secondary infections by allowing for extensive neutrophil infiltration readily upon microbial challenge.     

Closely related mycobacterial strains demonstrate contrasting levels of efficacy as antitumor vaccines and are processed for major histocompatibility complex class I presentation by multiple routes in dendritic cells

Mycobacteria expressing recombinant antigens are already being developed as vaccines against both infections and tumors. Little is known about how dendritic cells might process such antigens. Two different mycobacterial species, the fast-growing Mycobacterium smegmatis and the slow-growing M. bovis M. bovis BCG, were engineered to express a model tumor antigen, the K(b)-restricted dominant cytotoxic T-lymphocyte epitope OVA(257-264). Recombinant M. bovis BCG but not recombinant M. smegmatis conferred protection to mice challenged with the B16-OVA tumor cell line. We went on to investigate whether the contrast in antitumor efficacy could be due to differences in how dendritic cells process antigen from the two mycobacterial strains for class I presentation. Both strains of mycobacteria caused phenotypic maturation of dendritic cells, but recombinant M. smegmatis infection led to a greater degree of dendritic cell maturation than recombinant M. bovis BCG infection. Antigen from recombinant M. smegmatis was processed and presented as OVA(257-264) on K(b) molecules by the dendritic cell line DC2.4 but not by bone marrow-derived dendritic cells (BMDC) or splenic dendritic cells. In contrast, antigen from recombinant M. bovis BCG was presented by all three dendritic cell types as long as the mycobacteria were viable. Such presentation was dependent on proteasome function and nascent major histocompatibility complex (MHC) class I molecules in DC2.4 cells but independent of the proteasome and transporter associated with antigen processings (TAP) in BMDC and splenic dendritic cells. These data demonstrate for the first time that antigen vectored by the slow-growing M. bovis BCG but not that vectored by fast-growing, readily destroyed M. smegmatis is processed and presented on MHC class I by in vitro-generated dendritic cells, which has implications for recombinant microbial vaccine development.     

Mycobacterium bovis-infected cervine alveolar macrophages secrete lymphoreactive lipid antigens

Tuberculosis is caused by intracellular bacteria belonging to the genus Mycobacterium, including M. tuberculosis and M. bovis. Alveolar macrophages (AMs) are the primary host cell for inhaled mycobacteria. However, little is known about the mechanisms by which infected AMs can process and present mycobacterial antigens to primed lymphocytes and how these responses may affect ensuing protection in the host. In the present study, we sought to determine whether AMs from a naturally susceptible host for Mycobacterium bovis (red deer) could produce and secrete soluble immunoreactive antigens following mycobacterial infection in vitro. Confluent monolayers of deer AMs were infected with either heat-killed or live virulent M. bovis or M. bovis BCG at a multiplicity of infection of 5:1 and cultured for 48 h. Culture supernatants were collected, concentrated, and tested for the presence of mycobacterial antigens in a lymphocyte proliferation assay by using peripheral blood mononuclear cells from M. bovis-sensitized or naive deer. Supernatants derived from macrophages which had been infected with live bacilli stimulated the proliferation of antigen-sensitized, but not naive, lymphocytes. Supernatants derived from uninoculated AMs or AMs inoculated with heat-killed bacilli failed to stimulate lymphocyte proliferation. The lymphoproliferative activity was retained following lipid extraction of the supernatants, which were free of amino groups as determined by thin-layer chromatography. These results demonstrate that mycobacteria which are actively growing within AMs produce lipids which are secreted into the extracellular milieu and that these lipids are recognized by lymphocytes from mycobacterium-primed hosts. We suggest that mycobacterial lipids are released from AMs following aerosol infection in vivo and that they play an important role in the early immune response to tuberculosis.     

Gammadelta T cells in immunity induced by Mycobacterium bovis bacillus Calmette-Guérin vaccination

Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccination is efficacious for newborns or adults with no previous exposure to environmental mycobacteria. To determine the relative contribution and the nature of gammadelta T-cell receptor-positive T cells in newborns, compared to CD4(+) T cells, in immunity induced by M. bovis BCG vaccination, 4-week-old specific-pathogen-free pigs were vaccinated with M. bovis BCG and monitored by following the gammadelta T-cell immune responses. A flow cytometry-based proliferation assay and intracellular staining for gamma interferon (IFN-gamma) were used to examine gammadelta T-cell responses. Pigs were found to mount Th1-like responses to M. bovis BCG vaccination as determined by immunoproliferation and IFN-gamma production. The gammadelta T-cell lymphoproliferation and IFN-gamma production to stimulation with mycobacterial antigens were significantly enhanced by M. bovis BCG vaccination. The relative number of proliferating gammadelta T cells after stimulating peripheral blood mononuclear cells with Mycobacterium tuberculosis H37Rv culture filtrate protein was higher than that of CD4(+) T cells at an early time point after M. bovis BCG vaccination, but CD4(+) T cells were found to be more abundant at a later time point. Although the gammadelta T-cell responses were dependent on the presence of CD4(+) T cells for the cytokine interleukin-2, the enhanced gammadelta T cells were due to the intrinsic changes of gammadelta T cells caused by M. bovis BCG vaccination rather than being due solely to help from CD4(+) T cells. Our study shows that gammadelta T cells from pigs at early ages are functionally enhanced by M. bovis BCG vaccination and suggests an important role for this T-cell subset in acquired immunity conferred by M. bovis BCG vaccination.     

Transfer of macrophage-derived mycobacterial antigens to dendritic cells can induce naïve T-cell activation

Mycobacteria are capable of surviving and replicating in host macrophages, where they can release antigenic material into the environment. However, unlike dendritic cells (DCs), macrophages do not appear to be capable of activating naïve T cells. Therefore, this work investigated antigen transfer between macrophages and DCs. We generated culture supernatants from bacille Calmette–Guérin (BCG)-infected and uninfected macrophages and then determined whether DCs could present these extracellular mycobacterial antigens to T cells. Here, we show that DCs pulsed with antigens released from BCG-infected macrophages can stimulate primed T cells in vitro and initiate naïve T-cell responses in vivo. These results suggest that antigen transfer can occur between macrophages and DCs.     

Gene-based strategies for the immunotherapy of cancer

 T lymphocytes play a crucial role in the host’s immune response to cancer. Although there is ample evidence for the presence of tumor-associated antigens on a variety of tumors, they are seemingly unable to elicit an adequate antitumor immune response. Modern cancer immunotherapies are therefore designed to induce or enhance T cell reactivity against tumor antigens. Vaccines consisting of tumor cells transduced with cytokine genes in order to enhance their immunogenicity have been intensely investigated in the past decade and are currently being tested in clinical trials. With the development of novel gene transfer technologies it has now become possible to transfer cytokine genes directly into tumors in vivo. The identification of genes encoding tumor-associated antigens and their peptide products which are recognized by cytotoxic T lymphocytes in the context of major histocompatibility complex class I molecules has allowed development of DNA-based vaccines against defined tumor antigens. Recombinant viral vectors expressing model tumor antigens have shown promising results in experimental models. This has led to clinical trials with replication-defective adenoviruses encoding melanoma-associated antigens for the treatment of patients with melanoma. An attractive alternative concept is the use of plasmid DNA, which can elicit both humoral and cellular immune responses following injection into muscle or skin. New insights into the molecular biology of antigen processing and presentation have revealed the importance of dendritic cells for the induction of primary antigen-specific T cell responses. Considerable clinical interest has arisen to employ dendritic cells as a vehicle to induce tumor antigen-specific immunity. Advances in culture techniques have allowed the generation of large numbers of immunostimulatory dendritic cells in vitro from precursor populations derived from blood or bone marrow. Experimental immunotherapies which now transfer genes encoding tumor-associated antigens or cytokines directly into professional antigen-presenting cells such as dendritic cells are under evaluation in preclinical studies at many centers. Gene therapy strategies such as in vivo cytokine gene transfer directly into tumors as well as the introduction of genes encoding tumor-associated antigens into antigen-presenting cells hold considerable promise for the treatment of patients with cancer. Received: 20 January 1997 / Accepted: 17 February 1997     

Dendritic cells recruited to the lung shortly after intranasal delivery of Mycobacterium bovis BCG drive the primary immune response towards a type 1 cytokine production

We showed in a previous study that the intranasal (i.n) delivery of bacille Calmette-Guérin (BCG) to BP2 mice (H-2q) inhibits eosinophilia and bronchial hyperreactivity in a mouse model of asthma. The present work has been performed to characterize the leucocyte lineages recruited to the lungs of mice after i.n. delivery of BCG and potentially involved in the polarization of T lymphocytes. The different antigen-presenting cells (APC) recruited to bronchoalveolar lavage (BAL) and to lung tissue of mice shortly after the delivery of BCG were analysed in parallel as well as their capacity to drive the immune response towards a T helper type 1 cytokine production. Alveolar macrophages (AM) from the BAL were CD11c+, F4/80+ and CD11b-, and in the lung tissue two major populations of potential APC were detected: one CD11c-, F4/80+, CD11b+ and I-Aq- was identified as interstitial macrophages (IM) and a second expressing CD11c+ and I-Aq+ antigens, negative for CD11b and F4/80 markers as leucocytic dendritic cells (DC). Freshly isolated DC up-regulated CD11b and CD40 antigens after overnight culture, but remained negative for CD8alpha antigen, suggesting a myeloid origin. Lung DC which produced high amount of interleukin (IL)-12 were potent inducers of naive CD4+ T lymphocyte priming, as assessed by interferon-gamma (IFN-gamma) production by these naive CD4+ T cells. Lung explants recovered long term after BCG delivery produced sustained levels of IFN-gamma. Our results suggest that AM and particularly DC by secreting IL-12 shortly after BCG delivery induce the long-term persistence of IFN-gamma-secreting T cells percolating in BCG-loaded lung tissue.     

Subcultured human endothelial cells can function independently as fully competent antigen-presenting cells

Recent evidence has suggested that dendritic cells, epidermal Langerhan's cells and endothelial cells (EC) as well as macrophages, fulfill the requirements of antigen-presenting cells. Despite a variety of controls, one weakness in the evidence that these latter cell types can independently serve as antigen-presenting cells is that the cell preparations may contain small numbers of contaminating macrophages or other cell types. The experiments described in this paper are directed towards providing firm evidence that human EC are independently capable of presenting antigen to T cells. EC were isolated from human umbilical veins and maintained continuously by serial subculture for periods of up to 8 months. The subcultured EC displayed classic EC morphology and uniform immunofluorescent staining for Factor VIII-related antigen. The subcultured EC (tested to the 18th subculture) presented both particulate and soluble antigens to macrophage-depleted T cells with an efficiency equivalent to freshly isolated cells. Monoclonal antibodies to HLA-DR and HLA-DS determinants inhibited antigen presentation by either autologous macrophages or EC. In addition, antigen presentation by the subcultured EC was not affected by the macrophage-specific monoclonal antibody Mac-120, which inhibited antigen presentation by autologous macrophages in the same experiments. These results are consistent with human EC being able to independently function as fully competent antigen-presenting cells.     

Immunomodulation of mouse macrophage killing of Mycobacterium avium in vitro.

When C57BL/6 mice were infected intravenously with Mycobacterium avium, bacterial growth continued within the spleen until more than 10(8) CFU/g of tissue were attained. This contrasted with Mycobacterium bovis BCG infections where growth declined after 2 weeks. In vivo M. avium-infected splenic macrophages were harvested from chronically infected mice and cultured in vitro for 4 days at 37 degrees C. The number of viable mycobacteria within the resulting macrophage monolayers decreased when cultured in the presence of autologous sensitized T cells and an exogenous source of interleukin-2 (recombinant interleukin-2; 50 U/ml) compared with untreated controls (P less than 0.05). Incubation of the infected macrophages with autologous T cells and soluble M. avium antigens also significantly reduced the number of viable organisms. These results indicate that the mycobactericidal activity of M. avium-infected macrophages can be enhanced in a way that may have important therapeutic implications for patients infected with this opportunistic pathogen.     

Mycobacterial-induced cytotoxic T cells as well as nonspecific killer cells derived from healthy individuals and leprosy patients

Little information is available about the generation and specificity of the cytotoxic cells that eliminate human monocytes/macrophages infected with mycobacteria. To address this we have developed a cytotoxicity assay in which 51Cr-labeled monocytes pulsed with bacillus Calmette Guerin (BCG) or Mycobacterium leprae, were used as target cells in overnight cytotoxicity assays. As effector cells, peripheral blood mononuclear cells from healthy occupational contacts or from leprosy patients stimulated with antigen for 7 days were used. Cytotoxicity against antigen-pulsed monocytes that could be induced by mycobacterial antigens was proportional to the degree of antigen responsiveness in each individual, as measured in lymphocyte transformation tests. The lepromatous leprosy patients tested were often poor responders to BCG as well as M. leprae, both with regard to induction of cytotoxicity as well as in lympho-proliferation. Killing was significantly higher against antigen-pulsed vs. nonpulsed monocytes, although significant killing was induced against the latter as well and paralleled by induction of natural killer activity against the K-562 target cell. Cross-reactivity was observed between BCG and M. leprae, but not with unrelated antigen (tetanus toxoid) or with endogenous stress proteins induced by heat shock. M. leprae- and BCG-activated cytotoxic cells were found in both the CD4-CD8+ and CD4+CD8- populations, whereas in contrast the soluble antigen, purified protein derivative of M. tuberculosis, generated cytotoxic cells that were exclusively of the CD4+ phenotype. The involvement of both specific T cells as well as nonspecific cells in the killing of human macrophages may be important with respect to protection and immunopathology induced by mycobacterial antigens.     

Enhancement of delayed-type hypersensitivity to Trypanosoma cruzi in mice treated with Mycobacterium bovis BCG and cyclophosphamide.

C57BL/10J mice treated with Mycobacterium bovis BCG and cyclophosphamide were immunized with disrupted epimastigotes or with living blood trypomastigotes from Trypanosoma cruzi and assayed for delayed hypersensitivity by footpad testing with epimastigote antigens. Enhanced and lasting reactions were observed in mice pretreated with BCG or cyclophosphamide or both and immunized with epimastigotes. Whereas BCG pretreatment clearly reduced the mortality rates of mice immunized with living blood forms, no enhancement of the delayed hypersensitivity responses was observed in animals treated with BCG or cyclophosphamide or both before infection. The production of high levels of delayed hypersensitivity in the absence of infection and its adoptive transfer with cells could help to evaluate the participation of cell-mediated immunity in the protection against T. cruzi.     

Human NK cells directly recognize Mycobacterium bovis via TLR2 and acquire the ability to kill monocyte-derived DC

NK cells are important players of the early innate defense against various pathogens. In this study, we investigated the interaction between human NK cells and Mycobacterium bovis [bacille Calmette-Guérin (BCG)] and we determined whether and how such an interaction might impact on NK cell activation, cytokine production and cytotoxicity. We show that highly purified NK cells, upon short-term co-culture with BCG, expressed activation markers including CD69 and CD25. Moreover, these NK cells released IFN-gamma and tumor necrosis factor-alpha and killed more efficiently different targets including monocyte-derived immature dendritic cell. All these functions were strongly up-regulated in the presence of exogenous IL-12. Although more efficient responses were detected in NK cell populations displaying an NCR(bright) phenotype, no direct evidence of an involvement of triggering NK receptors in BCG recognition could be obtained. On the other hand, anti-toll-like receptor (TLR)2 mAb inhibited NK cell responses to BCG, suggesting that NK cells may express a functional TLR2, which plays a role in their mechanism of direct BCG recognition. Taken together, these data suggest that BCG, by inducing simultaneous activation of NK and antigen-presenting cells via their 'shared' TLR2, can promote efficient bidirectional NK-dendritic cell interactions necessary for subsequent priming of T(h)1 responses.     

Dendritic cells induce CD4+ and CD8+ T-cell responses to Mycobacterium bovis and M. avium antigens in Bacille Calmette Guérin vaccinated and nonvaccinated cattle

Few data are available regarding the induction of memory T-lymphocyte responses in cattle following Bacille Calmette Guérin (BCG) vaccination. Studies of the immune response induced by BCG vaccination provide an insight into the basis of antimycobacterial immunity that could be exploited for the development of more effective vaccination strategies. We used autologous dendritic cells (DC) infected with Mycobacterium bovis Bacille Calmette Guérin (BCG) or pulsed with purified protein derivative from M. bovis (PPD-B) or M. avium (PPD-A) to assess responses of CD4+, CD8+ and WC1+ gammadelta TCR+ lymphocytes from BCG vaccinated and nonvaccinated cattle. Mycobacteria-specific CD4+ and CD8+, but not WC1+ gammadelta TCR+, memory T lymphocytes were demonstrated in BCG-vaccinated cattle. CD4+ and CD8+ lymphocytes proliferated and produced interferon (IFN)-gamma in response to BCG-infected or PPD-B-pulsed DC. Proliferative responses were greater for CD4+ than CD8+ lymphocytes, although secretion of IFN-gamma was higher from the CD8+ T cells. Responses to PPD-A-pulsed DC were lower, with no CD8+ response. Lymphocytes from nonvaccinated calves were also stimulated to proliferate by BCG-infected DC, although the magnitude of proliferation was lower. The findings suggest that immunity to M. bovis induced by BCG vaccination in cattle may involve CD8+ memory T cells which produce IFN-gamma, as well as CD4+ memory T cells.     

T-cell responses to CD1-presented lipid antigens in humans with Mycobacterium tuberculosis infection

CD1-restricted presentation of lipid or glycolipid antigens derived from Mycobacterium tuberculosis has been demonstrated by in vitro experiments using cultured T-cell lines. In the present work, the frequency of T-cell responses to natural mycobacterial lipids was analyzed in ex vivo studies of peripheral blood lymphocytes from human patients with pulmonary tuberculosis, from asymptomatic individuals with known contact with M. tuberculosis documented by conversion of their tuberculin skin tests, and from healthy tuberculin skin test-negative individuals or individuals vaccinated with Mycobacterium bovis BCG. Proliferation and gamma interferon enzyme-linked immunospot assays using peripheral blood lymphocytes and autologous CD1(+) immature dendritic cells revealed that T cells from asymptomatic M. tuberculosis-infected donors responded with significantly greater magnitude and frequency to mycobacterial lipid antigen preparations than lymphocytes from uninfected healthy donors. By use of these methods, lipid-antigen-specific proliferative responses were minimally detectable or absent in blood samples from patients with active tuberculosis prior to chemotherapy but became detectable in blood samples drawn 2 weeks after the start of treatment. Lipid antigen-reactive T cells were detected predominantly in the CD4-enriched T-cell fractions of circulating lymphocytes, and anti-CD1 antibody blocking experiments confirmed the CD1 restriction of these T-cell responses. Our results provide further support for the hypothesis that lipid antigens serve as targets of the recall response to M. tuberculosis, and they indicate that CD1-restricted T cells responding to these antigens comprise a significant portion of the circulating pool of M. tuberculosis-reactive T cells in healthy individuals with previous exposure to M. tuberculosis.