Lymphocyte recruitment and protective efficacy against pulmonary mycobacterial infection are independent of the route of prior Mycobacterium bovis BCG immunization

Mycobacterium tuberculosis infects humans through the lung, and immunity to this chronic infection is mediated primarily by CD4(+) T lymphocytes. Recently we have demonstrated that the recruitment of lymphocytes to the lung during primary aerosol M. tuberculosis infection in mice occurs predominantly through the interaction of alpha(4)beta(1) integrin on CD4(+) T cells and vascular cell adhesion molecule-1 on the pulmonary endothelium. To investigate the effect of route of immunization with Mycobacterium bovis BCG on the pattern of T-cell recruitment to the lung, we have analyzed the differences in expression of integrins on activated memory CD4(+) T cells infiltrating the lung following primary BCG immunization by aerosol, intravenous, and subcutaneous routes and after subsequent aerosol challenge with M. tuberculosis. There were marked differences in the patterns of recruitment of activated CD4(+) T cells to the lung following primary immunization by the three routes. Expansion of CD44(hi) CD62L(low) CD4(+) T cells in the lung occurred following aerosol and intravenous BCG immunizations, and the lymphocyte recruitment was proportional to the pulmonary bacterial load. The majority of infiltrating CD4(+) T cells expressed alpha(4)beta(1) integrin. On subsequent exposure to aerosol BCG rapid expansion of gamma interferon-secreting alpha(4)beta(1)(+) CD4(+) T cells occurred to the same extent in all immunized mice, regardless of the route of immunization. Similar expansion of alpha(4)beta(1)(+) CD4(+) memory T cells occurred following M. tuberculosis challenge. The three routes of BCG immunization resulted in the same level of protection against aerosol M. tuberculosis or BCG challenge in both the lungs and spleen. Therefore, recruitment of effector T lymphocytes and protective efficacy against pulmonary mycobacterial infection are independent of the route of prior BCG immunization.     

Effects of antenatal and postnatal environments on CD4 T-cell responses to Mycobacterium bovis BCG in healthy infants in the Gambia

The Mycobacterium bovis BCG vaccine has a poor record of efficacy in low-income tropical settings. Against this background, we evaluated the immune response of infants to mycobacterial antigens over the 2 years following BCG vaccination at birth by measuring the gamma interferon (IFN-gamma), interleukin-2 (IL-2), and CD154 responses of CD4 T cells. Similar numbers of cells expressed IFN-gamma in infants, 4- to 5-year-old children, and adults, while CD154 was not expressed at comparable levels until the second year of infancy. The IL-2 response remained relatively low in infants, children, and adults but correlated negatively with mother's body mass index and was highest among infants born to Mandinka mothers. Similarly, infants born in the wet season had a stronger CD154 response than those born in the dry season throughout the 2 years of the study. We conclude that the prenatal and perinatal environments have a lasting effect on the response of infants to the BCG vaccine.     

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.     

Generation of CD8(+) T-cell responses to Mycobacterium bovis and mycobacterial antigen in experimental bovine tuberculosis

Protective immunity against tuberculosis is considered to be essentially cell mediated, and an important role for CD8(+) T lymphocytes has been suggested by several studies of murine and human infections. The present work, using an experimental model of infection with Mycobacterium bovis in cattle, showed that live M. bovis elicits the activation of CD8(+) T cells in vitro. However, a sonic extract prepared from M. bovis (MBSE) and protein purified derivative (PPDb) also induced a considerable degree of activation of the CD8(+) T cells. Analysis of proliferative responses of peripheral blood mononuclear cells, purified CD8(+) T cells, and CD8(+) T-cell clones to M. bovis and to soluble antigenic preparations (MBSE, PPDb) showed that the responses of all three types of cells were always superior for live mycobacteria but that strong responses were also obtained with complex soluble preparations. Furthermore, while cytotoxic capabilities were not investigated, the CD8(+) T cells were found to produce and release gamma interferon in response to antigen (live and soluble), which indicated one possible protective mechanism for these cells in bovine tuberculosis. Finally, it was demonstrated by metabolic inhibition with brefeldin A and cytochalasin D at the clonal level that an endogenous pathway of antigen processing is required for presentation to bovine CD8(+) cells and that presentation is also dependent on phagocytosis of the antigen.     

Quantitative studies of CD8+ T-cell responses during microbial infection

MHC class I tetramer staining, intracellular cytokine staining and ELISPOT assays have made it possible to quantify CD8(+) T-cell responses precisely during and following viral and bacterial infection. Although these quantitative methods are by now familiar and trusted components of the immunologist's toolbox, their application to models of microbial infection continues to provide surprising insights into mammalian adaptive immunity. In the past year there have been many exciting new findings on CD8(+) T-cell priming, expansion and memory formation in response to microbial infection.     

Evolution of cell types and T-cell subsets in the spleens of Mycobacterium bovis BCG-resistant and M. bovis BCG-susceptible strains of mice after infection with M. bovis BCG.

In mice, the early host response to intravenous infection with small doses of dispersed Mycobacterium bovis BCG is controlled by the Bcg gene. After infection with a low dose of M. bovis BCG, Lyt-1+ cells were generated in the spleens of BCG-susceptible mice (Bcgs) in parallel with an increase in the proportion of phagocytic cells. Very few changes occurred in the splenic cell types of BCG-resistant mice (Bcgr).     

Gamma interferon production in response to Mycobacterium bovis BCG and Mycobacterium tuberculosis antigens in infants born to human immunodeficiency virus-infected mothers

In utero sensitization to infectious pathogens can establish immunological memory and may influence the immune response to unrelated antigens. Little is known about the influence of intrauterine human immunodeficiency virus (HIV) exposure on the cellular immune response to mycobacterial antigens. Whole-blood culture gamma interferon (IFN-gamma) production in response to mycobacterial antigens was measured at birth and 6 weeks of age to determine the characteristics of the IFN-gamma response in HIV-exposed infants to Mycobacterium bovis BCG and mycobacterial antigens. At birth, we observed an increased immune activation in response to phytohemagglutinin among HIV-exposed, uninfected infants. In a proportion of these infants, we also observed an increased immune activation in response to purified protein derivative, BCG, and early secreted antigen target 6. Increases in the IFN-gamma response to the four antigens between birth and 6 weeks of age, observed in all HIV-unexposed infants, was absent in a substantial proportion of HIV-exposed, uninfected infants. The immunological differences persisted at 6 weeks of age, suggesting a sustained impact of in utero immune priming by HIV. Intrauterine exposure to HIV affects the infants' cellular immune response to mycobacterial antigens, either specifically or as a consequence of nonspecific, broadly reactive immune activation. Further studies will be important to help determine optimal vaccination and disease prevention strategies for this vulnerable population group.     

CD4(+) T-cell subsets that mediate immunological memory to Mycobacterium tuberculosis infection in mice

We have studied CD4(+) T cells that mediate immunological memory to an intravenous infection with Mycobacterium tuberculosis. The studies were conducted with a mouse model of memory immunity in which mice are rendered immune by a primary infection followed by antibiotic treatment and rest. Shortly after reinfection, tuberculosis-specific memory cells were recruited from the recirculating pool, leading to rapidly increasing precursor frequencies in the liver and a simultaneous decrease in the blood. A small subset of the infiltrating T cells was rapidly activated (<20 h) and expressed high levels of intracellular gamma interferon and the T-cell activation markers CD69 and CD25. These memory effector T cells expressed intermediate levels of CD45RB and were heterogeneous with regard to the L-selectin and CD44 markers. By adoptive transfer into nude mice, the highest level of resistance to a challenge with M. tuberculosis was mediated by CD45RB(high), L-selectin(high), CD44(low) cells. Taken together, these two lines of evidence support an important role for memory cells which have reverted to a naive phenotype in the long-term protection against M. tuberculosis.     

Modulation of naive CD4+ T-cell responses to an airway antigen during pulmonary mycobacterial infection

During pulmonary mycobacterial infection, there is increased trafficking of dendritic cells from the lungs to the draining lymph nodes. We hypothesized that ongoing mycobacterial infection would modulate recruitment and activation of antigen-specific naive CD4+ T cells after airway antigen challenge. BALB/c mice were infected by aerosol with Mycobacterium bovis BCG. At peak bacterial burden in the lungs (4 to 6 weeks postinfection), carboxy-fluorescein diacetate succinimidyl ester-labeled naive ovalbumin-specific DO11.10 T cells were adoptively transferred into infected and uninfected mice. Recipient mice were challenged intranasally with soluble ovalbumin (OVA), and OVA-specific T-cell responses were measured in the lungs, draining mediastinal lymph nodes (MLN), and spleens. OVA challenge resulted in increased activation and proliferation of OVA-specific T cells in the draining MLN of both infected and uninfected mice. However, only BCG-infected mice had prominent OVA-specific T-cell activation, proliferation, and Th1 differentiation in the lungs. BCG infection caused greater distribution of airway OVA to pulmonary dendritic cells and enhanced presentation of OVA peptide by lung CD11c+ cells. Together, these data suggest that an existing pulmonary mycobacterial infection alters the phenotype of lung dendritic cells so that they can activate antigen-specific naive CD4+ T cells in the lungs in response to airway antigen challenge.     

Distinct CD4 T-cell effects on primary versus recall CD8 T-cell responses during viral encephalomyelitis

CD4 T-cell help is not a universal requirement for effective primary CD8 T cells but is essential to generate memory CD8 T cells capable of recall responses. This study examined how CD4 T cells affect primary and secondary anti-viral CD8 T-cell responses within the central nervous system (CNS) during encephalomyelitis induced by sublethal gliatropic coronavirus. CD4 T-cell depletion before infection did not impair peripheral expansion, interferon-γ production, CNS recruitment or initial CNS effector capacity of virus-specific CD8 T cells ex vivo. Nevertheless, impaired virus control in the absence of CD4 T cells was associated with gradually diminished CNS CD8 T-cell interferon-γ production. Furthermore, within the CD8 T-cell population short-lived effector cells were increased and memory precursor effector cells were significantly decreased, consistent with higher T-cell turnover. Transfer of memory CD8 T cells to reduce viral load in CD4-depleted mice reverted the recipient CNS CD8 T-cell phenotype to that in wild-type control mice. However, memory CD8 T cells primed without CD4 T cells and transferred into infected CD4-sufficient recipients expanded less efficiently and were not sustained in the CNS, contrasting with their helped counterparts. These data suggest that CD4 T cells are dispensable for initial expansion, CNS recruitment and differentiation of primary resident memory CD8 T cells as long as the duration of antigen exposure is limited. By contrast, CD4 T cells are essential to prolong primary CD8 T-cell function in the CNS and imprint memory CD8 T cells for recall responses.     

Role of eosinophils in the pathogenesis of Mycobacterium bovis BCG infection in gamma interferon receptor-deficient mice

A profound eosinophil infiltration of granulomas is observed in the lungs of Mycobacterium bovis bacillus Calmette Guérin-infected gamma interferon receptor-deficient mice. Blockade of eosinophil proliferation and recruitment into the lung by treatment with anti-interleukin-5 monoclonal antibody marginally reduced mycobacterial growth within the lung but did not affect dissemination of the infection to other tissues.     

Mechanisms of T-lymphocyte accumulation during experimental pleural infection induced by Mycobacterium bovis BCG

Tuberculous pleurisy is a frequent extrapulmonary manifestation characterized by accumulation of fluid and inflammatory cells in the pleural space. Here, we investigated the mechanisms of T-lymphocyte accumulation in the pleural space by using a murine model of pleurisy induced by Mycobacterium bovis BCG. Intrathoracic (i.t.) injection of BCG (4.5 × 10⁵ bacteria/cavity) induced accumulation of T lymphocytes in the pleural cavities of C57BL/6 mice. We observed the presence of CFU in pleural washes conducted 1, 2, 3, 7, and 15 days after pleurisy induction. Pretreatment with fucoidan inhibited T-lymphocyte accumulation at 1 day, but not at 15 days, after BCG-induced pleurisy. Accordingly, adoptive transfer of fluorescein isothiocyanate-labeled blood mononuclear cells to infected mice showed that T lymphocytes migrated into the pleural cavity 1 day (but not 15 days) after BCG injection. Cell-free pleural wash fluids recovered from mice 1 day after BCG i.t. stimulation (day 1 BCG-PW), but not day 7 or day 15 BCG-PW, induced in vitro T-cell transmigration, which was dependent on L-, P-, and E-selectins. In contrast, day 7 BCG-PW (but not day 1 BCG-PW) induced in vitro T-lymphocyte proliferation via interleukin-2 (IL-2) and gamma interferon (IFN-γ). Accordingly, in vivo IL-2 or IFN-γ neutralization abolished T-lymphocyte accumulation 7 days after pleurisy induction. Our results demonstrate that pleural infection induced by BCG leads to T-lymphocyte accumulation in two waves. The acute phase depends on selectin-mediated migration, while the second wave of T-lymphocyte accumulation seems to depend on a local proliferation induced by cytokines produced in situ.     

Exposure to human alveolar lining fluid enhances Mycobacterium bovis BCG vaccine efficacy against Mycobacterium tuberculosis infection in a CD8+ T-cell-dependent manner

Current tuberculosis (TB) treatments include chemotherapy and preventative vaccination with Mycobacterium bovis Bacillus Calmette-Guérin (BCG). In humans, however, BCG vaccination fails to fully protect against pulmonary TB. Few studies have considered the impact of the human lung mucosa (alveolar lining fluid (ALF)), which modifies the Mycobacterium tuberculosis (M.tb) cell wall, revealing alternate antigenic epitopes on the bacterium surface that alter its pathogenicity. We hypothesized that ALF-induced modification of BCG would induce better protection against aerosol infection with M.tb. Here we vaccinated mice with ALF-exposed BCG, mimicking the mycobacterial cell surface properties that would be present in the lung during M.tb infection. ALF-exposed BCG-vaccinated mice were more effective at reducing M.tb bacterial burden in the lung and spleen, and had reduced lung inflammation at late stages of M.tb infection. Improved BCG efficacy was associated with increased numbers of memory CD8⁺ T cells, and CD8⁺ T cells with the potential to produce interferon-γ in the lung in response to M.tb challenge. Depletion studies confirmed an essential role for CD8⁺ T cells in controlling M.tb bacterial burden. We conclude that ALF modifications to the M.tb cell wall in vivo are relevant in the context of vaccine design.     

Evolution of CD4 T-cell subsets following infection of naive and memory immune mice with Mycobacterium tuberculosis.

We report here that during the course of an experimental infection of mice with Mycobacterium tuberculosis, the differential expression of the cell surface antigens CD44 and CD45RB could be used to delineate CD4+ T cells into four phenotypically distinct subsets. The major subset present was designated CD44lo/CD45RBhi and is associated with naive or resting T cells. The three remaining subsets expressed increased levels of the CD44 antigen as the infection progressed and could therefore be considered to be in an activated state. These activated populations could be further divided on the basis of their variable expression of the CD45RB antigen. These populations were designated CD44hi/CD45RBhi, CD44hi/CD45RBlo, and CD44hi/CD45RBneg. Kinetic studies of the emergence of these populations indicated that these subsets arose sequentially from the naive population at times associated with the peak expression of acquired specific resistance. In further studies, in an attempt to associate either the CD44hi/CD45RBlo or the CD44hi/CD45RBneg population with acquired immunologic memory of tuberculosis infection, draining lymph nodes of challenged memory immune animals were analyzed for the accumulation of the CD4+ subsets. The accumulation of both the CD44hi/CD45RBlo and the CD44hi/CD45RBneg populations was observed, but the CD44hi/CD45RBlo population was enriched in a manner consistent with the rapid accumulation of memory T cells during the anamnestic response. While functional roles for each of these subsets remain to be determined, these data provide the first evidence for the evolution of multiple, phenotypically distinct CD4+ T-cell subsets during the in vivo response to an experimental mycobacterial infection.     

Efficacy of Mycobacterium bovis BCG vaccination in mice undergoing prior pulmonary infection with atypical mycobacteria

The efficacy of Mycobacterium bovis BCG immunization in mice with established pulmonary infections caused by atypical mycobacteria was studied. In all four strains of Mycobacterium tested (M. kansasii, M. simiae, M. avium, and M. scrofulaceum), intravenous inoculation with 10(6) BCG had no discernible effect upon the course of atypical mycobacterial infection within the lungs; despite this, however, all BCG-vaccinated groups of mice were fully resistant to a subsequent acute aerogenic challenge with M. tuberculosis H37Rv, regardless of the presence of the pulmonary atypical mycobacterial infections. Furthermore, animals infected with M. kansasii, M. simiae, or M. avium but not vaccinated with BCG expressed considerable antituberculous resistance within the lungs, resulting in significant prolonged survival of these animals. The relevance of these findings to the expression of antituberculous resistance in human populations in areas in which atypical mycobacteria are endemic and the failure of these findings to support the hypothesis that prior contact with atypical mycobacteria might in some way jeopardize or interfere with the efficacy of subsequent BCG vaccination are discussed.     

Mycobacterium bovis BCG substrains confer different levels of protection against Mycobacterium tuberculosis infection in a BALB/c model of progressive pulmonary tuberculosis

Mycobacterium bovis BCG is the only available vaccine against tuberculosis. Reasons for why diverse BCG substrains induce different levels of protection in clinical trials remain unclear. The aim of this study was to compare the effectiveness of 10 BCG substrains in a mouse model of pulmonary tuberculosis. BALB/c mice were subcutaneously vaccinated and 2 months later were challenged with Mycobacterium tuberculosis H37Rv by intratracheal injection. Two and 4 months after challenge, delayed-type hypersensitivity (DTH) response, lung tissue affected by pneumonia, CFU, T-cell counts, and cytokine expression (interleukin-2 [IL-2], IL-4, IL-10, and gamma interferon) were determined. A differential protective effect of the diverse BCG substrains was found. BCG Phipps led to the largest and most persistent reduction of CFU counts and of the area of pneumonia at 2 and 4 months after challenge. This protection was accompanied by reduced IL-10-producing T cells. Contemporary BCG substrains induce a wide range of protection in this animal model. These data can help in the selection of the best vaccine for human immunization and for the development of novel recombinant BCG-based vaccine.     

Unimpaired clearance of Mycobacterium bovis BCG infection in selectively T-cell anergic TCR-V beta 8.2 transgenic mice.

The anergy induced in mice with staphylococcal enterotoxin B (SEB) has been shown to involve selective unresponsiveness in cytokine expression. While interleukin-2 (IL-2), IL-3 and IL-4 mRNA levels are substantially reduced in anergic T cells upon restimulation with SEB, mRNA for interferon-gamma (IFN-gamma) is expressed normally. On the other hand, infection with Nippostrongylus brasiliensis is known to break an established T-cell anergy. This knowledge prompted us to examine the effect of infection with an intracellular microbe, bacillus Calmett-Guérin (BCG), on the expression of anergy induced with SEB. We have demonstrated that while the SEB-induced anergy was not abrogated by BCG infection, the V beta 8.2 transgenic mice, in which almost all T cells were anergized with SEB, were capable of developing the effective acquired protective immunity, possibly through the preserved capacity to induce IFN-gamma leading to induction of nitric oxide synthase.     

Type I interferon supports primary CD8+ T-cell responses to peptide-pulsed dendritic cells in the absence of CD4+ T-cell help

CD8(+) T-cell responses to non-pathogen, cell-associated antigens such as minor alloantigens or peptide-pulsed dendritic cells (DC) are usually strongly dependent on help from CD4(+) T cells. However, some studies have described help-independent primary CD8(+) T-cell responses to cell-associated antigens, using immunization strategies likely to trigger natural killer (NK) cell activation and inflammatory cytokine production. We asked whether NK cell activation by MHC I-deficient cells, or administration of inflammatory cytokines, could support CD4(+) T-cell help-independent primary responses to peptide-pulsed DC. Injection of MHC I-deficient cells cross-primed CD8(+) T-cell responses to the protein antigen ovalbumin (OVA) and the male antigen HY, but did not stimulate CD8(+) T-cell responses in CD4-depleted mice; hence NK cell stimulation by MHC I-deficient cells did not replace CD4(+) T-cell help in our experiments. Dendritic cells cultured with tumour necrosis factor-α (TNF-α) or type I interferon-α (IFN-α) also failed to prime CD8(+) T-cell responses in the absence of help. Injection of TNF-α increased lymph node cellularity, but did not generate help-independent CD8(+) T-cell responses. In contrast, CD4-depleted mice injected with IFN-α made substantial primary CD8(+) T-cell responses to peptide-pulsed DC. Mice deficient for the type I IFN receptor (IFNR1) made CD8(+) T-cell responses to IFNR1-deficient, peptide-pulsed DC; hence IFN-α does not appear to be a downstream mediator of CD4(+) T-cell help. We suggest that primary CD8(+) T-cell responses will become help-independent whenever endogenous IFN-α secretion is stimulated by tissue damage, infection, or autoimmune disease.     

Comparison of immune responses of mice immunized with five different Mycobacterium bovis BCG vaccine strains.

Among the various parameters which may contribute to Mycobacterium bovis BCG vaccination efficiency, the choice of the vaccine strain may play an important role. In the present study, we therefore compared the immunogenicity of five different BCG strains that are commonly used for BCG vaccine production (Glaxo 1077, Japanese 172, Pasteur 1173P2, Prague, and Russian strains). The comparison of the growth capacity of these BCG strains in BALB/c and C3H mice demonstrated that a great difference exists between the capacity of various BCG strains to multiply and persist in target organs. A much lower recovery of BCG could be shown in mice immunized with Prague and Japanese BCG strains. T-cell responses of BCG-immunized mice were also examined by analyzing T-cell proliferative responses, cytokine production, delayed-type hypersensitivity responses, and cytotoxic activity. All these assays demonstrated that BCG immunization induced strong CD4+ T-cell responses, mostly of the Th1 type, as demonstrated by interleukin-2 and gamma interferon production. These studies also demonstrated that there are differences between BCG strains in stimulating these T-cell responses. A lack of induction of cytotoxic activity was observed following immunization with the Japanese strain. Lower anti-purified protein derivative antibody responses were also observed after intravenous or oral immunization with this BCG strain. Finally, the protective activity of these BCG strains was tested by measuring the capacity of immunized mice to eliminate recombinant Pasteur and Japanese BCG strains which expressed beta-galactosidase. The results of these experiments clearly demonstrated that the Prague and Japanese strains were unable to protect mice against a second mycobacterial challenge whereas mice immunized with the Glaxo, Pasteur, or Russian strain eliminated the recombinant BCG very efficiently. Altogether, the results of the present study strongly support the view that there are considerable differences in the immunogenicity of various BCG vaccine strains and that these differences may play a major role in BCG vaccination efficiency.