Role of chemokine ligand 2 in the protective response to early murine pulmonary tuberculosis

Chemokines play an important role in the development of immunity to tuberculosis. Chemokine ligand 2 (CCL2, JE, monocyte chemoattractant protein-1) is thought to be primarily responsible for recruiting monocytes, dendritic cells, natural killer cells and activated T cells, all of which play critical roles in the effective control of tuberculosis infection in mice. We show here that in mice in which the CCL2 gene was disrupted, low-dose aerosol infection with Mycobacterium tuberculosis resulted in fewer macrophages entering the lungs, but only a minor and transient increase in bacterial load in the lungs; these mice were still able to establish a state of chronic disease. Such animals showed similar numbers of activated T cells as wild-type mice, as determined by their expression of the CD44hi CD62lo phenotype, but a transient reduction in cells secreting interferon-gamma. These data indicate that the primary deficiency in mice unable to produce CCL2 is a transient failure to focus antigen-specific T lymphocytes into the infected lung, whereas other elements of the acquired host response are compensated for by different ligands interacting with the chemokine receptor CCR2.     

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.     

Protective effect of DNA immunization against mycobacterial infection is associated with the early emergence of interferon-gamma (IFN-gamma)-secreting lymphocytes

The development of more effective anti-tuberculosis (TB) vaccines would contribute to the global control of TB. Understanding the activated/memory T cell response to mycobacterial infection and identifying immunological correlates of protective immunity will facilitate the design and assessment of new candidate vaccines. Therefore, we investigated the kinetics of the CD4+ T cell response and IFN-gamma production in an intravenous challenge model of Mycobacterium bovis bacille Calmette-Guérin (BCG) before and after DNA immunization. Activated/memory CD4+ T cells, defined as CD44hiCD45RBlo, expanded following infection, peaking at 3-4 weeks, and decreased as the bacterial load fell. Activated/memory CD4+ T cells were the major source of IFN-gamma and the level of antigen-specific IFN-gamma-secreting lymphocytes, detected by ELISPOT, paralleled the changes in bacterial load. To examine the effects of a DNA vaccine, we immunized mice with a plasmid expressing the mycobacterial secreted antigen 85B (Ag85B). This led to a significant reduction in mycobacteria in the liver, spleen and lung. This protective effect was associated with the rapid emergence of antigen-specific IFN-gamma-secreting lymphocytes which were detected earlier, at day 4, and at higher levels than in infected animals immunized with a control vector. This early and increased response of IFN-gamma-secreting T cells may serve as a correlate of protective immunity for anti-TB vaccines.     

The expression of early resistance to an infection with Mycobacterium tuberculosis by old mice is dependent on IFN type II (IFN-gamma) but not IFN type I

Old mice can express a transient early resistance to infection with M. tuberculosis that requires the presence of CD8 T cells within the lungs. Further characterization of those CD8 T cells within the aged lung established that the majority of CD8 T cells from old mice expressed the IL-15 receptor (CD122) in combination with bright expression of CD44 (CD44(hi)), and were capable of producing IFN-gamma after T cell receptor cross-linking. It has been previously described that CD8 CD44(hi) T cells proliferate in response to IFN-I, acting via IL-15, and therefore we determined whether IFN-I signaling could be a participant in the response of CD8 T cells within the lungs of old mice infected with M. tuberculosis. We demonstrate here that IFN-I signaling was required for the expansion of CD8 T cells within the aging lung in response to infection with M. tuberculosis, but that IFN-I signaling had no influence on the capacity of old mice to express early resistance to an infection with M. tuberculosis. Resident CD8 T cells were still however capable of producing IFN-gamma, which we demonstrate here to be critical in the expression of early resistance, suggesting that the expression of early resistance requires the participation, but not expansion, of the CD8 T cell pool within the aging lung.     

Failure to recruit anti-inflammatory CD103+ dendritic cells and a diminished CD4+ Foxp3+ regulatory T cell pool in mice that display excessive lung inflammation and increased susceptibility to Mycobacterium tuberculosis

Susceptibility to Mycobacterium tuberculosis is characterized by excessive lung inflammation, tissue damage, and failure to control bacterial growth. To increase our understanding of mechanisms that may regulate the host immune response in the lungs, we characterized dendritic cells expressing CD103 (α(E) integrin) (αE-DCs) and CD4(+) Foxp3(+) regulatory T (T(reg)) cells during M. tuberculosis infection. In resistant C57BL/6 and BALB/c mice, the number of lung αE-DCs increased dramatically during M. tuberculosis infection. In contrast, highly susceptible DBA/2 mice failed to recruit αE-DCs even during chronic infection. Even though tumor necrosis factor alpha (TNF-α) is produced by multiple DCs and macrophage subsets and is required for control of bacterial growth, αE-DCs remained TNF-α negative. Instead, αE-DCs contained a high number of transforming growth factor beta-producing cells in infected mice. Further, we show that T(reg) cells in C57BL/6 and DBA/2 mice induce gamma interferon during pulmonary tuberculosis. In contrast to resistant mice, the T(reg) cell population was diminished in the lungs, but not in the draining pulmonary lymph nodes (PLN), of highly susceptible mice during chronic infection. T(reg) cells have been reported to inhibit M. tuberculosis-specific T cell immunity, leading to increased bacterial growth. Still, despite the reduced number of lung T(reg) cells in DBA/2 mice, the bacterial load in the lungs was increased compared to resistant animals. Our results show that αE-DCs and T(reg) cells that may regulate the host immune response are increased in M. tuberculosis-infected lungs of resistant mice but diminished in infected lungs of susceptible mice.     

The role of interleukin-12 in acquired immunity to Mycobacterium tuberculosis infection.

The early phase of acquired cellular immunity to Mycobacterium tuberculosis infection is mediated by the emergence of protective CD4 T lymphocytes that secrete cytokines including interferon-gamma (IFN-gamma), a molecule which is pivotal in the expression of resistance to tuberculosis. Recent evidence demonstrates that infection with M. tuberculosis induces peripheral blood mononuclear cells to release the cytokine interleukin-12 (IL-12), a molecule that promotes the emergence of T-helper type-1 (Th1), IFN-gamma-producing T cells. We demonstrate here that IL-12 mRNA expression was induced by M. tuberculosis infection both in vivo and in vitro and that exogenous administration of IL-12 to mice transiently resulted in increased resistance to the infection. IL-12 also increased the production of IFN-gamma by both splenocytes derived from infected animals treated in vivo and by antigen-stimulated CD4 cells from untreated infected animals, with maximal effects at times associated with the expansion of antigen-specific CD4 T cells in vivo. In the absence of a T-cell response, as seen in SCID mice or nude mice, IL-12 only slightly augmented the moderate bacteriostatic capacity of these immunocompromised mice. Neutralization of IL-12 by specific monoclonal antibodies resulted in a reduction in granuloma integrity and slowing of the capacity of the animal to control bacterial growth.     

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.     

CD27 contributes to the early systemic immune response to Mycobacterium tuberculosis infection but does not affect outcome

The development of a strong Th1-mediated adaptive immune response is considered of main importance for host defense against the intracellular pathogen Mycobacterium tuberculosis. The induction of a cellular immune response is not only dependent on the engagement of the TCR but also requires co-stimulation. In order to study the role of the co-stimulatory molecule of the tumor necrosis factor receptor family member CD27 during murine M. tuberculosis infection, we intranasally infected wild-type (WT) and CD27 knockout (KO) mice with 10(5) colony-forming units M. tuberculosis. Whereas there were no differences in bacterial growth, inflammation and IFNgamma production by CD4+ and CD8+ lymphocytes in the lungs early after infection, the number of splenic CD8+ T cells producing the key Th1 cytokine IFNgamma was lower in CD27 KO mice than in WT mice. After 6 weeks, CD27 KO mice had 3.6-fold higher mycobacterial counts in their lungs and displayed more pulmonary inflammation and increased numbers of infiltrated leukocytes. Despite these differences early in infection, an equal number of WT and CD27 KO mice died during a 43-week observation period and lung bacterial loads and inflammation were comparable in the surviving animals. Our data suggest that CD27 does not contribute to the local IFNgamma-mediated response and long-term protection against M. tuberculosis.     

Activation of NKT cells protects mice from tuberculosis

The T-cell immune response to Mycobacterium tuberculosis is critical in preventing clinical disease. While it is generally accepted that both major histocompatibility complex class I (MHC-I)-restricted CD8(+) and MHC-II-restricted CD4(+) T cells are important for the immune response to M. tuberculosis, the role of non-MHC-restricted T cells is still not clearly delineated. We have previously reported that CD1d(-/-) mice do not differ from CD1d(+/+) mice in their survival following infection with M. tuberculosis. We now show that, although CD1d-restricted NKT cells are not required for optimum immunity to M. tuberculosis, specific activation of NKT cells by the CD1d ligand alpha-galactosylceramide protects susceptible mice from tuberculosis. Treatment with alpha-galactosylceramide reduced the bacterial burden in the lungs, diminished tissue injury, and prolonged survival of mice following inoculation with virulent M. tuberculosis. The capacity of activated NKT cells to stimulate innate immunity and modulate the adaptive immune response to promote a potent antimicrobial immune response suggests that alpha-galactosylceramide administration could have a role in new strategies for the therapy of infectious diseases.     

SWR mice are highly susceptible to pulmonary infection with Mycobacterium tuberculosis

Inbred mice differ in their abilities to control the growth of Mycobacterium tuberculosis in the lung and can as a result be regarded as either resistant or susceptible strains. In this study we report that the SWR mouse is both highly susceptible and in addition appears incapable of establishing a characteristic state of chronic disease after low-dose aerosol infection. In comparison to C57BL/6 mice, SWR mice were unable to contain the bacterial load in the lungs, resulting in progressive fatal disease. Histologic analysis of the lung tissue revealed evidence of a florid inflammatory cell response in the SWR mice leading to degeneration and necrosis and consolidation of a large percentage of the lung surface area. Digestion of infected lungs and analysis by flow cytometry demonstrated an initially similar but eventually higher number of lymphocytes accumulating in the SWR mice. Additionally, in contrast to the C57BL/6 mice, SWR mice had a significantly lower percentage of CD4 T cells in the lungs showing evidence of proliferation and positive intracellular staining for gamma interferon during the first two months of infection, and a lower percentage of both CD4 and CD8T cells exhibiting differentiation to an effector/memory phenotype during the first month of infection. We propose that further investigation of the SWR mouse may provide a new animal model for immunocompetent individuals apparently unable to effectively control the growth of M. tuberculosis in the lung.     

CD4+ CD25+ transforming growth factor-beta-producing T cells are present in the lung in murine tuberculosis and may regulate the host inflammatory response

CD4(+) CD25(+) regulatory T cells produce the anti-inflammatory cytokines transforming growth factor (TGF)-beta or interleukin (IL)-10. Regulatory T cells have been recognized to suppress autoimmunity and promote self-tolerance. These cells may also facilitate pathogen persistence by down-regulating the host defence response during infection with Mycobacterium tuberculosis. We evaluated TGF-beta(+) and IL-10(+) lung CD4(+) CD25(+) T cells in a murine model of M. tuberculosis. BALB/c mice were infected with approximately 50 colony-forming units of M. tuberculosis H37Rv intratracheally. At serial times post-infection, lung cells were analysed for surface marker expression (CD3, CD4, CD25) and intracellular IL-10, TGF-beta, and interferon (IFN)-gamma production (following stimulation in vitro with anti-CD3 and anti-CD28 antibodies). CD4(+) lung lymphocytes were also selected positively after lung digestion, and stimulated in vitro for 48 h with anti-CD3 and anti-CD28 antibodies in the absence and presence of anti-TGF-beta antibody, anti-IL-10 antibody or rmTGF-beta soluble receptor II/human Fc chimera (TGFbetasrII). Supernatants were assayed for elicited IFN-gamma and IL-2. Fluorescence activated cell sorter analyses showed that TGF-beta- and IL-10-producing CD4(+) CD25(+) T cells are present in the lungs of infected mice. Neutralization of TGF-beta and IL-10 each resulted in increases in elicited IFN-gamma, with the greatest effect seen when TGFbetasrII was used. Elicited IL-2 was not affected significantly by TGF-beta neutralization. These results confirm the presence of CD4(+) CD25(+) TGF-beta(+) T cells in murine pulmonary tuberculosis, and support the possibility that TGF-beta may contribute to down-regulation of the host response.     

Genetic aspects and microenvironment affect expression of CD18 and VLA-4 in experimental tuberculosis

Control of infection by Mycobacterium tuberculosis is dependent on macrophage activation and efficient migration of effector T-cell populations. Lymphocyte differentiation is associated with changes in cell surface phenotype and alterations in the migratory pattern of these cells. In this study, we investigated the expression of adhesion receptors involved in activation and migration process in experimental tuberculosis. We observed that susceptible BALB/c mice infected with virulent M. tuberculosis by intraperitoneal route presented downmodulation of very late antigen 4 (VLA-4) and unchanged levels of CD18 and CD44hi on peritoneal lymphocytes. On the other hand, lymphocytes from resistant C57BL/6 mice infected by the same route showed unchanged levels of VLA-4 and upregulation of CD18 and CD44hi. However, when BALB/c mice were infected by intratracheal route, lung lymphocytes presented a different pattern of CD18, CD44hi and VLA-4 expression from that observed on peritoneal cells, characterized by unchanged levels of VLA-4 and upregulation of CD18 and CD44hi- coincidentally the same phenotype found on peritoneal cells from C57BL/6. These results suggest that susceptibility and resistance to M. tuberculosis infection, depending on the experimental model, are related to the expression of CD18, CD44hi and VLA-4. Moreover, the microenvironment at the site of infection seems to differentially regulate the expression of these receptors. Thus, the up- or downmodulation of these adhesion receptors is probably associated with differential recruitment of T cells at the site of infection, which may or may not mediate protection in experimental tuberculosis.     

CXCR6 is a marker for protective antigen-specific cells in the lungs after intranasal immunization against Mycobacterium tuberculosis

Convincing correlates of protective immunity against tuberculosis have been elusive. In BALB/c mice, intranasal immunization with a replication-deficient recombinant adenovirus expressing Mycobacterium tuberculosis antigen 85A (adenovirus-85A) induces protective lower respiratory tract immunity against pulmonary challenge with Mycobacterium tuberculosis, while intradermal immunization with adenovirus-85A does not. Here we report that intranasal immunization with adenovirus-85A induces expression of the chemokine receptor CXCR6 on lung CD8 T lymphocytes, which is maintained for at least 3 months. CXCR6-positive antigen-specific T cell numbers are increased among bronchoalveolar lavage-recoverable cells. Similarly, intranasal immunization with recombinant antigen 85A with adjuvant induces CXCR6 expression on lung CD4 cells in BALB/c and C57BL/6 mice, while a synthetic ESAT6(1-20) peptide with adjuvant induces CXCR6 expression in C57BL/6 mice. Parenteral immunization fails to do so. Upregulation of CXCR6 is accompanied by a transient elevation of serum CXCL16 after intranasal immunization, and lung cells cultured ex vivo from mice immunized intranasally show increased production of CXCL16. Administration of CXCL16 and cognate antigen intranasally to mice previously immunized parenterally increases the number of antigen-specific T lymphocytes in the bronchoalveolar lavage-recoverable population, which mediates inhibition of the early growth of Mycobacterium tuberculosis after challenge. We conclude that expression of CXCR6 on lung T lymphocytes is a correlate of local protective immunity against Mycobacterium tuberculosis after intranasal immunization and that CXCR6 and CXCL16 play an important role in the localization of T cells within lung tissue and the bronchoalveolar lavage-recoverable compartment.     

A novel nonclassic beta2-microglobulin-restricted mechanism influencing early lymphocyte accumulation and subsequent resistance to tuberculosis in the lung

In this study, we compared the course of a low-dose aerosol Mycobacterium tuberculosis infection in mice bearing gene disruptions for the beta2-microglobulin molecule, the CD8 molecule, and the CD1 molecule. Over the first 50 d of infection, the CD8- and CD1-disrupted mice were no more susceptible to infection than were the control mice. In contrast, the bacterial load in beta2-microglobulin gene-disrupted mice increased rapidly and attained much higher levels than that observed in the other gene-disrupted mice and in control mice. A second major difference between the beta2-microglobulin gene-disrupted mice and the other animals was the development of lung granulomas; both the CD8- and CD1-disrupted mice developed essentially normal granulomas except for an apparent increased lymphocyte influx in the CD8-disrupted mice. The beta2-microglobulin gene-disrupted mice, on the other hand, developed granulomas virtually devoid of lymphocytes, with these cells instead localized within prominent perivascular cuffing adjacent to the lesions. These data support the hypothesis that a beta2-microglobulin-dependent, non-CD8- and non-CD1-dependent mechanism controls the early and efficient influx of protective lymphocytes into infected lesions, and that the absence of this mechanism decreases the capacity of the animal to initially deal with pulmonary tuberculosis.     

Vaccine-elicited 10-kilodalton culture filtrate protein-specific CD8+ T cells are sufficient to mediate protection against Mycobacterium tuberculosis infection

The 10-kDa culture filtrate protein (CFP-10) and 6-kDa early secretory antigen of T cells (ESAT-6) are secreted in abundance by Mycobacterium tuberculosis and are frequently recognized by T cells from infected people. The genes encoding these proteins have been deleted from the genome of the vaccine strain Mycobacterium bovis bacillus Calmette-Guérin (BCG), and it is hypothesized that these proteins are important targets of protective immunity. Indeed, vaccination with ESAT-6 elicits protective CD4+ T cells in C57BL/6 mice. We have previously shown that M. tuberculosis infection of C3H mice elicits CFP-10-specific CD8+ and CD4+ T cells. Here we demonstrate that immunization with a CFP-10 DNA vaccine stimulates a specific T-cell response only to the H-2K(k)-restricted epitope CFP-10(32-39). These CFP-10(32-39)-specific CD8+ cells undergo a rapid expansion and accumulate in the lung following challenge of immunized mice with aerosolized M. tuberculosis. Protective immunity is induced by CFP-10 DNA vaccination as measured by a CFU reduction in the lung and spleen 4 and 8 weeks after challenge with M. tuberculosis. These data demonstrate that CFP-10 is a protective antigen and that CFP-10(32-39)-specific CD8+ T cells elicited by vaccination are sufficient to mediate protection against tuberculosis.     

Gamma interferon-producing CD4+ T lymphocytes in the lung correlate with resistance to infection with Mycobacterium tuberculosis

The human immune system efficiently limits the replication of Mycobacterium tuberculosis in most infected individuals. Only 5 to 10% of infected people develop clinical tuberculosis, a sign of the inability of the immune system to control the infection. We have studied the C3H/HeJ (C3H) and C57BL/6 (B6) inbred mouse strains, which differ in their susceptibility to tuberculosis, in order to ascertain the immunological determinants of a successful immune response against M. tuberculosis and to establish a system to identify genes that influence susceptibility to tuberculosis. We found that the resistant B6 mice were able to control infection in both the lung and spleen, while susceptible C3H mice were incapable of limiting bacteria growth, especially in the lung, and succumbed to infection within 4 weeks. We determined that the susceptibility of C3H mice was independent of the Toll-like receptor 4 (tlr4) genetic locus and allelic major histocompatibility complex differences. Although the splenic immune responses were similar in the two mouse strains, the local immune responses in the lungs of the infected mice differed greatly. The pulmonary immune response in resistant B6 mice was characterized by an early influx of both CD4+ and CD8+ lymphocytes that produced gamma interferon (IFN-gamma). In contrast, the immune response of C3H mice in the lung was characterized by a delayed and decreased influx of lymphocytes, which produced little IFN-gamma. These results suggest an important role for the early appearance of IFN-gamma-producing lymphocytes in the lung in resistance to infection with M. tuberculosis.     

The major histocompatibility complex haplotype affects T-cell recognition of mycobacterial antigens but not resistance to Mycobacterium tuberculosis in C3H mice

Both innate and adaptive immunity play an important role in host resistance to Mycobacterium tuberculosis infection. Although several studies have suggested that the major histocompatibility complex (MHC) haplotype affects susceptibility to infection, it remains unclear whether the modulation of T-cell immunity by the MHC locus determines the host's susceptibility to tuberculosis. To determine whether allelic differences in the MHC locus affect the T-cell immune response after M. tuberculosis infection, we infected inbred and H-2 congenic mouse strains by the respiratory route. The H-2 locus has a profound effect on the antigen-specific CD4+-T-cell response after M. tuberculosis infection. CD4+ T cells from infected mice of the H-2(b) haplotype produced more gamma interferon (IFN-gamma) after in vitro stimulation with mycobacterial antigens than mice of the H-2(k) haplotype. A higher level of IFN-gamma was also detected in bronchoalveolar lavage fluid from infected mice of the H-2(b) haplotype. Furthermore, C3.SW-H2(b)/SnJ mice generate and recruit activated T cells to the lung after infection. Despite a robust immune response, C3.SW-H2(b)/SnJ mice succumbed to infection early and were similarly susceptible to infection as other C3H (H-2(k)) substrains. These results suggest that although the MHC haplotype has a profound impact on the T-cell recognition of M. tuberculosis antigens, the susceptibility of C3H mice to infection is MHC independent.     

Expression of L-Selectin (CD62L), CD44, and CD25 on activated bovine T cells

Mycobacterium bovis infection of cattle represents a natural host-pathogen interaction and, in addition to its economic and zoonotic impact, represents a model for human tuberculosis. Extravasation and trafficking of activated lymphocytes to inflammatory sites is modulated by differential expression of multiple surface adhesion molecules. However, effects of M. bovis infection on adhesion molecule expression have not been characterized. To determine these changes, peripheral blood mononuclear cells from M. bovis-infected cattle were stimulated with M. bovis purified protein derivative (PPD) or pokeweed mitogen (PWM) and evaluated concurrently for proliferation and activation marker expression. Stimulation with PPD or PWM increased CD25 and CD44 mean fluorescence intensity (MFI) and decreased CD62L MFI on CD4(+) cells from infected animals. CD62L MFI on PPD- and PWM-stimulated gammadelta T-cell receptor-positive (TCR(+)) and CD8(+) cells was also reduced compared to that of nonstimulated gammadelta TCR(+) and CD8(+) cells. Using a flow cytometry-based proliferation assay, it was determined that proliferating cells, regardless of lymphocyte subset, exhibited increased expression of CD25 and CD44 and decreased expression of CD62L compared to cells that had not proliferated. In contrast to proliferation, activation-induced apoptosis of CD4(+) cells resulted in a significant down regulation of CD44 expression. Lymphocytes obtained from lungs of M. bovis-infected cattle also had reduced expression of CD44 compared to lymphocytes from lungs of noninfected cattle. These alterations in surface molecule expression upon activation likely impact trafficking to sites of inflammation and the functional capacity of these cells within tuberculous granulomas.     

Exposure to Mycobacterium avium can modulate established immunity against Mycobacterium tuberculosis infection generated by Mycobacterium bovis BCG vaccination

Mycobacterium bovis bacille Calmette Guerin (BCG), the current vaccine against infection with Mycobacterium tuberculosis, offers a variable, protective efficacy in man. It has been suggested that exposure to environmental mycobacteria can interfere with the generation of BCG-specific immunity. We hypothesized that exposure to environmental mycobacteria following BCG vaccination would interfere with established BCG immunity and reduce protective efficacy, thus modeling the guidelines for BCG vaccination within the first year of life. Mice were vaccinated with BCG and subsequently given repeated oral doses of live Mycobacterium avium to model exposure to environmental mycobacteria. The protective efficacy of BCG with and without subsequent exposure to M. avium was determined following an aerogenic challenge with M. tuberculosis. Exposure of BCG-vaccinated mice to M. avium led to a persistent increase in the number of activated T cells within the brachial lymph nodes but similar T cell activation profiles in the lungs following infection with M. tuberculosis. The capacity of BCG-vaccinated mice to reduce the bacterial load following infection with M. tuberculosis was impaired in mice that had been exposed to M. avium. Our data suggest that exposure to environmental mycobacteria can negatively impact the protection afforded by BCG. These findings are relevant for the development of a vaccine administered in regions with elevated levels of environmental mycobacteria.