Induction of IFN-gamma-producing CD4+ natural killer T cells by Mycobacterium bovis bacillus Calmette Guérin

The CD4+ natural killer (NK)T cells in the liver are potent IL-4 producers and hence may promote Th2 cell development. Following Mycobacterium bovis bacillus Calmette Guérin (BCG) infection, IL-4-producing CD4+ NKT cells become undetectable in liver mononuclear cells of normal density (interface between 40 and 70% Percoll) by flow cytometry. The present study shows that M. bovis BCG infection changes the density of liver CD4+ NKT cells and shifts cytokine production from IL-4 to IFN-gamma. The number of CD4+ NK1+ TCR alpha/beta(intermediate) cells increased in the low-density fraction (<40% Percoll density gradient) in parallel to the reduction of this cell population in the fraction of normal density. The number of IL-4-producing cells, however, was small and high frequencies of IFN-gamma-secreting cells were identified in the low-density fraction after TCR/CD3 ligation. Accordingly, selected low-density CD4+ NKT cells encompassed high numbers of IFN-gamma producers and minute numbers of IL-4-secreting cells. Induction of low-density CD4+ NKT cells by M. bovis BCG was abrogated by endogenous IL-12 neutralization which also caused increased bacterial growth in the liver. We assume that M. bovis BCG infection changes cytokine secretion by the CD4+ NKT cell population from IL-4 to IFN-gamma through IL-12 induction. Thus, CD4+ NKT cells may contribute to host resistance against intracellular bacteria prior to conventional IFN-gamma-producing Th1 cells.     

Accelerated induction of mycobacterial antigen-specific CD8+ T cells in the Mycobacterium tuberculosis-infected lung by subcutaneous vaccination with Mycobacterium bovis bacille Calmette–Guérin

Both CD4⁺ and CD8⁺ T cells are important in protection against Mycobacterium tuberculosis infection. To evaluate the effect of vaccination with Mycobacterium bovis bacille Calmette–Guérin (BCG) on the CD8⁺ T-cell response to pulmonary M. tuberculosis infection, we analyzed the kinetics of CD8⁺ T cells specific to the mycobacterial Mtb32a_309–318 epitope, which is shared by M. tuberculosis and M. bovis BCG, in the lung of mice infected with M. tuberculosis. The CD8⁺ T cells were detected by staining lymphocytes with pentameric major histocompatibility complex (MHC) class I H-2D^b–Mtb32a_209–318 peptide complex and were analysed by flow cytometry. Mtb32a-specific CD8⁺ T cells became detectable on day 14, and reached a plateau on day 21, in the lung of M. tuberculosis-infected unvaccinated mice. Subcutaneous vaccination with M. bovis BCG in the footpads induced Mtb32a-specific CD8⁺ T cells in the draining lymph nodes (LNs) on day 7 and their numbers further increased on day 14. When M. bovis BCG-vaccinated mice were exposed to pulmonaryinfection with M. tuberculosis 4 weeks after vaccination, the Mtb32a-specific CD8⁺ T cells in the infected lung became detectable on day 7 and reached a plateau on day 14, which was 1 week earlier than in the unvaccinated mice. The pulmonary CD8⁺ T cells from the BCG-vaccinated M. tuberculosis-infected mice produced interferon-γ in response to Mtb32a_209–318 peptide on day 7 of the infection, whereas those of unvaccinated mice did not. The results demonstrate that induction of mycobacterial antigen-specific protective CD8⁺ T cells in the M. tuberculosis-infected lung is accelerated by subcutaneous vaccination with M. bovis BCG.     

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.     

Can soluble antigens induce CD8+ cytotoxic T-cell responses? A paradox revisited

Cytotoxic T lymphocyte (CTL) induction usually follows processing of antigens via endogenous pathways before presentation on the cell surface in association with MHC class I molecules. Soluble antigens do not, in general, induce specific CTL responses. Here, Syamal Raychaudhuri and John Morrow suggest that a novel adjuvant formulation can elicit CTL responses to soluble antigens and discuss the implications for vaccine development.     

Double- and monofunctional CD4⁺ and CD8⁺ T-cell responses to Mycobacterium tuberculosis DosR antigens and peptides in long-term latently infected individuals

More than 2 billion individuals are latently infected with Mycobacterium tuberculosis (Mtb). Knowledge of the key Mtb antigens and responding T-cell subsets mediating protection against Mtb is critical for developing improved tuberculosis (TB) vaccines. We previously reported that Mtb DosR-regulon-encoded antigens are recognized well by human T cells in association with control of Mtb infection. The characteristics of the responding T-cell subsets, however, remained unidentified. We have therefore studied the cytokine production and memory phenotypes of Mtb DosR-regulon-encoded antigen-specific T cells from individuals who had been infected with Mtb decades ago, yet never developed TB (long-term latent Mtb-infected individuals). Using multi-parameter flow cytometry and intracellular cytokine staining for IFN-γ, TNF-α and IL-2, we found double and single cytokine-producing CD4(+) as well as CD8(+) T cells to be the most prominent subsets, particularly IFN-γ(+) TNF-α(+) CD8(+) T cells. The majority of these T cells comprised effector memory and effector T cells. Furthermore, CFSE labeling revealed strong CD4(+) and CD8(+) T-cell proliferative responses induced by several "immunodominant" Mtb DosR antigens and their specific peptide epitopes. These findings demonstrate the prominent presence of double- and monofunctional CD4(+) and CD8(+) T-cell responses in naturally protected individuals and support the possibility of designing Mtb DosR antigen-based TB vaccines.     

Comparative protective effects of recombinant DNA and Mycobacterium bovis bacille Calmette-Guérin vaccines against M. avium infection.

A range of strategies are being explored to develop more effective vaccines against mycobacterial infection, including immunization with DNA plasmids encoding single mycobacterial bacterial genes and the use of recombinant live vectors based on the current vaccine, Mycobacterium bovis bacille Calmette-Guérin (BCG). We have compared these two approaches using a model of virulent M. avium infection, and the gene for the immunodominant 35 kDa protein which is shared by M. avium and M. leprae, but absent from BCG. Recombinant BCG over-expressing the M. avium 35 kDa protein (BCG-35) induced strong antigen-specific proliferative and interferon-gamma (IFN-gamma)-secreting T cell responses. These were comparable to those induced by a single immunization with a plasmid expressing the same antigen (DNA-35); however, repeat DNA-35 immunization evoked the strongest IFN-gamma release. Immunization with BCG-35 significantly reduced the growth of virulent M. avium, although this effect was similar to that induced by wild-type BCG. Immunization with DNA-35 resulted in significantly greater (2 x log(10)) reduction in the growth of M. avium. Prime-boost strategies combining DNA-35 and BCG-35 increased the protective effect above that achieved by BCG-35, but they were not more protective than DNA-35 alone. Therefore, recombinant BCG-35 and BCG induced similar levels of protection in this model, and maximal protection against M. avium infection was attained by immunization with DNA encoding the 35 kDa protein.     

Characterization of human Mycobacterium bovis bacille Calmette-Guérin-reactive CD8+ T cells.

Gamma interferon (IFN-gamma)-secreting CD4+ T cells have long been established as an essential component of the protective immune response against Mycobacterium tuberculosis. It is now becoming evident from studies with the murine model of tuberculosis that an important role also exists for major histocompatibility complex (MHC) class I-restricted CD8+ T cells. These cells are capable of acting as both IFN-gamma secretors and cytotoxic T lymphocyte (CTL) effectors; however, their exact role in immunity against tuberculosis remains unclear. This study demonstrates the presence of Mycobacterium bovis BCG-reactive CD8+ T cells in healthy BCG-vaccinated donors and that these CD8+ T cells are potent cytokine producers as well as cytotoxic effector cells. Using FACScan analysis, we have shown that restimulation with live M. bovis BCG induced more CD8+-T-cell activation than the soluble antigen purified protein derivative and that these cells are actively producing the type 1 cytokines IFN-gamma and tumor necrosis factor alpha (TNF-alpha). These CD8+ T cells also contain the cytolytic granule perforin and are capable of acting as potent CTLs against M. bovis BCG-infected macrophages. The mycobacterial antigens 85A and B (Ag85A and Ag85B, respectively), and to a lesser extent the 19- and 38-kDa proteins, are major antigenic targets for these mycobacterium-specific CD8+ T cells, while whole-M. bovis BCG activated effector cells from these BCG-vaccinated donors, as expected, failed to recognize the 6-kDa ESAT-6 protein. The use of metabolic inhibitors and blocking antibodies revealed that the CD8+ T cells recognize antigen processed and presented via the classical MHC class I pathway. These data suggest that CD8+ T cells may play a critical role in the human immune response to tuberculosis infection.     

Mycobacterium bovis abdominal aortic and femoral artery aneurysms following intravesical bacillus Calmette–Guérin therapy for bladder cancer

BackgroundInfectious complications of intravesical bacillus Calmette–Guérin (BCG) therapy are rare, but these have included a handful of cases of mycotic aneurysm.Methods and ResultsWe present the case of a patient with a ruptured abdominal aortic aneurysm and a femoral artery aneurysm who had previously received intravesical BCG therapy for bladder carcinoma. Histopathologic examination of resected tissue revealed numerous acid-fast bacilli, and subsequent mycobacterial culture of blood and resected tissue revealed BCG strain Mycobacterium bovis.ConclusionsClinicians should be aware of the possible extravesical complications, albeit rare, of BCG therapy. Therapy should consist of combined medical and surgical management.     

Complex genetic control of susceptibility to Mycobacterium bovis (Bacille Calmette-Guérin) infection in wild-derived Mus spretus mice

Susceptibility to Mycobacterium bovis Bacille Calmette-Guérin (BCG) is genetically controlled by Nramp1 (Slc11a1). Inbred mouse strains harbor either the resistance (Nramp1(G169)) or the susceptibility (Nramp1(D169)) allele at Nramp1. Mus spretus (Nramp1(G169); SPRET/EiJ) is shown to display an intermediate level of BCG replication in the spleen (log(10) colony-forming units (CFU) approximately 5), compared to resistant A/J (log(10)CFU approximately 4.0) and susceptible C57BL/6J (log(10)CFU approximately 6.0) mice. The presence of genetic modifiers of Nramp1-dependent susceptibility to M. bovis (BCG) infection in Mus spretus was analyzed by whole-genome scanning in 175 mice of an informative (C57BL/6J x SPRET/EiJ) x C57BL/6J backcross. Nramp1 showed a major effect (D1Mcg4, P<1e(-4)), but additional single marker effects were identified on chromosomes 4 (D4Mit150) and x (DXMit249) in male mice, and on chromosome 9 (D9Mit77) and 17 (D17Mit81) in female mice. A strong interaction between Nramp1 and the major histocompatibility locus was also noted in female mice. The mapped loci may act as modifiers of Nramp1 action, and constitute novel entry points for the parallel search of loci regulating susceptibility to mycobacterial infections in humans.     

Bacillus Calmette-Guérin Suppresses Asthmatic Responses via CD4+CD25+ Regulatory T Cells and Dendritic Cells

Purpose

Bacillus Calmette-Guérin (BCG) is known to suppress the asthmatic responses in a murine model of asthma and to induce dendritic cells (DCs) maturation. Mature DCs play a crucial role in the differentiation of regulatory T cells (Tregs), which are known to regulate allergic inflammatory responses. To investigate whether BCG regulates Tregs in a DCs-mediated manner, we analyzed in a murine model of asthma.

Methods

BALB/c mice were injected intraperitoneally with BCG or intravenously with BCG-stimulated DCs and then sensitized and challenged with ovalbumin (OVA). Mice were analysed for bronchial hyperresponsiveness (BHR), the influx of inflammatory cells in the bronchoalveolar lavage (BAL) fluid, and histopathological changes in the lung. To identify the mechanisms, IgE, IgG₁ and IgG_2a in the serum were analysed and the CD25₊ Tregs in the mice were depleted with anti-CD25 monoclonal antibody (mAb).

Results

BCG and the transfer of BCG-stimulated DCs both suppressed all aspects of the asthmatic responses, namely, BHR, the production of total IgE and OVA-specific IgE and IgGs, and pulmonary eosinophilic inflammation. Anti-CD25mAb treatment reversed these effects.

Conclusions

BCG can attenuate the allergic inflammation in a mouse model of asthma by a Tregs-related mechanism that is mediated by DCs.     

From crucial to negligible: functional CD8⁺ T-cell responses and their dependence on CD4⁺ T-cell help

CD8(+) T cells play an important role in controlling pathogenic infections and are therefore key players in the immune response. It has been shown that among other factors CD4(+) T cells can shape the magnitude as well as the quality of primary and/or secondary CD8(+) T-cell responses. However, due to the complexity and the differences among diverse immunization or infection models, the overall requirement, the time points, as well as the specific mechanism(s) of CD4(+) T-cell help may differ substantially. Here, we summarize current knowledge about the differential requirement of CD4(+) T-cell help in promoting primary CD8(+) T-cell responses as well as establishing functional memory CD8(+) T cells in various experimental settings.     

CD103−CD11b+ dendritic cells regulate the sensitivity of CD4 T-cell responses to bacterial flagellin

Toll-like receptor 5 (TLR5) has been widely studied in an inflammatory context, but the effect of TLR5 on the adaptive response to bacterial flagellin has received considerably less attention. Here, we demonstrate that TLR5 expression by dendritic cells (DCs) allows a 1,000-fold enhancement of T-cell sensitivity to flagellin, and this enhancement did not require the expression of NLRC4 or Myd88. The effect of TLR5 on CD4 T-cell sensitivity was independent of the adjuvant effect of flagellin and TLR5 ligation did not alter the sensitivity of ovalbumin (OVA)-specific T cells to OVA. In the spleen, the exquisite T-cell sensitivity to flagellin was regulated by CD4−CD8α− DCs and was blocked by a monoclonal antibody to TLR5. In the mesenteric lymph nodes, flagellin-specific T-cell activation was regulated by a population of CD103−CD11b+ DCs. Thus, TLR5 expression by mucosal and systemic DC subsets controls the sensitivity of the adaptive immune response to flagellated pathogens.     

Human CD56bright and CD56dim natural killer cell subsets respond differentially to direct stimulation with Mycobacterium bovis bacillus Calmette-Guérin

Mycobacterium bovis bacillus Calmette-Guérin (BCG) is capable of directly stimulating several effector functions of human natural killer (NK) cells in the absence of interleukin-12 and professional antigen presenting cells. To assess the contribution of two main human NK-cell subsets (CD56(dim) and CD56(bright)) to the overall in vitro NK-cell response to BCG, peripheral blood mononuclear cells depleted of nylon wool-adherent cells or purified NK cells were stimulated with live BCG. By combining intranuclear bromodeoxyuridine (BrdU) staining and analysis of CD56 marker intensity, statistically higher percentages of BrdU(+) cells were found among the CD56(bright) subset than the CD56(dim) subset after 6 days of stimulation with BCG. Similarly, evaluation of intracellular interferon-gamma (IFN-gamma) revealed that CD56(bright) cells were those mainly involved in IFN-gamma production in response to BCG. In contrast, the CD56(dim) subset contained higher levels of perforin and granzyme A, two key molecules for exocytosis-mediated cytotoxicity, than the CD56(bright) subset. Although 16-20-h stimulation with BCG did not substantially alter the expression of cytotoxic molecules by the two subsets, a decrease in perforin content was observed in the CD56(dim), but not in the CD56(bright) subset, following 4-h incubation with the NK-sensitive target K562 cell line. This decrease in perforin content correlated with the induction by BCG-stimulated NK cells, of early markers of apoptosis on target cells to a greater extent than unstimulated cells suggesting a major role for the CD56(dim) subset in cytotoxic activity in response to BCG. Taken together, these results demonstrate that CD56(bright) and CD56(dim) human NK-cell subsets exert different functional activities in response to a live bacterial pathogen.     

Splenic CD8α⁺ dendritic cells undergo rapid programming by cytosolic bacteria and inflammation to induce protective CD8⁺ T-cell memory

Memory CD8⁺ T lymphocytes are critical effector cells of the adaptive immune system mediating long‐lived pathogen‐specific protective immunity. Three signals – antigen, costimulation and inflammation – orchestrate optimal CD8⁺ T‐cell priming and differentiation into effector and memory cells and shape T‐cell functional fate and ability to protect against challenge infections. While among the conventional spleen DCs (cDCs), the CD8α⁺ but not the CD8α^? cDCs most efficiently mediate CD8⁺ T‐cell priming, it is unclear which subset, irrespective of their capacity to process MHC class I‐associated antigens, is most efficient at inducing naïve CD8⁺ T‐cell differentiation into pathogen‐specific protective memory cells in vivo. Moreover, the origin of the required signals is still unclear. Using mice infected with the intracellular bacterium Listeria monocytogenes, we show that splenic CD8α⁺ cDCs become endowed with all functional features to optimally prime protective memory CD8⁺ T cells in vivo within only a few hours post‐immunization. Such programming requires both cytosolic signals resulting from bacterial invasion of the host cells and extracellular inflammatory mediators. Thus, these data designate these cells as the best candidates to facilitate the development of cell‐based vaccine therapy.     

Robust interferon-α and IL-12 responses by dendritic cells are related to efficient CD4+ T-cell recovery in HIV patients on ART

Amongst HIV patients with successful virological responses to antiretroviral therapy (ART), poor CD4(+) T-cell recovery is associated with low nadir CD4(+) T-cell counts and persistent immune activation. These factors might be influenced by dendritic cell (DC) function. Interferon-α-producing plasmacytoid DC and IL-12-producing myeloid DC were quantified by flow cytometry after stimulation with agonists to TLR7/8 (CL075) or TLR9 (CpG-ODN). These were compared between patients who achieved CD4(+) T-cell counts above or below 200 cells/μL after 6 months on ART (High vs. Low groups). High Group patients had more DC producing interferon-α or IL-12 at Weeks 6 and 12 on ART than Low Group patients. The frequencies of cytokine-producing DC at Week 12 were directly correlated with CD4(+) T-cell counts at baseline and at Week 12. Patients with good recovery of CD4(+) T-cells had robust TLR-mediated interferon-α responses by plasmacytoid DC and IL-12 responses by myeloid DC during early ART (1-3 months).     

Differential sensitivity of naïve and subsets of memory CD4+ and CD8+ T cells to hydrogen peroxide-induced apoptosis

CD4+ and CD8+ memory T cells are identified into central and effector memory subsets, which are characterized by distinct homing patterns and functions. In this investigation, we show that na?ve and central memory CD4+ and CD8+ T cells are sensitive to hydrogen peroxide (H2O2)-induced apoptosis, whereas effector memory CD4+ and CD8+ T cells are relatively resistant to H2O2-induced apoptosis. Apoptosis in na?ve and central memory CD4+ and CD8+ is associated with the release of cytochrome c and activation of caspase-9 and caspase-3, upregulation of Bax and voltage-dependent anion channel (VDAC) expression, and decreased intracellular glutathione (GSH). In vitro GSH and a superoxide dismutase mimetic Mn(III) tetrakis (1-methyl-4-pyridyl) porphyrin inhibited H2O2-induced apoptosis in both na?ve and central memory CD4+ and CD8+ T cells. Furthermore, VDAC inhibitor 4,4'-diisothiocynostilbene-2,2'-disulfonic acid blocked H2O2-induced apoptosis. These data demonstrate that H2O2 induces apoptosis preferentially in human na?ve and central memory CD4+ and CD8+ T cells via the mitochondrial pathway by regulating intracellular GSH and the expression of Bax and VDAC.     

The capacity of the natural ligands for CD28 to drive IL-4 expression in naïve and antigen-primed CD4+ and CD8+ T cells

The B7/CD28 costimulatory pathway plays a critical role in T cell activation including Th1/Th2 differentiation. However, little is known about whether CD28 costimulation favors polarization of either Th1 and Th2 or both. Here, we show a critical role of the natural ligands for CD28 molecules (B7.2-Ig or B7.1-Ig fusion proteins), particularly in the induction of type 2 T cell polarization. Upon TCR-triggering with suboptimal doses of anti-CD3, costimulation of na?ve CD4+ T cells with anti-CD28 mAb or B7-Ig fusion proteins led to comparable levels of IFN-gamma production. Na?ve T cells could produce IL-4 when CD28 costimulation was done with B7-Ig, but not with anti-CD28. IL-4-selective upregulation was also observed when T cells from anti-OVA TCR transgenic mice were stimulated with OVA in the presence of B7-Ig. Correlating with IL-4 expression, GATA-3 expression was induced much more potently by costimulation with B7-Ig than with anti-CD28 mAb, while T-bet induction by these two costimulatory reagents was comparable. This B7 effect was also applied for na?ve and antigen-primed CD8+ T cells: IL-4-expressing CD8+ T cells were generated when na?ve and alloantigen-primed T cells were stimulated with anti-CD3 and recall antigens, respectively, in the presence of B7-Ig costimulation. Importantly, such CD8+ T cell differentiation required the coexistence of CD4+ T cells during the initial TCR stimulation. These observations indicate that both type 2 CD4 and CD8 T cell polarizations are efficiently induced via costimulation of CD28 with its natural ligands, although the differentiation of CD8+ T cells is dependent on CD4+ cells.