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.     

Mycobacterium bovis bacillus Calmette-Guérin (BCG) induces human CC- and CXC-chemokines in vitro and in vivo

Both CC- and CXC-chemokines are known to be potent leucocyte activators and chemoattractants and play important roles in inflammatory responses. However, chemokine response to bacillus Calmette-Guérin (BCG) infection remains incompletely defined. In this study, we investigated human CC- [macrophage-derived chemokine (MDC), monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1alpha and eosinophil chemoattractant activity (eotaxin)] and CXC-interferon-inducible protein (IP)-10 chemokine production in response to BCG stimulation. BCG efficiently induced all chemokines tested in the urine of four bladder cancer patients undergoing intravesical BCG immunotherapy. The peak urinary chemokine responses occurred generally between the fourth and sixth weekly treatment, except eotaxin, which was less predictable. To evaluate the effect of BCG on induction of chemokines in vitro, urothelial cell lines and peripheral blood mononuclear cells (PBMCs) were used. Although BCG induced no or marginal chemokines from urothelial SV-HUC-1, RT4 and T24 cells, BCG-derived cytokines [interleukin (IL)-1beta, interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha] induced all chemokines tested except eotaxin from these cell lines. BCG also efficiently induced all chemokines tested except eotaxin from PBMCs of both BCG-naive and BCG-vaccinated subjects. MCP-1 and MIP-1alpha emerged at 4-5 h post-BCG exposure (early chemokines); IP-10 elevated at day 1 and peaked at day 2 (intermediate chemokine); and MDC elevated at day 1 and peaked at day 7 (late chemokine). This kinetic pattern was paralleled with that of BCG-induced cytokines [early: TNF-alpha; intermediate: IL-6 and IL-10; and late: IFN-gamma and granulocyte-macrophage colony-stimulating factor (GM-CSF)]. Taken together, these results indicate that BCG directly or indirectly induces human CC- and CXC-chemokine production, which may represent one of the mechanisms by which BCG exerts its anti-tumour activity.     

Interferon-alphabeta mediates partial control of early pulmonary Mycobacterium bovis bacillus Calmette-Guérin infection

The role of type I interferon (IFN-alphabeta) in modulating innate or adaptive immune responses against mycobacterial infection in the lung is unclear. In this study we investigated the susceptibility of IFN-alphabeta-receptor-deficient (IFN-alphabetaR-/-) mice to pulmonary infection with aerosolized Mycobacterium bovis bacillus Calmette-Guérin (BCG). During early infection (2-3 weeks), enhanced growth of BCG was measured in the lungs of IFN-alphabetaR-/- mice compared to wild-type mice. However, during late infection the burden of BCG was similar in the lungs of IFN-alphabetaR-/- and wild-type mice. Although control of BCG growth was delayed, recruitment and activation of T and natural killer cells, production of IFN-gamma, and cytokine expression were all similar in wild-type and IFN-alphabetaR-/- mice. However, decreased expression of nitric oxide in bronchoalveolar lavage fluids from IFN-alphabetaR-/- mice correlated with enhanced growth of BCG. Bone marrow-derived macrophages from IFN-alphabetaR-/- mice also produced less nitric oxide following infection with BCG in vitro. These findings suggest that IFN-alphabeta contributes to innate immunity to pulmonary mycobacterial infection by augmenting production of nitric oxide.     

Cytologic features of disseminated bacillus Calmette-Guérin (BCG) infection

Disseminated bacillus Calmette-Guérin (BCG) infection is an unusual complication of immunization against Mycobacterium tuberculosis with the bacillus Calmette-Guérin. We report on 4 such cases in which the diagnosis was suspected at the fine-needle aspiration biopsy (FNAB) procedure. Participants were 4 males (mean age, 21.5 mo; range, 8-36 mo) in good general condition, in whom epidemiology data favoring tuberculosis and presence of pulmonary tuberculosis were lacking. Cases 1 and 2 presented with a deep-seated subcutaneous nodule located near the left mamilla and lower aspect of the left scapula, respectively, resulting from lymph node involvement by BCG. Cases 3 and 4 presented as an osteolytic lesion of the ninth right rib and right iliac bone, respectively. FNAB findings showed poorly to moderately cellular smears. Epithelioid histiocytes in a granuloma pattern with occasional multinucleated Langerhans-type giant cells, lymphocytes, and polymorphonuclear leukocytes in a finely granular background with necrotic debris were found in all cases. The presence of isolated calcified spherules interspersed among the cells was found to be a useful finding for diagnosis. When dealing with disseminated BCG infections, clinical and cytological pictures must be evaluated as a whole in order to arrive at an accurate diagnosis.     

Recombinant Mycobacterium bovis bacillus Calmette-Guérin (BCG) expressing mouse IL-18 augments Th1 immunity and macrophage cytotoxicity

Interleukin-18 (IL-18) has been demonstrated to synergize with BCG for induction of a T-helper-type 1 (Th1) immune response. Since successful treatment of superficial bladder cancer with BCG requires proper induction of Th1 immunity, we have developed a recombinant (r) BCG strain that functionally secretes mouse (m) IL-18. This rBCG-mIL-18 strain significantly increased production of the major Th1 cytokine IFN-gamma in splenocyte cultures, at levels comparable to that elicited by control BCG plus exogenous rIL-18. IFN-gamma production by splenocytes was eliminated by addition of neutralizing anti-IL-18 antibody. Endogenous IL-12 played a favourable role whereas IL-10 played an adverse role in rBCG-mIL-18-induced IFN-gamma production. Enhanced host antimycobacterial immunity was observed in mice infected with rBCG-mIL-18 which showed less splenic enlargement and reduced bacterial load compared to control mice infected with BCG. Further, splenocytes from rBCG-mIL-18-infected mice, in response to BCG antigen, displayed increased production of IFN-gamma and GMCSF, decreased production of IL-10, elevated cellular proliferation and higher differentiation of IFN-gamma-secreting cells. rBCG-mIL-18 also enhanced BCG-induced macrophage cytotoxicity against bladder cancer MBT-2 cells in a dose-dependent manner. Neutralizing all endogenous macrophage-derived cytokines tested (IL-12, IL-18 and TNF-alpha) as well as IFN-gamma severely diminished the rBCG-mIL-18-induced macrophage cytolytic activity, indicating a critical role for these cytokines in this process. Cytokine analysis for supernatants of macrophage-BCG mixture cultures manifested higher levels of IFN-gamma and TNF-alpha in rBCG-mIL-18 cultures than in control BCG cultures. Taken together, this rBCG-mIL-18 strain augments BCG's immunostimulatory property and may serve as a better agent for bladder cancer immunotherapy and antimycobacterial immunization.     

Improved immunogenicity of recombinant Mycobacterium bovis bacillus Calmette-Guérin strains expressing fusion protein Ag85A-ESAT-6 of Mycobacterium tuberculosis

Early secretory antigen target 6 (ESAT-6) is a dominant target for cell-mediated immunity in the early phase of tuberculosis (TB) in patients with TB, causing T-cell proliferation and gamma interferon (IFN-γ) production, which has been considered to be a protective antigen that can be used for future vaccine development. Ag85A is the most essential component for bacterial survival within macrophages and has been used in numerous vaccine preparations, which can induce strong cellular immune responses. In this study, we constructed a new recombinant bacilli Calmette-Guérin (BCG) strain (rBCG-AE) that could express fusion protein Ag85A-ESAT-6 of Mycobacterium tuberculosis and evaluated its immunogenicity in BALB/c mice. There was no evidence for increased virulence of this rBCG. Our experiments illustrated that the rBCG-AE was able to induce higher titer of antibody and elicit more long-lasting and stronger Th1 type cellular immune responses than the parental BCG strain, or rBCG-A (expressing Ag85A) strain, or rBCG-E (expressing ESAT-6) strain, which are characterized by the strong antibody response, the proliferation rate of splenocytes, the ratio of CD4(+) T and CD8(+) T cells stimulated by tuberculin-purified protein derivative and elevated levels of IFN-γ in antigen-stimulated splenocyte cultures. The results show that rBCG-AE is an improved TB vaccine for further study.     

Interleukin-15 as an immune adjuvant to increase the efficacy of Mycobacterium bovis bacillus Calmette-Guérin vaccination

Interleukin-15 (IL-15) transgenic mice which had been inoculated with Mycobacterium bovis bacillus Calmette-Guérin (BCG) 24 weeks previously showed resistance against airborne infection with Mycobacterium tuberculosis H37Rv accompanied by an increased CD8(+)-Tc1-cell response. IL-15 may be used as an immune adjuvant given with BCG vaccination to enhance its biologic efficacy.     

Human leucocytes response to viable, extended freeze-drying or heat-killed Mycobacterium bovis bacillus Calmette-Guérin

We investigated the effects of viable, extended freeze-drying (EFD) or heat-killed (HK) Mycobacterium bovis bacillus Calmette-Guérin (BCG) in respiratory burst activity, gene expression of CYBB and NCF1 encoding components of the human phagocyte nicotinamide adenine dinucleotide (NADPH) oxidase, TLR2 expression, and in IL-10 and TNF-α cytokine production by human peripheral blood mononuclear cells (PBMCs). Viable BCG significantly inhibited TLR2 and CYBB gene expression, as well as superoxide release by human PBMC. All BCG stimuli augmented IL-10 release, but only HK BCG or viable BCG increased TNF-α release by PBMCs. Our studies show that viable BCG can impair the NADPH oxidase system activation and the TLR2 route in human PBMCs. As well, different BCG preparations can distinctly influence cytokine production by human PBMCs.     

Simultaneous blocking of human Toll-like receptors 2 and 4 suppresses myeloid dendritic cell activation induced by Mycobacterium bovis bacillus Calmette-Guérin peptidoglycan

The Mycobacterium bovis bacillus Calmette-Guérin (BCG) cell wall skeleton (CWS) consists of mycolic acids, arabinogalactan, and peptidoglycan (PGN) and activates Toll-like receptor 2 (TLR2) and TLR4. Here we investigated the ability of the essential portion of highly purified BCG CWS to support the TLR agonist function by using the following criteria: myeloid dendritic cell (DC) maturation, i.e., tumor necrosis factor alpha (TNF-alpha) production and CD83/CD86 up-regulation. The purified PGN region was sufficient to activate TLR2 and TLR4 in mouse DCs and macrophages; in TLR2 and TLR4 double-knockout cells the BCG PGN-mediated TNF-alpha production ability was completely impaired. Likewise, stimulation with BCG CWS of HEK293 cells expressing either human TLR2 or TLR4, MD-2, and CD14 resulted in NF-kappa B activation as determined by a reporter assay. Notably, specific blockers of extracellular human TLR2 (an original cocktail of monoclonal antibodies TLR2.45 and TH2.1) and TLR4 (E5531) inhibited BCG CWS-mediated NF-kappa B activation by 80%. Using this human TLR blocking system, we tested whether human myeloid DC maturation was TLR2 and TLR4 dependent. BCG PGN-mediated DC maturation was blocked by 70% by suppression of both TLR2 and TLR4 and by 30 to 40% by suppression of either of these TLRs. Similar but less profound suppression of BCG CWS-mediated DC maturation was observed. Hence, the presence of BCG PGN is a minimal requirement for activation of both TLR2 and TLR4 in human DCs, unlike the presence of PGNs of gram-positive bacteria, which activate only TLR2. Unexpectedly, however, BCG PGN, unlike BCG CWS, barely activated NF-kappa B in HEK293 cells coexpressing TLR2 plus TLR1, TLR2 plus TLR4, TLR2 plus TLR6, or TLR2 plus TLR10, suggesting that PGN receptors other than TLR2 and TLR4 present on human DCs but not on HEK293 cells are involved in TLR signaling for DC activation.     

Maturation of human dendritic cells by cell wall skeleton of Mycobacterium bovis bacillus Calmette-Guérin: involvement of toll-like receptors

The constituents of mycobacteria are an effective immune adjuvant, as observed with complete Freund's adjuvant. In this study, we demonstrated that the cell wall skeleton of Mycobacterium bovis bacillus Calmette-Guérin (BCG-CWS), a purified noninfectious material consisting of peptidoglycan, arabinogalactan, and mycolic acids, induces maturation of human dendritic cells (DC). Surface expression of CD40, CD80, CD83, and CD86 was increased by BCG-CWS on human immature DC, and the effect was similar to those of interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha), heat-killed BCG, and viable BCG. BCG-CWS induced the secretion of TNF-alpha, IL-6, and IL-12 p40. CD83 expression was increased by a soluble factor secreted from BCG-CWS-treated DC and was completely inhibited by monoclonal antibodies against TNF-alpha. BCG-CWS-treated DC stimulated extensive allogeneic mixed lymphocyte reactions. The level of TNF-alpha secreted through BCG-CWS was partially suppressed in murine macrophages with no Toll-like receptor 2 (TLR 2) or TLR4 and was completely lost in TLR2 and TLR4 double-deficient macrophages. These results suggest that the BCG-CWS induces TNF-alpha secretion from DC via TLR2 and TLR4 and that the secreted TNF-alpha induces the maturation of DC per se.     

Detection of bacillus Galmette-Guérin (Mycobacterium bovis BCG) DNA in urine and blood specimens after intravesical immunotherapy for bladder carcinoma

A real-time PCR targeting IS6110 was employed for the detection of Mycobacterium tuberculosis DNA in specimens collected from 10 patients treated with intravesical M. bovis bacillus Galmette-Guérin (BCG) immunotherapy for bladder malignancy. BCG DNA was detected in all urine specimens taken 24 h after the instillations, as well as in 24% of the specimens collected 7 days after the instillations; it was also detected in a single specimen taken 6 weeks after the last instillation. BCG DNA was detected in 8.3% of the blood specimens taken 1 day after instillation, and its amplification was associated with cases of self-limiting fever. These findings give indications that this real-time PCR is helpful to recognize BCG bacteremic cases, which may lead to mycobacterial infection.     

Interleukin-10 inhibits Mycobacterium bovis bacillus Calmette�CGu��rin (BCG)-induced macrophage cytotoxicity against bladder cancer cells

The mechanisms underlying bacillus Calmette–Guérin (BCG) immunotherapy of bladder cancer currently remain elusive. Previously, we demonstrated that macrophages were cytotoxic to bladder cancer cells upon BCG stimulation in vitro. However, macrophages from C57BL/6 mice were less potent than those from C3H/HeN mice for the killing of bladder cancer cells. This study was to determine whether interleukin (IL)-10 produced by macrophages in response to BCG is a causative factor for the reduced cytotoxicity in BCG-stimulated C57BL/6 macrophages. Thioglycollate-elicited peritoneal macrophages were prepared and analysed for the BCG induction of cytotoxicity, cytokines and nitric oxide (NO) in vitro. Compared to BCG-stimulated C3H/HeN macrophages, BCG-stimulated C57BL/6 macrophages exhibited reduced killing of bladder cancer MBT-2 cells and MB49 cells. Studies demonstrated further that BCG-stimulated C57BL/6 macrophages produced a high level of IL-10, which correlated with reduced production of tumour necrosis factor (TNF)-α, IL-6 and NO. Neutralizing endogenous IL-10 during BCG stimulation increased C57BL/6 macrophage cytotoxicity against MB49 cells by 3·2-fold, along with increased production of TNF-α by 6·4-fold and NO by 3·6-fold, respectively. Macrophages from C57BL/6 IL-10^−/− mice also exhibited increased killing of MB49 cells and production of TNF-α and NO upon BCG stimulation. In addition, supplementation of exogenous recombinant IL-10 reduced BCG-induced C3H/HeN macrophage cytotoxicity against both MBT-2 cells and MB49 cells in a dose-dependent manner. These results reveal the inhibitory role of IL-10 in BCG-induced macrophage cytotoxicity, suggesting that blockage of IL-10 may potentially enhance the effect of BCG in the treatment of bladder cancer patients.     

Identification of distinct lymphocyte subsets responding to subcellular fractions of Mycobacterium bovis bacille calmette-Guérin (BCG)

In order to investigate the ability of Mycobacterium bovis BCG vaccination to induce immune responses toward different classes of mycobacterial antigens and the cell populations involved in such responses, proliferation of distinct human lymphocyte subsets from BCG-vaccinated donors in response to different subcellular fractions of BCG was analysed and compared with that of not sensitized subjects. Proliferation of different cell subsets was evaluated by flow cytometric determination of bromodeoxyuridine incorporated into DNA of dividing cells and simultaneous identification of cell surface markers. Although a certain degree of variability was observed among different donors, after 6 days of in vitro stimulation BCG-vaccinated subjects displayed, as a mean, a stronger blastogenic response to all the classes of antigens compared with non-sensitized ones. PPD, culture filtrates and membrane antigens induced a predominant proliferation of CD4+ T cells. In contrast, preparations enriched in cytosolic antigens elicited strong proliferation of gammadelta+ T cells which, as a mean, represented 55% of the proliferating cells. Although to a lesser extent, proliferation of gammadelta+ T cells was also elicited by preparations enriched in membrane and cell wall antigens. In response to the latter preparation proliferation of CD4+ T cells and CD16+/CD3- (natural killer (NK)) cells was observed, as well. In particular, cell wall antigens were found to induce significantly higher levels of proliferation of NK cells compared with all the other classes of antigens.     

Functional characterization of human natural killer cells responding to Mycobacterium bovis bacille Calmette-Guérin

The kinetics of activation and induction of several effector functions of human natural killer (NK) cells in response to Mycobacterium bovis bacille Calmette-Guérin (BCG) were investigated. Owing to the central role of monocytes/macrophages (MM) in the initiation and maintenance of the immune response to pathogens, two different experimental culture conditions were analysed. In the first, monocyte-depleted nylon wool non-adherent (NW) cells from healthy donors were stimulated with autologous MM preinfected with BCG (intracellular BCG). In the second, the NW cells were directly incubated with BCG, which was therefore extracellular. In the presence of MM, CD4+ T lymphocytes were the cell subset mainly expressing the activation marker, CD25, and proliferating with a peak after 7 days of culture. In contrast, in response to extracellular BCG, the peak of the proliferative response was observed after 6 days of stimulation, and CD56+ CD3- cells (NK cells) were the cell subset preferentially involved. Such proliferation of NK cells did not require a prior sensitization to mycobacterial antigens, and appeared to be dependent upon contact between cell populations and bacteria. Following stimulation with extracellular BCG, the majority of interferon-gamma (IFN-gamma)-producing cells were NK cells, with a peak IFN-gamma production at 24-30 hr. Interleukin (IL)-2 and IL-4 were not detectable in NK cells or in CD3+ T lymphocytes at any time tested. IL-12 was not detectable in the culture supernatant of NW cells stimulated with extracellular BCG. Compared to the non-stimulated NW cells, the NW cells incubated for 16-20 hr with BCG induced the highest levels of expression of apoptotic/death marker on the NK-sensitive K562 cell line. BCG also induced expression of the activation marker, CD25, and proliferation, IFN-gamma production and cytotoxic activity, on negatively selected CD56+ CD3- cells. Altogether, the results of this study demonstrate that extracellular mycobacteria activate several NK-cell functions and suggest a possible alternative mechanism of NK-cell activation as the first line of defence against mycobacterial infections.     

Restricted cyclooxygenase-2 expression in the central nervous system following acute and delayed-type hypersensitivity responses to bacillus Calmette-Guérin.

The expression of cyclooxygenase-2, a key enzyme in prostaglandin and thromboxane synthesis in inflammation, was studied immunohistochemically in in vivo models of acute and chronic inflammatory responses in rat central nervous system. In the acute inflammatory response to intracranial injection of heat-killed bacillus Calmette-Guérin as well as in the immune-mediated, delayed-type hypersensitivity response to the same pathogen, cyclooxygenase-2 expression was restricted to major infiltrating haematogenous cell populations such as neutrophils and mononuclear phagocytes, while the expression of the enzyme by brain non-neuronal resident cells (astrocytes, microglia, perivascular cells) appeared to be limited to perivascular cells of the blood vessels in the vicinity of the lesion and in the surrounding area. On the basis of their morphology and location, these perivascular cells were identified as perivascular macrophages, but we could not rule out the possibility that some endothelial cells also expressed cyclooxygenase-2. The constitutive neuronal cyclooxygenase-2 was not affected by the ongoing inflammation. Interestingly, in spite of the extensive astrocyte and microglial reaction occurring over a broad area surrounding the inflammatory lesions, there was no obvious cyclooxygenase-2 staining in these cells. These data indicate that the up-regulation of cyclooxygenase-2 expression in acute and chronic, immune-mediated lesions in the brain parenchyma is remarkably restricted to the lesion site. Since cyclooxygenase metabolites can regulate important functions of resident as well as infiltrating cells, the increased synthesis of prostaglandins and thromboxanes, which is likely to occur as a consequence of the expression of cycloxygenase-2 at the lesion site, might represent an important component of the inflammatory processes within the brain.     

Virulence,immunogenicity, and protective efficacy of two recombinant Mycobacterium bovis bacillus Calmette-Guérin strains expressing the antigen ESAT-6 from Mycobacterium tuberculosis

We constructed two recombinant Mycobacterium bovis BCG (rBCG) strains expressing ESAT-6 of Mycobacterium tuberculosis, named rBCG-1 and rBCG-2. rBCG-1 contained the ESAT-6 gene linked to BCG hsp60 and expressed a fusion protein, while rBCG-2, with a secretory sequence, could secret ESAT-6 into the culture medium. There was no evidence for increased virulence of the two rBCG strains when we made a comparison between them and BCG with regard to organ bacterial loads, lung histology, and survival time. rBCG-1 induced significantly higher specific antibody titers and stronger cellular immune response than BCG, whereas rBCG-2 had immunogenicity similar to that of the parental BCG strain. Both rBCG-1 and rBCG-2 conferred marked protection against M. tuberculosis infection, yet in terms of protective efficacy, they showed no significant improvements upon conventional BCG vaccine.     

PD-1-PD-L1 pathway impairs T(h)1 immune response in the late stage of infection with Mycobacterium bovis bacillus Calmette-Guérin

A major concern still prevails as to the reason why various mycobacteria are able to persist within infected host in which protective immunity is generated. To address this question, we monitored the generation of protective T cells during infection with Mycobacterium bovis bacillus Calmette-Guérin (BCG). CD4(+) T cells obtained 3 weeks after infection conferred protection against Mycobacterium tuberculosis challenge and produced IFN-γ and tumor necrosis factor (TNF)-α upon antigen stimulation. However, these abilities were decreased after 6 weeks of infection even though BCG was not thoroughly eliminated from the host. We analyzed the expression of ligands for the CD28/CTLA-4 family receptors on antigen-presenting cells and found that the expression of PD-L1, a ligand for programmed cell death-1 (PD-1), was up-regulated later than 3 weeks of infection. We also found that bacterial numbers in the spleen of PD-1-deficient mice were significantly reduced compared with wild-type mice at 6 and 12 weeks after BCG infection. Furthermore, CD4(+) T cells of PD-1-deficient mice showed a higher ability to confer protection and produce IFN-γ and TNF-α even at 12 weeks after infection. These results indicate that the PD-1-PD-L1 pathway impairs T(h)1 immunity in the late stage of BCG infection, thereby facilitating the bacterial persistence in the host.