Interleukin-12 primes CD4+ T cells for interferon-γ production and protective immunity during Mycobacterium avium infection

Interleukin-12 (IL-12) is a crucial cytokine for the generation of a protective immune response against Mycobacterium avium infection. In contrast to infected control mice, IL-12-deficient mice were unable to control bacterial proliferation and their spleen T cells were almost unresponsive in vitro to specific antigens of M. avium. Susceptibility of mice deficient in IL-12 was similar to that of interferon-gamma (IFN-gamma)-deficient mice. These data indicate a crucial role of IL-12 in the development of a T-cell population able to produce IFN-gamma and to mediate protection against M. avium infection. Treatment of M. avium-infected mice with IL-12 induced CD4+ T cells with enhanced capacity to produce IFN-gamma as well as to confer increased protection against M. avium.     

Infection of mice with Mycobacterium avium primes CD8+ lymphocytes for apoptosis upon exposure to macrophages

Mycobacterial infection is associated with granuloma formation in which the presence of apoptosis has been recognized. The role of CD4+ T and CD8+ T cells in host protection against mycobacterial infections has been demonstrated. Previous studies, however, have shown that CD8+ T cells have a limited role in host defense against Mycobacterium avium infection, and we hypothesize that M. avium infection could lead to T cell apoptosis. To investigate this hypothesis, C57BL/6 mice were infected with M. avium strain 101, and the rate of apoptosis of splenic lymphocytes cultured ex vivo with peritoneal macrophages was determined and compared with that of controls. When exposed to infected macrophages ex vivo, splenic lymphocytes from M. avium-infected mice underwent apoptosis, as determined by the TUNEL assay. This increased T cell apoptosis above the control level was observed after 3 weeks but not after only 1 week of infection in mice. No splenic T cell apoptosis was observed when lymphocytes from Mycobacterium smegmatis-infected mice were cultured in the presence of M. smegmatis-infected peritoneal macrophages. Likewise, macrophages infected in vitro with heat-killed M. avium did not trigger T cell apoptosis. Culture of macrophages in different chamber from lymphocytes, separated by a transwell membrane, was not associated with increase of apoptosis compared with uninfected control, suggesting a requirement for direct cell-cell interactions to trigger lymphocyte apoptosis. Using a double staining TUNEL followed by anti-mouse CD4 or anti-mouse CD8 monoclonal antibodies, it was observed that only CD8+ T cells but not CD4+ T cells underwent apoptosis at 3 weeks of infection. In conclusion, M. avium infection in C57/BL6 mice for 3 weeks renders CD8+ T cells prone to apoptosis when exposed ex vivo to macrophages infected with M. avium.     

Direct evidence for a critical role of CD30 in the development of allergic asthma

BACKGROUND: CD30 is a costimulatory molecule belonging to the TNF receptor superfamily that is expressed on activated T and B cells. Several studies have demonstrated a positive correlation between expression of CD30 or increased levels of soluble CD30 and the development and severity of allergic diseases. However, thus far, the evidence for a role of CD30 in allergic diseases, such as asthma, is only indirect. OBJECTIVE: The aim of the study was to directly investigate the role of CD30 in a murine asthma model. METHODS: CD30-deficient (B6.129P2-Tnfrsf8(tm1Mak)/J) and wild-type (WT) mice were immunized to ovalbumin (OVA) to induce an asthma-like phenotype and compared in our murine asthma model. Moreover, CD30/CD30 ligand signaling was blocked in OVA-immunized WT animals by using mAbs against CD30 receptor and its ligand, CD153. RESULTS: The absence of CD30 in OVA-immunized CD30-deficient mice resulted in significantly reduced airway inflammation, serum IgE levels, and TH2 cytokine levels. The same effect was observed when CD30/CD153 signaling was blocked in OVA-immunized WT animals with mAbs against CD30 or CD30 ligand. CONCLUSION: Our results directly demonstrate that CD30/CD153 interaction plays an important role in the induction of TH2 cell-mediated allergic asthma. CLINICAL IMPLICATIONS: These findings provide evidence for a role of the costimulatory molecule CD30 in allergic asthma.     

CD40 is required for the optimal induction of protective immunity to Mycobacterium avium

C57Bl/6 mice and mice deficient in the CD40 molecule were infected with three strains of Mycobacterium avium. Two of the M. avium strains proliferated more extensively in CD40-deficient (CD40-/-) mice than in control mice. The increased susceptibility to infection of CD40-/- mice was associated with the generation of poorer interleukin-12 (IL-12) p40 and interferon-gamma (IFN-gamma) responses as compared to the controls, suggesting a role for CD40 in the development of protective immunity. In contrast, direct triggering of CD40 on infected macrophages failed to induce any anti-mycobacterial activity in infected macrophages.     

Endogenous Inhibition of Antimycobacterial Immunity by IL-10 Varies between Mycobacterial Species

Interleukin (IL)-10 is an immunoregulatory cytokine that inhibits both Th1-like T cell responses and macrophage activation. Deficiency of IL-10 has been associated with increased Th1-like CD4+ T-cell responses and increased clearance of some intracellular pathogens, however, its role in mycobacterial infections is controversial. In order to examine the effects of mycobacterial virulence on the outcome of infection we compared infection with Mycobacterium avium and virulent Mycobacterium tuberculosis in C57Bl/6 IL-10-/- mice. M. avium infection in IL-10-/- mice resulted in sustained increases in interferon (IFN)-gamma-secreting T-cell responses and was associated with the increased clearance of M. avium from the liver and lung. By contrast, M. tuberculosis infection in IL-10-/- mice led to a transient increase in IFN-gamma T-cell responses at 4 weeks postinfection, with reduced bacterial burden in the lungs. This was not sustained so that by 8 weeks there was no difference to wild-type (WT) mice. In vitro infection of IL-10-/- macrophages with M. avium, but not M. tuberculosis, led to an increased IL-12 production. Therefore, endogenous IL-10 exerts a significant inhibition on specific IFN-gamma T-cell responses to M. avium infection, however, this effect is short lived during the M. tuberculosis infection, and fails to influence the long-term course of infection.     

Role of CD40 Ligand in Mycobacterium avium Infection

Mycobacterium avium is a common opportunistic pathogen in immunocompromised patients such as those infected with human immunodeficiency virus. Although M. avium is an intracellular organism replicating predominantly in macrophages, disseminated M. avium infection is seen in AIDS patients with CD4(+) cell counts of <50 cells/microliters, suggesting a possible involvement of a T cell-macrophage interaction for the elimination of M. avium. To determine whether CD40-CD40 ligand (CD40L) interactions play a role in M. avium infection, we studied the ability of CD40L to restrict M. avium replication in human monocyte-derived macrophages (MDM) in vitro. MDM were infected with M. avium and cocultured with CD40L-transfected 293 cells for 7 days. Intracellular growth of M. avium in these MDM was assessed by colony counting. CD40L-expressing cells inhibited growth of M. avium in MDM by 86.5% +/- 4.2% compared to MDM cultured with control cells. These findings were verified by assays using purified, soluble recombinant human CD40L (CD40LT). CD40LT (5 micrograms/ml) inhibited intracellular growth of M. avium by 76.9% +/- 18.0% compared to cells treated with medium alone. Inhibition by CD40LT was reduced by monoclonal antibodies (MAbs) against CD40 and CD40L. The inhibitory effect of CD40LT was not accompanied by enhancement of interleukin-12 (IL-12) production by M. avium-infected MDM, while CD40L-expressing cells stimulated IL-12 production by these cells. Treatment of M. avium-infected mice with MAb against murine CD40L resulted in recovery of larger numbers of organisms (0.8 to 1.0 log) from the spleens, livers, and lungs of these animals compared to infected mice which received normal immunoglobulin G. These results indicate that CD40-CD40L signaling may be an important step in host immune response against M. avium infection.     

Mycobacterium bovis BCG-induced granuloma formation depends on gamma interferon and CD40 ligand but does not require CD28

Progressive granuloma formation is a hallmark of chronic mycobacterial infection. Granulomas are localized, protective inflammatory reactions initiated by CD4+ T cells, which contribute to control of bacterial growth and blockade of bacterial dissemination. In order to understand the costimulatory requirements that allow CD4+ T cells to directly or indirectly induce granulomas, we studied granuloma formation after 6 weeks in Mycobacterium bovis BCG-infected CD28- and CD40 ligand (CD40L)-deficient mice and compared it to granuloma formation in infected wild-type inbred mice and infected cytokine-deficient mice. We characterized granulomas morphologically in liver sections, analyzed granuloma infiltrating cells by flow cytometry, and measured cytokine production by cultured granuloma cells. CD28-deficient mice have no defect at the local inflammatory site, inasmuch as they form protective granulomas and control bacterial growth. However, there are fewer activated T cells in the spleen compared to infected wild-type animals, and quantitative differences in the cellular composition of the granuloma are observed by flow cytometry. In CD40L-deficient mice, the granuloma phenotype is very similar to the phenotype in gamma interferon (IFN-gamma)-deficient mice. Both IFN-gamma-deficient and CD40L-deficient mice form granulomas which prevent bacterial dissemination, but control of bacterial growth is significantly impaired. The relative proportion of CD4+ T cells in granulomas from both CD28(-/-) and CD40L(-/-) mice is significantly decreased compared with wild-type animals. Both models demonstrate that the phenotype and activation stage of systemic T cells do not always correlate with the phenotype and activation stage of the localized granulomatous response.     

Mycobacterium avium infection in CD14-deficient mice fails to substantiate a significant role for CD14 in antimycobacterial protection or granulomatous inflammation

CD14 is a pattern-recognition receptor implicated in the inflammatory response to microbial components such as lipopolysaccharide, peptidoglycan and lipoarabinomannan. In this work, we made use of CD14-deficient (CD14-/-) mice to evaluate the relative importance of CD14 in response to infection with viable, intact cells of Mycobacterium avium in vitro and in vivo. Following co-incubation of either bone marrow-derived macrophages (Mphi) or thioglycollate-elicited peritoneal Mphi from CD14-/- mice with viable M. avium, tumour necrosis factor (TNF) production was significantly reduced and delayed compared to TNF secretion by infected CD14+/+ Mphi. However, following intravenous infection with a M. avium strain of either high virulence (TMC724) or intermediate virulence (SE01), there was no difference in the bacterial loads of lungs, livers or spleens at 3, 5 and 8 weeks postinfection in CD14-/- mice when compared with syngeneic CD14+/+ mice. At these time-points, TNF and interferon-gamma (IFN-gamma) mRNA expression in the liver was similar in infected CD14+/+ and CD14-/- mice, and granuloma formation and expression of inducible nitric oxide synthase within granuloma Mphi was the same in both mouse groups. In conclusion, although the absence of CD14 results in significantly reduced and delayed TNF production in response to stimulation with M. avium in vitro, there is no evidence that CD14 plays a significant role in either the antibacterial defence or the chronic granulomatous reaction to M. avium infection in vivo.     

A role for CD45RBlow CD38+ T cells and costimulatory pathways of T-cell activation in protection of non-obese diabetic (NOD) mice from diabetes

Non-obese diabetic (NOD) mice spontaneously develop autoimmune insulin-dependent diabetes mellitus (IDDM). Infection of the animals with mycobacteria, or immunization with mycobacteria-containing adjuvant, results in permanent protection of NOD mice from diabetes and we have recently reported that the phenomenon is associated with increased numbers of interferon-gamma-producing T cells, possessing increased cytotoxic activity, and also with augmented numbers of activated immunoglobulin M-positive (IgM+) B cells. Here, we have investigated whether protection of NOD mice from IDDM was associated with changes on costimulatory pathways of T and B cells, namely CD28/CTLA-4-B7 and CD40-CD40 ligand (CD40L) and we also further characterized protective T helper (Th) cells with regards to the expression of the differentiation markers CD45RB and CD38. We report that Th cells involved in diabetes vaccination of NOD mice by mycobacterial infection seem to belong to CD45RBlo CD38+ phenotype. The protective effect of Mycobacterium avium infection is also associated with increased CD40L and CTLA-4- expressing Th cells and with the generation of a CD40- IgG+ B cells. Our data are consistent with induction by mycobacterial infection of regulatory CD45RBlo CD38+ Th cells with the ability to trigger deletion or anergy of peripheral self-reactive lymphocytes, with shutting down of IgG+ B-cell response. They also implicate a role for IgG+ B cells in the autoimmune aggression of the endocrine pancreas of NOD mice.     

Transgenic mice expressing human interleukin-10 in the antigen-presenting cell compartment show increased susceptibility to infection with Mycobacterium avium associated with decreased macrophage effector function and apoptosis

Interleukin-10 (IL-10) is thought to play an important role in the regulation of microbial immunity. While T-cell-derived IL-10 has been shown to suppress cell-mediated immunity, there has been debate as to whether antigen presenting cell (APC)-derived cytokine can perform the same function in vivo. To assess the influence of APC-produced IL-10 on host resistance to mycobacterial infection, transgenic mice expressing human IL-10 under the control of the major histocompatibility complex class II promoter (hu10Tg) were infected with Mycobacterium avium, and bacterial burdens and immune responses were compared with those observed in wild-type (wt) animals. Hu10Tg mice harbored substantially higher numbers of M. avium and succumbed 16 to 18 weeks postinfection. The granulomas in infected hu10Tg mice showed marked increases in both acid-fast bacilli and host macrophages. In addition, these animals displayed a dramatic increase in hepatic fibrosis. The increased susceptibility of the hu10Tg mice to M. avium infection is independent of T-cell-produced endogenous murine IL-10, since bacterial burdens in mice derived by crossing hu10Tg mice with murine IL-10-deficient mice were not significantly different from those in hu10Tg mice. Importantly, gamma interferon (IFN-gamma) responses were not decreased in the infected transgenic animals from those in wt animals, suggesting the normal development of Th1 effector cells. In contrast, mycobacterium-induced macrophage apoptosis as well as production of TNF, nitric oxide, and IL-12p40 were strongly inhibited in hu10Tg mice. Together, these data indicate that APC-derived IL-10 can exert a major inhibitory effect on control of mycobacterial infection by a mechanism involving the suppression of macrophage effector function and apoptosis.     

TLR2 deficiency by compromising p19 (IL-23) expression limits Th 17 cell responses to Mycobacterium tuberculosis

CD4(+) T(h)1 cells producing IFN-γ are of extreme importance in controlling infections by Mycobacterium tuberculosis both in mice and in men. In addition to IFN-γ-producing T cells, IL-17-producing T cells (T(h)17) have been observed during mycobacterial infections. Nevertheless, their contribution for the host immune response to mycobacteria as well as the signals triggering M. tuberculosis -specific T(h)17 cell differentiation and maintenance are not fully understood. We show that signaling via Toll-like receptor (TLR) 2 has a major impact on the regulation of p19 (IL-23) expression in response to M. tuberculosis and therefore on the establishment of T(h)17 cell responses to M. tuberculosis infection. Diminished T(h)17 responses in the lung of M. tuberculosis -infected TLR2-deficient animals were not caused by defective cell differentiation in the draining lymph node (LN) but rather by reduced maintenance at the site of infection. Consistent with the decreased numbers of T(h)17 cells in the lungs of infected TLR2-deficient animals, we observed reduced expression of CXCL9, CXCL10 and CXCL11, chemokines involved in recall responses to M. tuberculosis. Our data provides insights into the TLR2 role in infection with M. tuberculosis, with implications in pathophysiology of the disease and vaccine design.     

Granuloma necrosis during Mycobacterium avium infection does not require tumor necrosis factor

The infection of tumor necrosis factor (TNF)-deficient mice with low doses of the virulent Mycobacterium avium strain 25291 led to the appearance of necrotic granulomas at 93 days of infection, i.e., sooner than necrotic granulomas appeared in C57BL/6 animals. Additionally, TNF-deficient mice exhibited higher mycobacterial loads in the infected organs, had extremely exacerbated gamma interferon responses as evaluated in the sera of infected animals, and showed reduced survival. Thus, TNF is not required for granuloma necrosis.     

Chronic pneumonia despite adaptive immune response to Mycobacterium bovis BCG in MyD88-deficient mice

To assess the role of Toll-like receptor (TLR) signalling in host response to mycobacterial infection, mice deficient in the TLR adaptor molecule myeloid differentiation factor 88 (MyD88) were infected with the vaccine strain Mycobacterium bovis (BCG), and the immune response and bacterial burden were investigated. Macrophages and dendritic cells from MyD88-deficient mice stimulated in vitro with BCG mycobacterial antigens produced very low levels of proinflammatory cytokines, while the expression of costimulatory molecules such as CD40 and CD86 was preserved. Upon systemic infection with BCG (2 x 10(6) CFU i.v.) MyD88-deficient mice developed confluent chronic pneumonia with two log higher CFU than wild-type mice. Interestingly, the infection was controlled in liver and spleen and there was efficient systemic T-cell priming with high IFNgamma production by CD4+ splenic T cells in MyD88-deficient mice. Lung infiltrating cells showed IFNgamma production by pulmonary CD4+ T cells upon specific restimulation, and a reduced capacity to produce nitric oxide and IL-10. In summary, despite the dramatic reduction of the innate immune response, MyD88-deficient mice were able to mount an efficient T-cell response to mycobacterial antigens, which was however insufficient to control infection in the lung, resulting in chronic pneumonia in MyD88-deficient mice.     

The involvement of the chemokine receptor CXCR2 in neutrophil recruitment in LPS-induced inflammation and in Mycobacterium avium infection

Knockout mice for CXC receptor 2 (CXCR2) chemokine receptor were used to study the recruitment of neutrophils during acute and chronic inflammatory responses. When treated with lipopolysaccharide (LPS), either intraperitoneally or intratracheally, these animals had a significant reduction in the neutrophil recruitment in the first 24-48 h as compared with control mice. During 15 days of intraperitoneal infection by Mycobacterium avium, the knockout mice showed significantly reduced numbers of neutrophils in the peritoneal cavity as compared with the control mice. In contrast, the recruitment of neutrophils to the lungs during an aerogenic M. avium infection was not affected by the CXCR2 mutation throughout the 60 days of the study. Finally, we could not find any impact of the mutation on the mycobacterial growth of the infected animals. These findings indicate that CXCR2 may be essentially involved in acute inflammatory responses where an early and rapid recruitment of neutrophils is observed.     

CD4+ T cells but Not CD8+ or gammadelta+ lymphocytes are required for host protection against Mycobacterium avium infection and dissemination through the intestinal route

Disseminated Mycobacterium avium infection is common in AIDS patients that do not receive anti-AIDS therapy and in patients for whom therapy fails. M. avium is commonly acquired by ingestion, and a large number of AIDS patients have M. avium in their intestinal tracts. To better understand the dynamics of the infection in patients with AIDS, we studied orally infected mice. To determine if immunocompetent mice challenged orally with M. avium can develop protection against the infection, and if so, which cell population(s) is responsible for the protection, we exposed wild-type as well as CD4(-/-), CD8(-/-), and gammadelta(-/-) knockout mice to low concentrations of M. avium strain 101 given orally, followed by treatment with azithromycin. After 1 month, the mice were challenged with kanamycin-resistant M. avium 104. Only CD4(+) T cells appeared to be required for protection against the second challenge. Both CD4(+) and CD8(+) T cells produced comparable amounts of gamma interferon after the first exposure to the bacterium. Tumor necrosis factor alpha was elevated in CD4(+) T cells but not in CD8(+) T cells. Following exposure to a small inoculum of mycobacteria orally, wild-type mice did not develop disseminated infection for approximately 4 months, although viable bacteria could be observed in the mesenteric lymph nodes. The ingestion of small numbers of M. avium cells induces a protective immune response in the intestines against subsequent infection. However, the bacteria remain viable in intestinal lymph nodes and might disseminate later.     

Role of complement in Mycobacterium avium pathogenesis: in vivo and in vitro analyses of the host response to infection in the absence of complement component C3.

We investigated the importance of the host complement system in the pathogenesis of disease mediated by the intramacrophage pathogen Mycobacterium avium. Mycobacteria opsonized with complement are efficiently ingested by macrophages through various complement receptors. Furthermore, unlike other bacteria, mycobacteria can activate both the alternative and classical complement pathways in the absence of specific antibodies. Therefore, to examine the role of complement in the mycobacterial infection process in vivo, mice deficient in complement component C3 were infected with M. avium. Surprisingly, C3-deficient mice infected intravenously with M. avium displayed no difference in bacterial burden or granulomatous response compared to wild-type control mice. C3-sufficient mice and C3-deficient mice were equally susceptible to infection by M. avium regardless of the genotype at the bcg locus, a locus known to confer susceptibility to infection with intracellular pathogens. In vitro studies using mouse bone marrow-derived macrophages resulted in significant M. avium invasion of macrophages in the absence of C3; however, the kinetics of infection were delayed compared to complement-mediated invasion. The data indicate that complement does not play an essential role in mediating M. avium infections in the mouse and suggest either that other invasion mechanisms can compensate for the absence of complement-mediated entry or that complement is not a major mycobacterial opsonin in vivo.     

Toll-like receptor 2-deficient mice succumb to Mycobacterium tuberculosis infection

Recognition of Mycobacterium tuberculosis by the innate immune system is essential in the development of an adaptive immune response. Mycobacterial cell wall components activate macrophages through Toll-like receptor (TLR) 2, suggesting that this innate immune receptor plays a role in the host response to M. tuberculosis infection. After aerosol infection with either 100 or 500 live mycobacteria, TLR2-deficient mice display reduced bacterial clearance, a defective granulomatous response, and develop chronic pneumonia. Analysis of pulmonary immune responses in TLR2-deficient mice after 500 mycobacterial aerosol challenge showed increased levels of interferon-gamma, tumor necrosis factor-alpha, and interleukin-12p40 as well as increased numbers of CD4(+) and CD8(+) cells. Furthermore, TLR2-deficient mice mounted elevated Ag-specific type 1 T-cell responses that were not protective because all deficient mice succumb to infection within 5 months. Taken together, the data suggests that TLR2 may function as a regulator of inflammation, and in its absence an exaggerated immune inflammatory response develops.     

Stimulation of dendritic cells via CD40 enhances immune responses to Mycobacterium tuberculosis infection

The resolution of pulmonary tuberculosis (TB) critically depends on the development of the Th1 type of immune responses, as exemplified by the exacerbation of TB in IL-12-deficient mice. Therefore, vaccination strategies optimizing IL-12 production by antigen-presenting cells (APC) in response to mycobacteria may have enhanced protective efficacy. Since dendritic cells (DC) are the critical APC for activation of CD4+ and CD8+ T cells, we examined whether stimulation of Mycobacterium bovis bacillus Calmette Guérin (BCG)-infected DC via CD40 increased their ability to generate Th1-oriented cellular immune responses. Incubation of DC with an agonistic anti-CD40 antibody activated CD40 signaling in DC, as shown by increased expression of major histocompatibility complex class II and costimulatory molecules, mRNA production for proinflammatory cytokines and interleukin 12 (IL-12) p40. This activation pattern was maintained when DC were stimulated with anti-CD40 antibody and infected with BCG. Importantly, CD40-stimulated BCG-infected DC displayed increased capacity to release bioactive IL-12 and to activate gamma interferon (IFN-gamma) producing T cells in vitro. Moreover, when C57BL/6 mice were immunized with these DC and challenged with aerosol Mycobacterium tuberculosis, increased levels of mRNA for IL-12 p40, IL-18, and IFN-gamma were present in the draining mediastinal lymph nodes. However, the mycobacterial burden in the lungs was not reduced compared to that in mice immunized with BCG-infected non-CD40-stimulated DC. Therefore, although the manipulation of DC via CD40 is effective for enhancing immune responses to mycobacteria in vivo, additional strategies are required to increase protection against virulent M. tuberculosis infection.     

Immune response to Mycobacterium bovis bacille Calmette Guérin infection in major histocompatibility complex class I- and II-deficient knock-out mice: contribution of CD4 and CD8 T cells to acquired resistance

Knock-out mice with defined major histocompatibility complex (MHC) deficiencies were infected intravenously with Mycobacterium bovis bacille Calmette Guérin (M. bovis BCG) to assess the relative impact of MHC class I- and II-dependent immune responses. Heterozygous control mice were capable of controlling growth of M. bovis BCG, although infection progressed chronically, as assessed by determination of colony-forming units. Furthermore, infected controls developed granulomatous lesions at the site of mycobacterial growth and delayed-type hypersensitivity (DTH) reactions after challenge with purified protein derivative of tuberculin. In vitro, spleen cells from heterozygous control mice produced high concentrations of interferon-γ (IFN-γ) after restimulation with mycobacterial antigens. In contrast, the MHC class II-deficient Aβ^?/? mice, which are virtually devoid of functional CD4 T cells, succumbed to M. bovis BCG infection. Furthermore, Aβ^?/? mice lacked DTH reactions to tuberculin and only few minute picnotic lesions were formed in livers of infected mice. Finally, spleen cells from infected Aβ^?/? mice failed to produce measurable IFN-γ concentrations after restimulation in vitro with various mycobacterial antigen preparations. The capacity of β2-microglobulin (β2m)-deficient mice, which are devoid of CD8α/β T cells, to inhibit growth of M. bovis BCG was only slightly affected at low inocula, although significantly higher colony-forming units were detected in spleens. These knock-out mice developed strong DTH responses to tuberculin and their spleen cells produced high levels of IFN-γ once reactivated by mycobacterial antigens. Furthermore, in livers of infected β2m-deficient mice, extravascular infiltrates developed which were more diffuse than those in infected control littermates. Remarkably, the β2m-deficient mice were substantially more susceptible to higher inocula of M. bovis BCG than their control littermates. Our data formally prove the essential role of MHC class II-dependent immune mechanisms in all relevant aspects of immunity to M. bovis BCG. In addition, our findings emphasize an important contribution of MHC class I-dependent immunity to effective anti-mycobacterial protection. We assume that CD4 T cells are highly effective in containing M. bovis BCG within distinct granulomatous lesions, but fail to eradicate their intracellular pathogens. It appears most likely that CD8 T cells are also required to achieve this goal.     

Protection by CD4 or CD8 T Cells against Pulmonary Mycobacterium bovis Bacillus Calmette-Guérin Infection

Mice deficient in CD8 T cells demonstrated levels of Th1 cytokines and granulomatous responses in the lungs very similar to those demonstrated by normal control mice and were fully capable of controlling pulmonary mycobacterial infection by Mycobacterium bovis BCG as assessed at day 37 postinfection. In comparison, mice deficient in CD4 T cells had similar levels of interleukin-12 (IL-12) and tumor necrosis factor alpha but lower levels of gamma interferon in the lungs and were still able to mount tissue granulomatous responses and control pulmonary mycobacterial infection. In contrast, IL-12^−/− mice with impaired CD4 and CD8 T-cell responses had a markedly weakened control of infection, whereas SCID mice deficient in all T cells succumbed to such pulmonary mycobacterial infections.