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Abstract |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The role of CD8 T lymphocytes in the immune response to Mycobacterium tuberculosis infection remains enigmatic, with persuasive reports of both cytolytic and noncytolytic (cytokine-mediated) responses to infection. To address the importance
of the cytolytic mechanisms, mice with targeted disruptions
for CD8 and perforin or with gene mutations in the CD95/
CD95L signaling pathway were exposed to pulmonary infection. All mice tested showed no differences in their ability to
contain the growth of infection during the early phase of disease. As the chronic phase of the disease ensued, however,
both CD8- and CD95/CD95L-deficient mice gradually lost their
ability to limit bacterial growth. This was associated with a
tendency toward pyogenic inflammation in the lung. This tendency was not seen in the perforin gene-disrupted mice. In
CD8 gene-disrupted mice, the ability to generate interferon-
secreting T cells was unimpaired. Although these cells were
capable of entering the lung they were unable to influence
the increasing bacterial load in this organ.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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It is estimated that approximately one-third of the world's population has been exposed to the bacillus Mycobacterium tuberculosis; this organism is the leading killer among infectious diseases. It is thought that the majority of infected individuals harbor infection for some time before recrudescence results in clinical disease. This pattern of chronic infection followed by recrudescent disease can be modeled in the mouse using a low-dose aerosol exposure form of infection (1). In the lungs of these chronically infected mice the disease is limited within the organized mononuclear granulomas for a considerable period of time before regrowth of the bacteria is seen (1, 2).
It has been established that the emergence of interferon (IFN)- secreting CD4 T cells is crucial to the expression of the protective immune response (3), and it is
obvious that in the absence of these cells both mice and
humans are highly susceptible to disease (4, 5). On the
other hand, the contribution of other T-cell populations to
the control of M. tuberculosis infection is less clear. In particular, despite much enthusiasm directed toward the
study of the CD8 T-cell response to mycobacteria, there is
as yet no direct evidence that this T-cell subset contributes to the control and containment of pulmonary M. tuberculosis infection. The literature does, however, contain many
reports that CD8 T cells respond to infection with M. tuberculosis.
In one of the first reports, the ability of adoptively
transferred immune ly2+ (i.e., CD8+) cells to prolong the
survival of infected irradiated recipients was shown (6).
Later studies postulated that cytokine production was a
prime mediator of this protection inasmuch as CD8 T cells
from infected mice could make IFN- in response to mycobacterial antigens (7). In one of the first reports of mycobacterial infection of a gene-disrupted mouse, Flynn and
colleagues reported a dramatic increase in susceptibility of
2 microglobulin-deficient mice when infected systematically with M. tuberculosis (8). The role of 2 microglobulin
in expression of major histocompatibility complex (MHC)
class I and therefore CD8 T-cell maturation led to the conclusion that MHC class I-restricted T cells were critical in
controlling mycobacterial disease. More recent use of gene-deleted mice has, however, demonstrated distinct differences
between the response of mice lacking the 2-microglobulin molecule, the class I molecule, and the CD8 molecule
(9). The key observation of these studies was that CD8
gene-disrupted (CD8-KO) mice were able to adequately
control and contain a pulmonary M. tuberculosis infection
for a period of time (10).
Other investigators have formally demonstrated that
the protective effect of CD8 T cells in an intraperitoneal
model of bacterial challenge lies in the ability of the cells
to produce IFN- (12). Indeed, CD8 cells are activated to
produce IFN- and can be recruited to the lung during infection (4, 7, 13).
In addition to reports showing cytokine production, there is no shortage of literature supporting a direct cytolytic activity for CD8 T cells against target cells pulsed with mycobacterial antigens (14). Both of the mechanisms expressed by CD8 T cells that lead to target cell death (i.e., apoptotic mechanisms mediated by CD95 and direct cytolysis mediated by granule exocytosis [19]) have been shown to be active in mycobacteria-specific human CD8 T cells (20).
The studies discussed above are primarily in vitro in nature and as such do not directly address the role of CD8-mediated protective mechanisms in controlling pulmonary disease. To address this issue, therefore, the necessity and role of CD8 T cells in the control of pulmonary M. tuberculosis infection was assessed using CD8-KO and perforin gene-disrupted (perforin-KO) mice, and lpr (CD95) and gld (CD95L) mutant mice. In the present report we show that a requirement for the optimum expression of CD8 T cell-mediated immunity was not needed during the early stages of infection, but instead was important during chronic M. tuberculosis disease. Moreover, in the absence of the CD95/ CD95L signaling pathway there was also no increased early susceptibility but, as in the CD8-KO mice, an increase in bacterial load was seen as the infection moved into the chronic phase. In contrast, despite the absence of perforin-mediated mechanisms, no increased susceptibility was noted during the chronic phase of disease. These data therefore establish important roles for both CD8 T cells and a CD95-dependent mechanism in the maintenance of the chronic state of M. tuberculosis disease.
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Materials and Methods |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Mice
Control C57BL/6, CD8-KO, and CD95 signaling pathway mutant mice were purchased from the Jackson Laboratory (Bar Harbor, ME). The B6Smn.C3H-FasLgld (CD95L mutant) mice have a mutation in the CD95 ligand gene on chromosome 1 whereas the B6.MRL-Faslpr (CD95 mutant) mice have a mutation in the CD95 gene on chromosome 19. These CD95 pathway-deficient mice are susceptible to several autoimmune diseases that can begin as early as 3 mo of age. All experiments were, therefore, initiated at 6 wk of age and mice were analyzed by histology and clinical assessment at the later time points to determine their levels of autoimmune disease. The most common consequence of the gld and lpr mutations was lymphadenopathy; however, mice showed little sign of distress and were capable of controlling a systematic M. tuberculosis infection as well as the control mice did (35). Subject animals were maintained under specific-pathogen-free conditions on sterile bedding with water and mouse chow administered ad libitum.
Experimental Infection
The Erdman (TMCC 107) strain of M. tuberculosis was grown in
Proskauer Beck medium containing 0.05% Tween 80 to mid-log
phase and frozen in 1-ml aliquots at 
70°C. For aerosol infections, subject animals were infected using a Glas-Col (Terre Haute, IN) airborne infection system as previously described (21).
Bacterial Load Determination
As previously described (21), infected mice were killed by CO2 asphyxiation and the lungs aseptically excised. Each of these organs was individually homogenized in physiologic saline and serial dilutions of the organ homogenate were plated on nutrient 7H11 agar. Bacterial colony formation was counted after 3 wk of incubation at 37°C.
Histologic Preparation
The lower right lobe of each lung was inflated with 10% formalin in neutral buffered saline and processed routinely for light microscopy. Sections were stained with hematoxylin and eosin. Slides were examined without knowledge of experimental group and subjectively graded for both quantity and quality of cellular accumulation. Repeat evaluations were performed to confirm that grading was reproducible.
Flow Cytometry
A single-cell suspension was prepared from the spleen as described (22). Cells from individual mice were incubated with specific antibody for 30 min at 4°C and in the dark. After two washes in D-RPMI lacking biotin and phenol red (Irvine Scientific, Santa Ana, CA), cells were incubated with excess streptavidin Red670 (GIBCO BRL, Grand Island, NY) for 30 min, washed twice, and analyzed on a FACSCalibur flow cytometer (Becton Dickinson, San Diego, CA). Antibodies used were fluorescein-labeled CD4 clone RM4-5, phycoerythrin CD44 clone IM7, biotinylated CD11a clone 2D7, and biotinylated CD54 clone 3E2. All cell-surface antibodies were purchased from BDPharmingen (San Diego, CA), including appropriate isotype control antibodies that were included throughout the analysis. Data were collected and analyzed using CellQuest software (Becton Dickinson). Lung cells were stained as described earlier using a single-cell suspension prepared as described later.
Culture of Lung-Derived Lymphocytes
Mice were infected via the aerosol route with 102 viable M. tuberculosis and the lungs were harvested at indicated times after exposure. The lung lobes from each of four individual mice were perfused with Collagenase IX (0.7 mg/ml; Sigma, St. Louis, MO) and deoxyribonuclease (30 µg/ml; Sigma) and incubated at 37°C for 30 min to digest the lung tissue. Tissue was then gently disrupted and passed through a mesh sieve, washed twice, and resuspended at 5 × 106 cells/ml before incubation with culture filtrate proteins (CFPs) (10 µg/ml) from M. tuberculosis. After 5 d
incubation at 37°C with 5% CO2, plates were frozen at 70°C to
await enzyme-linked immunosorbent assay (ELISA) analysis.
CD4 Cell Assays
Bone marrow-derived macrophages were prepared as described previously. At 1 d before CD4 T-cell purification, bone marrow- derived macrophages were pulsed with either M. tuberculosis- derived CFPs or cell-wall (CW) antigens at 10 µg/ml (obtained from Dr. John Belisle, Colorado State University, Fort Collins, CO, under NIH contract AI-75320). The following day spleens were harvested from infected mice and a single-cell suspension was obtained by passing the organ gently through a 70-µm sieve. Red blood cells were lysed using Gey's lysis buffer and resuspended in phosphate-buffered saline (PBS) plus 2.5% bovine serum albumin (BSA). Cells were incubated with anti-CD4 microbeads (Miltenyi Biotech, Auburn, CA). CD4 T cells were purified using a magnetic column (Miltenyi Biotech) and resuspended at 1 × 106 cells/ml in Dulbecco's modified Eagle's medium (Sigma), containing 10% fetal calf serum, L-glutamine, and nonessential amino acid supplement. Pure CD4 T cells (> 98% pure, assessed by flow cytometry) were added to the antigen prepulsed macrophages. Cultures were incubated for 72 h at 37°C and 5% CO2 before cytokine analysis.
IFN- ELISA
Supernatants were assayed for the presence of IFN- by ELISA
using antibody pairs purchased from BDPharmingen. Briefly, the primary antibody (clone R4-6A2) was incubated overnight in 96-well flat bottom Immulon 2 plates in carbonated coating buffer.
Excess antibody was washed away using PBS-Tween 20 (PBS-T).
The wells were blocked with 3% BSA in PBS-T and samples dispensed in triplicate into the wells. The presence of cytokine was detected by the addition of a secondary biotinylated antibody (clone
XMG1.2), followed by avidin peroxidase and substrate (2,2'-azino-
bis[3-ethylbenz-thiazline-6-sulfonic acid]).
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Results |
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Introduction
Materials and Methods
Results
Discussion
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CD8-KO Mice Gradually Lose Control of Pulmonary M. tuberculosis
CD8-KO mice have previously been shown to be able to adequately control an aerosol infection with M. tuberculosis for at least 55 d (10). To test whether prolonged control of bacterial growth occurred in the absence of CD8+ cells, CD8-KO mice were infected with M. tuberculosis Erdman via the respiratory route and followed for 150 d. Figure 1 confirms the previous finding that CD8 T cells are not necessary for the initial containment of the infection, in that bacterial growth in the lungs was halted after approximately 20 d and was not statistically different during the early stages of the chronic disease up to 60 d. In the control mice, bacterial load did not increase from the Day 20 time point to the Day 150 time point (as has been shown previously [1]) and thus remained truly chronic. Although the CD8-KO mice did not demonstrate rapid susceptibility to infection, bacterial load increased progressively over time such that by Day 150 these mice had 10-fold more bacteria (n = 14 from three separate experiments, P < 0.001).
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CD95/CD95 Ligand Interactions, but Not Perforin, Are Important in the Maintenance of Chronic M. tuberculosis Infection
To determine whether the cytotoxic mechanisms observed in vitro were important in pulmonary infections in vivo, perforin-KO, or CD95 mutant and CD95L mutant, mice were infected by low-dose aerosol with M. tuberculosis. As seen in CD8-KO mice, both the CD95 mutant (Figure 2A) and the CD95L mutant mice (not shown, but identical result to CD95 mutant) showed exacerbated disease after about 45 d of infection. This exacerbation again resulted in an increase in bacterial number compared with controls (Figure 2A). To control for the effect of the increasingly prevalent autoimmunity in the CD95 mutant mice, aged CD95 mice were also infected aerogenically. These aged mice exhibited an identical phenotype to the young CD95 and CD95L mutant mice (data not shown), suggesting that the autoimmunity is not the cause of the reduced ability to control chronic infection. In contrast, the perforin-KO mice showed only a very mild, yet statistically significant, increase in bacterial numbers (0.25 logs) which persisted over time (Figure 2B).
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0.05, Student's t test). (B) Mice lacking
the perforin pathway of cell lysis are able to control a chronic
pulmonary infection with M. tuberculosis. Control ( filled circles)
and perforin-KO (open triangles) mice were infected aerogenically as in Figure CD8-KO and CD95/CD95L Mutant Mice Have Altered Granuloma Formation within the Lung
We have previously extensively characterized the granuloma formation in immunologically intact, aerogenically-infected mice (1, 23) and using the same criteria we have recently reported the increased lymphocytic recruitment to the lungs of CD8-KO mice (10). In the present report we document that the lymphocytic recruitment continued even as bacterial numbers increased (Table 1). As the disease progressed, however, neutrophils appeared and the lung tissue became necrotic even before detectable differences in bacterial numbers (Table 1 and Figure 3B). Histologic examination of the CD95L mutant (Figure 3C) and CD95 mutant (not shown but identical to CD95L mutant) infected lungs also showed an increase in neutrophil recruitment to the mycobacterial granulomas. Perforin-KO mice produced granulomas identical to C57BL/6 mice after infection (data not shown).
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In the Absence of CD8 T Cells the CD4 T-cell
Population Is Highly Active and Can Produce
Mycobacterial-Specific IFN-
To assess whether CD4 cells were able to compensate for
the absence of CD8 T cell-derived IFN- in the CD8-KO
mice, CD4 T cells from either infected control or CD8-KO
mice were cultured with antigen-pulsed bone marrow-derived macrophages. As shown in Figure 4, the CD4 T cells
from the CD8-KO mice were capable of producing IFN-
in response to the secreted CFP (Figure 4A) and CW (Figure 4B) antigens of M. tuberculosis, and these responses were significantly larger than those from infected control
mice. Lung cells from infected CD8-KO mice were also
able to express IFN- secretion after culture with CFP antigens (6,454 and 1,646 pg/ml at Days 90 and 120, respectively). In addition, the CD4 T-cell population within the
lungs of infected CD8-KO mice was substantially more activated as shown by an increase in the number of CD4 cells
with high- (as opposed to moderate-) level expression of the
activation markers CD44, CD11a, and CD54 (Figure 5).
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Discussion |
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Abstract
Introduction
Materials and Methods
Results
Discussion
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The results of this study show that optimal CD8 T-cell responses are a necessary component of the acquired immune response generated against pulmonary M. tuberculosis infection. By comparing the course of the infection in
control and gene-KO mice it was shown that the role of
the CD8 molecule occurred not during the early phase of
infection when the bacterial load is initially contained but
during the ensuing chronic stage of disease. This gradual
loss of resistance observed in CD8-KO mice was associated with an increased lymphocytic influx that consisted of
highly activated CD4 T cells. However, despite this influx
of lymphocytes, and despite the capacity of these cells to
produce IFN-, the bacterial load remained elevated.
The data support the hypothesis that the CD8 molecule is required to optimize CD8 T-cell responses during the chronic infection. It is possible, however, that the absence of the CD8 molecule does not preclude the presence of functional CD8-like MHC class I-restricted T cells (analogous to those seen in the CD4-KO mouse [24]). These cells may play a role in controlling early infection; however, this study was designed to assess the role of the CD8 molecule in optimizing the protective T-cell response in the lung and as such did not address the role of these atypical CD8-like cells.
Perhaps the most interesting aspect of the results reported here is the failure of CD8-KO mice to maintain
control of the infection while still capable of expressing a
strong antigen-specific CD4 IFN- response in the lung.
This observation suggests that during the chronic phase of
the disease there are lung cells harboring viable mycobacteria that cannot be detected by class II-restricted mechanisms. These bacteria may be residing within IFN- nonresponsive but class I-positive cells or within macrophages
that are damaged or degenerating within lesions and which
are refractory to IFN- activation. Sequestration within
such cells would provide a niche for bacterial survival and
would require recognition by CD8 T cells for their elimination (25).
After recognition of potential targets, CD8 T cells can kill infected cells via two mechanisms; direct lysis by granules (perforin, granzymes), or CD95/CD95 ligand-induced apoptosis (16, 19, 20, 26). Both mechanisms have been demonstrated in vitro, but the relative importance of each in a pulmonary infection has not been addressed. One could hypothesize that due to the nature of lung tissue, the specific induction of apoptosis by CD95 signaling rather than the release of cytolytic granules would be the preferred cytotoxic mechanism. In support of this hypothesis we clearly demonstrate that a specific mediator of cell death by apoptosis (i.e., the CD95/CD95L pathway) is crucial for the containment of chronic pulmonary M. tuberculosis infection, whereas a specific mediator of cell lysis (perforin) plays a lesser role.
The absence of a strong phenotype for the perforin-KO extends previous studies that also failed to demonstrate a phenotype for the perforin-KO during the early stages of M. tuberculosis infection (27, 28). Recent elegant studies, however, using CD8 cells taken directly from the lung, show that these cells can mediate lysis of infected macrophages using perforin (29). Thus, although the absence of perforin in the pulmonary model does not result in a dramatic phenotype, it is possible that this pathway may yet play a role in mycobacterial control (29). A recent publication showing decreased survival of perforin-KO mice after systematic infection suggests that this role may be important when disease has progressed to its terminal, disseminated phase (11).
The very nature of the two forms of cytotoxic activity predisposes them to induce different levels of inflammation and tissue damage. Thus, whereas apoptosis results in the cytoplasm being sequestered within discrete vesicles, perforin-mediated membrane damage results in the release of cellular contents into the tissue milieu (30). We would hypothesize, on the basis of our current data, that cytotoxic T cells (probably CD8) seek out infected cells within lung granulomas and destroy them by inducing apoptosis. Because these cells are not directly lysed they may be engulfed by surrounding macrophages or imprisoned by the developing fibrotic response (1). In the absence of this mechanism, however, these cells degenerate and release their contents, resulting in local tissue damage. This damage may then attract neutrophils, resulting in a pyogenic granuloma, as was observed in both the CD95/CD95L mutant and the CD8-KO mice.
Whichever way bacteria are released from their sequestered niche, rapid uptake by IFN--activated macrophages
would result in improved bacterial control (25) and again
the apoptotic event would be preferable over the lytic response because apoptotic vesicles express markers which
increase uptake by phagocytes (31). In addition, control of
mycobacterial growth by non-IFN--mediated pathways
has recently been suggested (16) and may be dependent upon apoptotic mechanisms. In this regard, apoptosis of
the host cell can be detrimental to mycobacterial survival
in itself (32, 33). Conversely, other recent reports have
identified a novel antibacterial mechanism that involves
the direct killing of bacteria by CD8 cells capable of releasing granulysin granules (16, 20). Although this may be
a potent mechanism it has yet been demonstrated only in
vitro, and one might imagine that these granules might be
detrimental to tissue integrity if released in the lung.
Although CD8 T cells have been implicated in many studies as a protective cell in tuberculosis, this report is the first to use the pulmonary model to demonstrate the need for an optimal CD8 T-cell response in mycobacterial infection in the lung. In addition, this report also highlights for the first time that CD95-mediated mechanisms are important in controlling the chronic pulmonary infection. Accordingly, vaccines targeted at CD8- and CD95-mediated effector mechanisms in the lungs may prove a new design strategy. Such vaccines could be given in both a prophylactic and postinfection mode (34) with the potential for further bacterial clearance rather than establishment of chronic disease, or alternatively preventing the transition from the chronic disease state to reactivation disease.
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Footnotes |
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Address correspondence to: Andrea Cooper, Dept. of Microbiology, Colorado State University, 200 W. Lake, Fort Collins, CO 80523. E-mail: acooper{at}cvmbs.colostate.edu
(Received in original form September 13, 2000 and in revised form November 1, 2000).
Acknowledgments: The authors thank Dr. John Belisle and Marc Keen for supplying mycobacterial components under NIH contract AI-75320. This work was supported by U.S. Public Health Service grants AI-40488, AI-44072, and AG-06946.
Abbreviations
CD8-KO, CD8 gene-disrupted;
CFP, culture filtrate protein;
CW, cell-wall;
ELISA, enzyme-linked immunosorbent assay;
IFN, interferon;
KO, knockout;
MHC, major histocompatibility complex;
perforin-KO, perforin gene-disrupted.
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References |
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Materials and Methods
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Discussion
References
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