help button home button
AJRCCM
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lazarevic, V.
Right arrow Articles by Flynn, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lazarevic, V.
Right arrow Articles by Flynn, J.
American Journal of Respiratory and Critical Care Medicine Vol 166. pp. 1116-1121, (2002)
© 2002 American Thoracic Society

Pulmonary Perspective

CD8+ T Cells in Tuberculosis

Vanja Lazarevic and JoAnne Flynn

Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania

Correspondence and requests for reprints should be addressed to JoAnne Flynn, Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, W1157 Biomedical Science Tower, Pittsburgh, PA 15261. E-mail: joanne{at}pitt.edu

Tuberculosis is caused by the bacterium Mycobacterium tuberculosis. Although M. tuberculosis primarily infects the respiratory tract, the bacterium can disseminate and cause disease in virtually every organ system in the body. This pathogen is responsible for an estimated 8 million cases of tuberculosis and at least 1.5 million deaths each year (1, 2). Most infected persons are asymptomatic; however, the bacteria are probably not eliminated and these persons are believed to have a subclinical infection, often referred to as latent tuberculosis. Approximately 10% of latently infected individuals will undergo reactivation and present with active tuberculosis. Immunosuppression, such as human immunodeficiency virus infection, leads to an increased incidence of reactivation of latent tuberculosis. The annual risk for subjects who tested positive for human immunodeficiency virus and purified protein derivative (PPD) of developing active tuberculosis is 8 to 10%, compared with a 10% lifetime risk for individuals who tested positive for PPD and negative for human immunodeficiency virus (3). Considering that one-third of the world's population is estimated to be infected with M. tuberculosis, this pathogen represents an enormous global health threat.

M. tuberculosis infection is most commonly acquired through the inhalation of aerosolized droplets produced by a person with active disease. The inhaled bacteria reach the alveoli of the lung, where they enter and replicate within resident macrophages. Initially, unactivated macrophages are unable to eliminate bacilli, allowing bacteria to establish infection in the lungs. During this early stage, M. tuberculosis–infected macrophages secrete proinflammatory cytokines, interleukin-1, interleukin-6, interleukin-12, and tumor necrosis factor-{alpha} (TNF-{alpha}), as well as chemokines, which help recruit monocytes, T cells, B cells, and neutrophils to the sites of infection (reviewed in [4]). The recruited cells form granulomas, which are composed of centrally located macrophages surrounded by T and B lymphocytes. Formation of such organized structures helps contain and prevent dissemination of infection and also allows for the close T cell–macrophage contact necessary for the induction of effective antimycobacterial mechanisms.

Although the effective immune response against tuberculosis is primarily due to cell-mediated immunity, the mechanisms by which T cells participate in the control of infection are still not completely defined. Production of cytokines, interferon-{gamma} (IFN-{gamma}), and TNF-{alpha}, as well as activation of macrophages (Figure 1) , are essential for the control of M. tuberculosis infection, but the relative contribution from different T cell subsets as well as additional mechanisms of protection are the subjects of intense study.



View larger version (51K):
[in this window]
[in a new window]
  		Figure 1. CD8+ T cells may contribute to the control of M. tuberculosis infection through four mechanisms: (1) cytokine release, (2) cytotoxicity via granule-dependent exocytosis pathway, (3) cytotoxicity mediated through Fas/Fas ligand interaction, and (4) direct microbicidal activity. (A) Cytokine production. Both CD4+ and CD8+ T cells are important producers of Th1-type cytokines, such as IFN-{gamma} and TNF-{alpha}, during M. tuberculosis infection. Both cytokines act synergistically to activate M. tuberculosis–harboring macrophages. Activation of macrophages results in upregulation of inducible nitric oxide synthase, which leads to the production of reactive nitrogen intermediates, such as nitric oxide (NO). Reactive nitrogen intermediates, together with reactive oxygen intermediates (O2), exerts antimycobacterial effects, which lead to the reduction in viable mycobacteria. (B) Cytotoxicity (i) Granule-dependent exocytosis pathway. On recognition of M. tuberculosis–infected cells, CD8+ T cells release perforin-containing granules. Perforin polymerizes on the cell membrane of the target cells allowing the entry of effector molecules such as granzyme A and granzyme B (serine proteases), leading to apoptosis or lysis of the target cell. The lysis of unresponsive macrophages infected with M. tuberculosis releases the pathogen into the extracellular environment to be taken up by freshly activated macrophages, which are better equipped for killing them. (ii) Fas/Fas ligand–mediated cytotoxicity. Cross-linking of Fas ligand (expressed on activated CD8+ T cell) and Fas (expressed on target cell) leads to recruitment of Fas–associated death domain and activation of caspase 8 leading to apoptosis of target cell. (iii) Direct microbicidal activity. Granules within human CD8+ T cells contain a newly identified molecule, granulysin, which has direct microbicidal effect on intracellular bacteria. It is a saposin-like protein, which interacts with lipid membranes, and activates lipid-degrading enzymes. Only in the presence of perforin, granulysin dramatically decreased the viability of intracellular M. tuberculosis. Granulysin induced discrete lesion on bacterial surface, increased permeability of bacterial membrane, and triggered fluid accumulation in the periplasm of M. tuberculosis (47). A murine homologue of granulysin has not been identified and may not exist.

 
DO CD8+ T CELLS CONTRIBUTE TO PROTECTIVE IMMUNITY AGAINST M. TUBERCULOSIS?

Murine Studies
It is generally accepted that CD4+ T cells are an essential component of protective immunity against tuberculosis, and this has been demonstrated convincingly in the murine model (reviewed in [4]). The enhanced susceptibility of subjects who tested positive for human immunodeficiency virus to tuberculosis supports the fact that CD4+ T cells are also important in human disease.

The evidence for an essential role of CD8+ T cells is not quite as compelling. For years, many researchers in the tuberculosis field ignored this T cell subset. CD4+ T cells recognize antigens presented by major histocompatibility complex (MHC) Class II molecules on antigen presenting cells, such as dendritic cells and macrophages. MHC Class II is loaded with antigenic peptide in a vacuole that is a part of the endocytic system. Conversely, MHC Class I molecules present antigens to CD8+ T cells; MHC Class I molecules are loaded with antigens transported from the cytoplasm into the endoplasmic reticulum, such as viral antigens. Because M. tuberculosis lives primarily within a vacuole inside the cell, rather than in the cytoplasm, it seemed unlikely that antigens would be effectively presented to CD8+ T cells by MHC Class I molecules.

However, early studies using antibody-mediated T cell subset depletion suggested that CD8+ T cells, in addition to CD4+ T cells, were necessary for the control of M. tuberculosis infection (5) and that adoptive transfer of purified immune CD8+ T cells reduced the numbers of M. tuberculosis bacteria in the spleens of infected mice, albeit at a lower efficiency compared with CD4+ T cells (6, 7). The development of gene-disrupted mice provided stronger data supporting the role of CD8+ T cells in the control of M. tuberculosis infection.

Mice genetically deficient in ß2-microglobulin (ß2m), which lack functional MHC Class I molecules and consequently CD8+ T cells, failed to control infection, particularly in the lung, and succumbed prematurely to tuberculosis (8). Although it was reasonable to assume at that time (10 years ago) that susceptibility of ß2m-/- mice was due to a drastically reduced total number of peripheral CD8+ T cells, the defect in ß2m-/- mice is broad. In addition to the absence of classical MHC Ia molecules, which present peptides to CD8+ T cells, these mice also lack functional CD1 and other nonclassical MHC Ib molecules, which present lipid antigens and N-formylated peptides derived from bacteria. Thus, even though CD8+ T cells were shown to be important for the control of infection, the molecules used to present antigens to the protective T cell subset remained unclear.

To determine the relative contributions of classical or nonclassical MHC Class I–dependent CD8+ T cell populations in protection against tuberculosis, a series of gene-disrupted mouse strains were compared for susceptibility to intravenous M. tuberculosis infection, as measured by survival time and bacterial loads. Among the strains tested, the most susceptible mice were the ß2m-/-, followed by TAP1-/- (transporter associated with antigen processing), followed by CD8{alpha}-/-, perforin-/-, and CD1d-/- mice (9). The conclusion was that classically restricted (which are TAP1-dependent) CD8+ T cells contribute to in vivo protection against M. tuberculosis; however, the role of CD8+ T cells in protective immunity was not limited to perforin-dependent cytotoxicity (9).

The absence of CD8+ T cells did not fully account for the increased susceptibility of ß2m-/- mice to infection, suggesting that there are additional protective components associated with the ß2m molecule (9). The ß2m-/- mice developed granulomas that were initially devoid of lymphocytes (9, 10). As infection progressed, lymphocytes did accumulate, but they failed to infiltrate the macrophage-dominated lesions, implicating a novel and undefined ß2m-dependent mechanism influencing early lymphocyte accumulation (10). In addition, ß2m-/- mice have defects in iron metabolism. These mice have high levels of iron in all organs tested, which seemed to promote the growth of M. tuberculosis, as administration of iron-chelating agents significantly reduced the bacterial load in ß2m-/- mice (Schaible and Kaufmann, personal communication).

This confusion has been clarified in a recent study where mice specifically deficient in classical MHC Ia molecules were more susceptible to M. tuberculosis infection than were wild-type mice, although not as susceptible as ß2m-/-, providing strong evidence for the role of MHC I classically restricted CD8+ T cells in resistance to M. tuberculosis (11).

It is well established that CD4+ T cell response is crucial to control infection, and it must be targeted in vaccine development. Although many investigators believe that CD8+ T cells are important and should be considered in the design of new vaccines, others are not as convinced. In a recent study, wild-type and gene-knockout mice deficient in both CD4+ and CD8+ T cells, MHC I (lack CD8+ T cells) or MHC II (lack CD4+ T cells) were infected via aerosol and monitored for survival and ability to control infection (12). The conclusion of this study was that in contrast to CD4+ T cells, CD8+ T cells were dispensable and not essential for the control of infection. However, data from this study do not fully support these conclusions. Although mice devoid of CD4+ T cells died earlier from tuberculosis than did CD8+ T cell–deficient mice, the absence of both CD4+ and CD8+ T cells resulted in even greater susceptibility. One interpretation is that the presence of CD8+ T cells in CD4+ T cell–deficient mice resulted not only in increased survival time but also prevented excessive immunopathology when compared with mice without both T cell subsets (12). The fact that mice devoid of CD8+ T cells still succumb to M. tuberculosis infection, despite the development of fully functional CD4+ T cell responses, argues that CD8+ T cells may play an important role in controlling chronic infection. Furthermore, depletion of CD8+ T cells resulted in reactivation of latent tuberculosis in a murine model, suggesting that this T cell subset may also be essential for controlling latent tuberculosis (13).

EFFECTOR FUNCTIONS OF CD8+ T CELLS

Murine Studies
CD8+ T cells have the potential to affect antimycobacterial immunity in a number of ways. These cells may function as a source of type-1 cytokines such as IFN-{gamma} and TNF-{alpha} or they may exert their protective effect by killing infected macrophages within the tissues (Figure 1). IFN-{gamma} and TNF-{alpha} are important for the activation of macrophages; both CD4+ and CD8+ T cells have the potential to produce these cytokines at the site of infection (14) (Figure 1). CD8+ T cells can kill infected macrophages via a perforin-mediated mechanism. Perforin, a protein in the granules of CD8+ T cells, forms a pore in the membrane of infected macrophages and allows the entry of toxic proteins, such as granzymes or granulysin, leading to the apoptosis of infected macrophages. Apoptosis (programmed cell death) of macrophages can also be induced by ligation of Fas ligand on activated CD8+ T cells, with Fas on infected macrophages (Figure 1).

Results from experiments using gene-deficient mice have generated controversy regarding which of the CD8+ T cell–mediated mechanisms are responsible for protection against tuberculosis. Intracellular cytokine staining indicated that comparable numbers of activated CD4+ and CD8+ T cells in the lungs of infected mice were primed to produce IFN-{gamma} after brief, nonspecific stimulation (14). However, intracellular cytokine staining of unstimulated CD4+ and CD8+ T cells from the lungs of infected mice, which is more reflective of an in vivo situation, suggested an important difference between the two T cell subsets. Early in infection, at least 13% of CD4+ T cells produced IFN-{gamma} directly ex vivo, in contrast to < 5% of IFN-{gamma}–producing CD8+ T cells (14). The presence of large numbers of activated CD8+ T cells in the lungs, with moderate cytokine production, suggests that the cytotoxic functions of CD8+ T cells may be important in the response against acute M. tuberculosis infection (14).

Early in infection, M. tuberculosis–specific CD8+ T cells from the lungs expressed perforin in vivo and lysed M. tuberculosis–infected macrophages in a perforin-dependent and MHC Class I–dependent manner (15). Additional evidence for the importance of CD8+ cytotoxic effector functions comes from studies in CD4+ T cell–deficient mice, which succumbed to acute or chronic infection of M. tuberculosis, despite compensatory IFN-{gamma} production by CD8+ T cells resulting in wild-type levels of this cytokine in the lungs (16, 17). Subsequently, it was shown that CD8+ T cells from CD4+ T cell–deficient mice have impaired cytotoxic function in the lungs of M. tuberculosis–infected mice (18). These results suggest that the susceptibility to tuberculosis seen in CD4+ T cell–deficient mice may be partly due to defective cytotoxic effector functions of CD8+ T cells. Furthermore, the level of protection against M. tuberculosis, conferred by the adoptive transfer of CD8+ T cell clones into recipient mice, correlated with the level of cytotoxicity rather than with the level of IFN-{gamma} secretion (19).

On the other hand, several studies suggest that cytokine secretion may be the major effector function of CD8+ T cells (Figure 1). In experiments using IFN-{gamma}-/- mice as CD8+ T cell donors, production of IFN-{gamma} was required for CD8+ T cells to exert a modest antimycobacterial effect in CD4+ T cell–deficient mice (20). Moreover, mice with targeted disruptions in the genes for Fas, perforin, or granzyme were no more susceptible to acute infection with M. tuberculosis than were their wild-type littermates (21, 22). The fact that perforin-/- mice succumb later in infection suggests that perforin-mediated cytotoxic activity of CD8+ T cells may be more important during the chronic stage of infection (9). However, interpretation of these results is complicated by the fact that perforin gene disruption is associated with a compensatory activation of T cells and expression of increased levels of cytokines even in the absence of experimental infection (22, 23). Perforin deficiency also affects the function of CD8+ T cells during acute M. tuberculosis infection (15). Perforin -/- mice had increased numbers of CD8+ T lymphocytes, which were in a state of hyperactivation, with 4.5-fold increased IFN-{gamma} production compared with wild-type mice (15). This could mask any effect of the lack of perforin on the control of infection.

Human Studies
There is one confounding factor for addressing the importance of CD8+ T cells in the control of M. tuberculosis in the murine model. Recent studies demonstrated that human CD8+ T cells recognizing M. tuberculosis–infected macrophages had the ability to directly kill intracellular mycobacteria (24) (Figure 1). This killing was due to a granule-associated protein, granulysin (25). The purified molecule was toxic to mycobacteria but required perforin pore formation to enter an infected cell. This was an important demonstration of how CD8+ T cells could be playing a direct role in the control of M. tuberculosis infection. Unfortunately, mice do not have a granulysin homolog, and at this time it is not possible to test the true contribution of this mechanism to the control of infection in the mouse model. Therefore, data from murine studies suggesting that CD8+ T cells are not necessary for the control of infection must take into account the absence of what may be the key mechanism by which CD8+ T cells participate in antituberculosis immunity. Nonetheless, questions on CD8+ T cell induction, kinetics of migration to the lungs, cytokine production, and cytotoxic activity can be and have been addressed in the murine model and have provided important insights into the role of these cells in protection against tuberculosis.

ANTIGEN SPECIFICITY OF CLASSICAL AND NONCLASSICAL MHC CLASS I–RESTRICTED CD8+ T CELLS IN HUMAN IMMUNE RESPONSES TO M. TUBERCULOSIS

Each T cell is specific for a single epitope, or short stretch of amino acids, within a protein antigen. Identification of antigens (and epitopes) that are protective is an important step in designing an effective vaccine. Inclusion of that antigen in a vaccine would induce expansion of T cells specific for that antigen and would lead to increased protection against infection with M. tuberculosis.

Classically restricted CD8+ T cells recognize peptides bound in the groove of MHC Ia molecules. CD8+ T cells can also recognize antigens that are not proteinaceous in nature, such as lipids and glycolipids, presented by nonclassical MHC I–like molecules, e.g., CD1 and MHC Ib. As a great proportion of the mycobacterial genome is dedicated to the synthesis of lipids and glycolipids, studies have focused on the identification of both classically and nonclassically restricted CD8+ T cells and the antigens recognized by these cells.

Human Studies
Identification of CD8+ T cells that could recognize M. tuberculosis antigens from infected mice or humans took many years and culture conditions needed to be carefully controlled. However, now both classically (MHC Class Ia) and nonclassically (MHC Class Ib) restricted CD8+ T cells are found in humans with active tuberculosis and also in subjects who tested positive for PPD without active disease (26–33). Mycobacteria-specific CD8+ T cells are induced in response to M. tuberculosis infection; however, the proportion of classically and nonclassically restricted CD8+ T cells may differ among subjects.

IFN-{gamma}–based enzyme–linked immunospot assays were used to determine the frequency of M. tuberculosis–reactive CD8+ T cells directly from peripheral blood mononuclear cells of naive (PPD negative) and M. tuberculosis–exposed (PPD positive) individuals (27). This study showed that the frequency of IFN-{gamma}–producing CD8+ effector cells among five healthy subjects who tested positive for PPD was 1/7,600 ± 4,300, compared with that of 1/16,000 ± 7,000 in six individuals who tested negative for PPD. Detailed characterization of CD8+ T cell clones from one subject who tested positive for PPD revealed that 4% were classically restricted and 96% were nonclassically restricted (although not CD1-restricted), suggesting that classically restricted CD8+ T cell clones may represent a small component of the overall M. tuberculosis–specific CD8+ T cell responses in some individuals (27). These results also suggest presentation of mycobacterial antigens by other nonpolymorphic MHC Ib molecules such as human leukocyte antigen (HLA)-E, -F, or -H.

The specific mycobacterial antigens to which human or mouse CD8+ T-cells respond are in the process of being characterized. Strong reactivity of human CD8+ T cells against several M. tuberculosis antigens has been reported, including early secretory antigenic target 6, Ag85A, Ag85B, 38 kD protein, heat shock protein 65, and 19 kD lipoprotein (29, 30, 34–36).

CD8+ T cells specific for epitopes in the early secretory antigenic target 6 protein, which is absent from the vaccine M. bovis Bacille Calmette-Guérin (BCG) strain, were detected in peripheral blood mononuclear cells from patients with active tuberculosis, after antibiotic therapy, and in healthy contacts who tested positive for PPD (34). The early secretory antigenic target 6–reactive CD8+ T cells produced IFN-{gamma}, recognized endogenously processed antigen, and were cytolytic (34). The frequency of the circulating early secretory antigenic target 6–specific effectors (1/23,000) in the peripheral blood of a patient with active tuberculosis was relatively high and comparable with the frequency of effectors specific for an influenza virus matrix epitope (1/14,000) (34). A prominent CD8+ T cell response against early secretory antigenic target 6 was also detected in patients with active pulmonary tuberculosis from Gambia, which was virtually absent from healthy, BCG-vaccinated control subjects (35).

Other important targets of the human CD8+ T cell responses against M. tuberculosis are the proteins of the Ag85 complex, a major component of M. tuberculosis culture filtrate (30). Ag85 complex induces strong T cell proliferation and IFN-{gamma} production in individuals who tested positive for PPD (37), and in BCG-vaccinated mice and humans (38, 39). The presence of Ag85-specific CD8+ T cells in BCG-vaccinated healthy subjects was demonstrated 10 to 30 years after vaccination, suggesting that mycobacterial Ag85-secreted proteins may be potent inducers of CD8+ T cells (35).

Because antigen recognition by CD8+ T cells is tightly controlled by polymorphic alleles of MHC Class I genes (HLA), screening of potential vaccine candidates needs to be conducted in the context of HLA, to ensure antigen recognition by a majority of the population (29). HLA-A*0201 is one of the most prevalent MHC-I alleles, with a frequency of over 30% in most populations (29). Two Ag85A and two Ag85B peptide epitopes for HLA-A*0201–restricted CD8+ T cells were identified (29, 35). These CD8+ T cells were present at a high frequency in peripheral blood mononuclear cells of healthy individuals who tested positive for PPD and produced IFN-{gamma} and TNF-{alpha}. In addition, the peptide-specific CD8+ T cells recognized and lysed M. tuberculosis–infected or BCG-infected HLA-A*0201 macrophages, suggesting that these two epitopes could be potential subunit components of future vaccines (29, 35).

A single HLA-A*0201–binding epitope derived from mycobacterial 19 kD lipoprotein was recognized by circulating CD8+ T cells from both healthy individuals who tested positive for PPD and in patients with active tuberculosis but not by subjects who tested negative for PPD (36). Nineteen kilodalton–specific CD8+ T cells were able to recognize and lyse M. tuberculosis–infected macrophages, implying that this antigen is endogenously processed and recognized during in vivo infection (36). In addition to secreted antigens, several HLA-A*0201–restricted epitopes derived from somatic proteins of M. tuberculosis (thymidylate synthase, RNA polymerase ß subunit, permease protein A-1) induced strong IFN-{gamma} production and cytotoxicity by CD8+ T cells from patients recovering from tuberculosis (31).

Whereas most of these studies concentrated on HLA-A*0201–restricted responses, two peptides from culture filtrate protein 10 (or Mtb11) were identified that were restricted by different MHC Class I alleles, HLA-B44, and HLA-B14 (28). In a single donor who tested positive for PPD, the effector CD8+ T cell frequency for the HLA-B44–restricted peptide was 1/700, and that for the HLA-B14–restricted peptide was 1/2,100 (28). Such high effector frequencies imply that most of the CD8+ T cell response against Mtb11 culture filtrate protein in this individual is directed toward these two epitopes. The results from studies mentioned previously are consistent with a model of immunodominance, which is used to describe strong and focused immune responses directed against only a few epitopes (reviewed in [40]). However, it remains to be seen whether the patterns of potential immunodominance apply to a larger study population.

A very limited number of antigens recognized by M. tuberculosis–reactive CD8+ T cells have been identified. However, it is promising that epitope-specific CD8+ T cells are present in the circulation of M. tuberculosis–exposed individuals at a relatively high frequency, suggesting a long-lived memory response to certain antigens. Single cell analyses, such as tetramer staining and IFN-{gamma}–based enzyme–linked immunospot assay, revealed that the frequency of CD8+ effector T cells against immunogenic epitopes is comparable with the frequency of CD8+ T cells seen during viral infections. Such antigens should be considered as potential candidates for subunit vaccines because they may be important in controlling M. tuberculosis infection.

CONCLUSION

Experimental evidence in the murine model supports the conclusion that CD8+ T cells are important for the in vivo control of M. tuberculosis infection. The isolation of M. tuberculosis–specific CD8+ T cells from infected mice and humans clearly shows that this subset is induced during infection. Reports on epitope-specific, M. tuberculosis–reactive CD8+ T cells, which are present at very high frequencies in the circulation of individuals who tested positive for PPD and in patients with active tuberculosis, support the importance of CD8+ T cells in immunity to M. tuberculosis and emphasize that the CD8+ T cell subset should be considered in the design of new antituberculosis vaccines.

Perhaps, even more importantly, CD8+ T cells from infected mice and humans can recognize M. tuberculosis–infected macrophages, and in response produce cytokines or kill infected macrophages. The infected macrophage is the relevant target cell in the lungs, and a subset of cells that can recognize this target and elaborate effector functions has the potential to control the infection.

A strong CD4+ T cell response is induced and is necessary, but apparently not sufficient, to resolve an infection. Many of the tuberculosis vaccine strategies tested have induced strong CD4+ T cell responses but have not been more effective than the current BCG vaccine in animal models. BCG also induces a strong CD4+ T cell response; however, it is not generally effective against adult tuberculosis. Therefore, vaccine development should be expanded to focus on generating both strong CD8+ T cell and CD4+ T cell responses. This can be achieved through a variety of strategies developed for other diseases, including DNA immunization with boosting from viral vectors expressing CD8+ T cell antigens (41–43) or alternative vectors for CD8+ T cell induction, such as attenuated viruses or bacteria (44, 45). These vaccine strategies for tuberculosis are under development now and may succeed in generating a robust and long-lived CD8+ T cell responses that, in conjunction with strong CD4+ T cell responses, could contribute substantially to the control or clearance of the infection.

The questions of whether or not CD8+ T cells are "required" for the control of M. tuberculosis and which type of CD8+ T cell (classically or nonclassically restricted) is important cannot be resolved in the current mouse system. The mouse does not express CD1a-c, the molecules that present mycobacterial lipids to T-cells; thus, the contribution of this system to protection against tuberculosis cannot be addressed. The recent description of CD1a-c molecules in the guinea pig will provide a better model for studying the protective role of CD1-restricted CD8+ T cells in tuberculosis (46). Guinea pigs can be immunized with lipids that are presented by CD1 molecules, then challenged to assess protection by these lipids. In addition, mouse CD8+ T cells do not produce granulysin, and because this is a molecule known to kill M. tuberculosis, it is likely to be quite important in the outcome of infection.

The importance of CD8+ T cells in the immune response to tuberculosis has been recognized by many scientists. However, many questions still remain such as processing and presentation of M. tuberculosis–specific antigens for recognition by CD8+ T cells; antigens and epitopes important for protection; the importance of these cells in initial and long-term control of infection; the memory of CD8+ T cell responses; the interaction of CD8+ T cells with CD4+ T cells; and how to vaccinate to induce protective and long-lasting CD8+ T cell responses.

Acknowledgments

The authors thank Jeffrey Mai for the illustration (Figure 1) and are also grateful to the members of the Flynn laboratory for helpful discussion and to Dr. Kaufmann for sharing results before publication.

FOOTNOTES

Supported by grants from the National Institutes of Health (AI37859, AI40310; J.L.F.) and the American Lung Association (CI-016-N; J.L.F.).

Received in original form April 17, 2002; accepted in final form July 22, 2002

REFERENCES

  1. WHO. The World Health Report. 1999. http://www.who.int/whr/.
  2. Dye C, Scheele S, Dolin P, Pathania V, Raviglione MC. Consensus statement. Global burden of tuberculosis: estimated incidence, prevalence, and mortality by country. WHO Global Surveillance and Monitoring Project. JAMA 1999;282:677–686.[Abstract/Free Full Text]
  3. Selwyn PA, Hartel D, Lewis VA, Schoenbaum EE, Vermund SH, Klein RS, Walker AT, Friedland GH. A prospective study of the risk of tuberculosis among intravenous drug users with human immunodeficiency virus infection. N Engl J Med 1989;320:545–550.[Abstract]
  4. Flynn JL, Chan J. Immunology of tuberculosis. Annu Rev Immunol 2001; 19:93–129.[CrossRef][Medline]
  5. Muller I, Cobbold SP, Waldmann H, Kaufmann SH. Impaired resistance to Mycobacterium tuberculosis infection after selective in vivo depletion of L3T4+ and Lyt-2+ T cells. Infect Immun 1987;55:2037–2041.[Abstract/Free Full Text]
  6. Orme IM. The kinetics of emergence and loss of mediator T lymphocytes acquired in response to infection with Mycobacterium tuberculosis. J Immunol 1987;138:293–298.[Abstract]
  7. Orme IM, Collins FM. Adoptive protection of the Mycobacterium tuberculosis-infected lung: dissociation between cells that passively transfer protective immunity and those that transfer delayed-type hypersensitivity to tuberculin. Cell Immunol 1984;84:113–120.[CrossRef][Medline]
  8. Flynn JL, Goldstein MM, Triebold KJ, Koller B, Bloom BR. Major histocompatibility complex class I-restricted T cells are required for resistance to Mycobacterium tuberculosis infection. Proc Natl Acad Sci USA 1992;89:12013–12017.[Abstract/Free Full Text]
  9. Sousa AO, Mazzaccaro RJ, Russell RG, Lee FK, Turner OC, Hong S, Van Kaer L, Bloom BR. Relative contributions of distinct MHC class I-dependent cell populations in protection to tuberculosis infection in mice. Proc Natl Acad Sci USA 2000;97:4204–4208.[Abstract/Free Full Text]
  10. D'Souza CD, Cooper AM, Frank AA, Ehlers S, Turner J, Bendelac A, Orme IM. A novel nonclassic beta2-microglobulin-restricted mechanism influencing early lymphocyte accumulation and subsequent resistance to tuberculosis in the lung. Am J Respir Cell Mol Biol 2000; 23:188–193.[Abstract/Free Full Text]
  11. Rolph MS, Raupach B, Kobernick HH, Collins HL, Perarnau B, Lemonnier FA, Kaufmann SH. MHC class Ia-restricted T cells partially account for beta2-microglobulin-dependent resistance to Mycobacterium tuberculosis. Eur J Immunol 2001;31:1944–1949.[CrossRef][Medline]
  12. Mogues T, Goodrich M, Ryan L, LaCourse R, North R. The relative importance of T cell subsets in immunity and immunopathology of airborne Mycobacterium tuberculosis infection in mice. J Exp Med 2001;193:271–280.[Abstract/Free Full Text]
  13. van Pinxteren LA, Cassidy JP, Smedegaard BH, Agger EM, Andersen P. Control of latent Mycobacterium tuberculosis infection is dependent on CD8 T cells. Eur J Immunol 2000;30:3689–3698.[CrossRef][Medline]
  14. Serbina NV, Flynn JL. Early emergence of CD8(+) T cells primed for production of type 1 cytokines in the lungs of Mycobacterium tuberculosis-infected mice. Infect Immun 1999;67:3980–3988.[Abstract/Free Full Text]
  15. Serbina NV, Liu CC, Scanga CA, Flynn JL. CD8+ CTL from lungs of Mycobacterium tuberculosis-infected mice express perforin in vivo and lyse infected macrophages. J Immunol 2000;165:353–363.[Abstract/Free Full Text]
  16. Caruso AM, Serbina N, Klein E, Triebold K, Bloom BR, Flynn JL. Mice deficient in CD4 T cells have only transiently diminished levels of IFN-gamma, yet succumb to tuberculosis. J Immunol 1999;162:5407–5416.[Abstract/Free Full Text]
  17. Scanga CA, Mohan VP, Yu K, Joseph H, Tanaka K, Chan J, Flynn JL. Depletion of CD4(+) T cells causes reactivation of murine persistent tuberculosis despite continued expression of interferon gamma and nitric oxide synthase 2. J Exp Med 2000;192:347–358.[Abstract/Free Full Text]
  18. Serbina NV, Lazarevic V, Flynn JL. CD4(+) T cells are required for the development of cytotoxic CD8(+) T cells during Mycobacterium tuberculosis infection. J Immunol 2001;167:6991–7000.[Abstract/Free Full Text]
  19. Silva CL, Lowrie DB. Identification and characterization of murine cytotoxic T cells that kill Mycobacterium tuberculosis. Infect Immun 2000; 68:3269–3274.[Abstract/Free Full Text]
  20. Tascon RE, Stavropoulos E, Lukacs KV, Colston MJ. Protection against Mycobacterium tuberculosis infection by CD8+ T cells requires the production of gamma interferon. Infect Immun 1998;66:830–834.[Abstract/Free Full Text]
  21. Cooper AM, D'Souza C, Frank AA, Orme IM. The course of Mycobacterium tuberculosis infection in the lungs of mice lacking expression of either perforin- or granzyme-mediated cytolytic mechanisms. Infect Immun 1997;65:1317–1320.[Abstract]
  22. Laochumroonvorapong P, Wang J, Liu CC, Ye W, Moreira AL, Elkon KB, Freedman VH, Kaplan G. Perforin, a cytotoxic molecule which mediates cell necrosis, is not required for the early control of mycobacterial infection in mice. Infect Immun 1997;65:127–132.[Abstract]
  23. Matloubian M, Suresh M, Glass A, Galvan M, Chow K, Whitmire JK, Walsh CM, Clark WR, Ahmed R. A role for perforin in downregulating T cell responses during chronic viral infection. J Virol 1999;73:2527–2536.[Abstract/Free Full Text]
  24. Stenger S, Mazzaccaro RJ, Uyemura K, Cho S, Barnes PF, Rosat JP, Sette A, Brenner MB, Porcelli SA, Bloom BR, et al. Differential effects of cytolytic T cell subsets on intracellular infection. Science 1997;276:1684–1687.[Abstract/Free Full Text]
  25. Stenger S, Hanson DA, Teitelbaum R, Dewan P, Niazi KR, Froelich CJ, Ganz T, Thoma-Uszynski S, Melian A, Bogdan C, et al. An antimicrobial activity of cytolytic T cells mediated by granulysin. Science 1998;282:121–125.[Abstract/Free Full Text]
  26. Rosat JP, Grant EP, Beckman EM, Dascher CC, Sieling PA, Frederique D, Modlin RL, Porcelli SA, Furlong ST, Brenner MB. CD1-restricted microbial lipid antigen-specific recognition found in the CD8+ alpha beta T cell pool. J Immunol 1999;162:366–371.[Abstract/Free Full Text]
  27. Lewinsohn DM, Briden AL, Reed SG, Grabstein KH, Alderson MR. Mycobacterium tuberculosis-reactive CD8+ T lymphocytes: the relative contribution of classical versus nonclassical HLA restriction. J Immunol 2000;165:925–930.[Abstract/Free Full Text]
  28. Lewinsohn DM, Zhu L, Madison VJ, Dillon DC, Fling SP, Reed SG, Grabstein KH, Alderson MR. Classically restricted human CD8+ T lymphocytes derived from Mycobacterium tuberculosis-infected cells: definition of antigenic specificity. J Immunol 2001;166:439–446.[Abstract/Free Full Text]
  29. Geluk A, van Meijgaarden KE, Franken KL, Drijfhout JW, D'Souza S, Necker A, Huygen K, Ottenhoff TH. Identification of major epitopes of Mycobacterium tuberculosis AG85B that are recognized by HLA-A*0201-restricted CD8+ T cells in HLA-transgenic mice and humans. J Immunol 2000;165:6463–6471.[Abstract/Free Full Text]
  30. Smith SM, Brookes R, Klein MR, Malin AS, Lukey PT, King AS, Ogg GS, Hill AV, Dockrell HM. Human CD8+ CTL specific for the mycobacterial major secreted antigen 85A. J Immunol 2000;165:7088–7095.[Abstract/Free Full Text]
  31. Cho S, Mehra V, Thoma-Uszynski S, Stenger S, Serbina N, Mazzaccaro RJ, Flynn JL, Barnes PF, Southwood S, Celis E, et al. Antimicrobial activity of MHC class I-restricted CD8+ T cells in human tuberculosis. Proc Natl Acad Sci USA 2000;97:12210–12215.[Abstract/Free Full Text]
  32. Lewinsohn DM, Alderson MR, Briden AL, Riddell SR, Reed SG, Grabstein KH. Characterization of human CD8+ T cells reactive with Mycobacterium tuberculosis-infected antigen-presenting cells. J Exp Med 1998;187:1633–1640.[Abstract/Free Full Text]
  33. Canaday DH, Ziebold C, Noss EH, Chervenak KA, Harding CV, Boom WH. Activation of human CD8+ alpha beta TCR+ cells by Mycobacterium tuberculosis via an alternate Class I MHC antigen-processing pathway. J Immunol 1999;162:372–379.[Abstract/Free Full Text]
  34. Lalvani A, Brookes R, Wilkinson RJ, Malin AS, Pathan AA, Andersen P, Dockrell H, Pasvol G, Hill AV. Human cytolytic and interferon gamma-secreting CD8+ T lymphocytes specific for Mycobacterium tuberculosis. Proc Natl Acad Sci USA 1998;95:270–275.[Abstract/Free Full Text]
  35. Smith SM, Klein MR, Malin AS, Sillah J, Huygen K, Andersen P, McAdam KP, Dockrell HM. Human CD8(+) T cells specific for Mycobacterium tuberculosis secreted antigens in tuberculosis patients and healthy BCG-vaccinated controls in The Gambia. Infect Immun 2000; 68:7144–7148.[Abstract/Free Full Text]
  36. Mohagheghpour N, Gammon D, Kawamura LM, van Vollenhoven A, Benike CJ, Engleman EG. CTL response to Mycobacterium tuberculosis: identification of an immunogenic epitope in the 19-kDa lipoprotein. J Immunol 1998;161:2400–2406.[Abstract/Free Full Text]
  37. Launois P, DeLeys R, Niang MN, Drowart A, Andrien M, Dierckx P, Cartel JL, Sarthou JL, Van Vooren JP, Huygen K. T cell-epitope mapping of the major secreted mycobacterial antigen Ag85A in tuberculosis and leprosy. Infect Immun 1994;62:3679–3687.[Abstract/Free Full Text]
  38. Huygen K, Lozes E, Gilles B, Drowart A, Palfliet K, Jurion F, Roland I, Art M, Dufaux M, Nyabenda J, et al. Mapping of TH1 helper T cell epitopes on major secreted mycobacterial antigen 85A in mice infected with live Mycobacterium bovis BCG. Infect Immun 1994;62:363–370.[Abstract/Free Full Text]
  39. Thole JE, Janson AA, Cornelisse Y, Schreuder GM, Wieles B, Naafs B, de Vries RR, Ottenhoff TH. HLA-class II-associated control of antigen recognition by T cells in leprosy: a prominent role for the 30/31-kDa antigens. J Immunol 1999;162:6912–6918.[Abstract/Free Full Text]
  40. Yewdell JW, Bennink JR. Immunodominance in major histocompatibility complex class I-restricted T lymphocyte responses. Annu Rev Immunol 1999;17:51–88.[CrossRef][Medline]
  41. Feng CG, Blanchard TJ, Smith GL, Hill AV, Britton WJ. Induction of CD8+ T-lymphocyte responses to a secreted antigen of Mycobacterium tuberculosis by an attenuated vaccinia virus. Immunol Cell Biol 2001;79:569–575.[CrossRef][Medline]
  42. Lowrie DB, Tascon RE, Bonato VL, Lima VM, Faccioli LH, Stavropoulos E, Colston MJ, Hewinson RG, Moelling K, Silva CL. Therapy of tuberculosis in mice by DNA vaccination. Nature 1999;400:269–271.[CrossRef][Medline]
  43. McShane H, Brookes R, Gilbert SC, Hill AV. Enhanced immunogenicity of CD4(+) T cell responses and protective efficacy of a DNA-modified vaccinia virus Ankara prime-boost vaccination regimen for murine tuberculosis. Infect Immun 2001;69:681–686.[Abstract/Free Full Text]
  44. Hess J, Grode L, Hellwig J, Conradt P, Gentschev I, Goebel W, Ladel C, Kaufmann SH. Protection against murine tuberculosis by an attenuated recombinant Salmonella typhimurium vaccine strain that secretes the 30-kDa antigen of Mycobacterium bovis BCG. FEMS Immunol Med Microbiol 2000;27:283–289.[CrossRef][Medline]
  45. Mollenkopf HJ, Groine-Triebkorn D, Andersen P, Hess J, Kaufmann SH. Protective efficacy against tuberculosis of ESAT-6 secreted by a live Salmonella typhimurium vaccine carrier strain and expressed by naked DNA. Vaccine 2001;19:4028–4035.[CrossRef][Medline]
  46. Dascher CC, Hiromatsu K, Naylor JW, Brauer PP, Brown KA, Storey JR, Behar SM, Kawasaki ES, Porcelli SA, Brenner MB, et al. Conservation of a CD1 multigene family in the guinea pig. J Immunol 1999; 163:5478–5488.[Abstract/Free Full Text]
  47. Stenger S. Cytolytic T cells in the immune response to Mycobacterium tuberculosis. Scand J Infect Dis 2001;33:483–487.[CrossRef][Medline]



This article has been cited by other articles:


Home page
 J. Leukoc. Biol.Home page
D. Ordway, D. M. Higgins, J. Sanchez-Campillo, J. S. Spencer, M. Henao-Tamayo, M. Harton, I. M. Orme, and M. Gonzalez Juarrero
XCL1 (lymphotactin) chemokine produced by activated CD8 T cells during the chronic stage of infection with Mycobacterium tuberculosis negatively affects production of IFN-{gamma} by CD4 T cells and participates in granuloma stability
J. Leukoc. Biol., November 1, 2007; 82(5): 1221 - 1229.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
A. Kamath, J. S.M. Woodworth, and S. M. Behar
Antigen-Specific CD8+ T Cells and the Development of Central Memory during Mycobacterium tuberculosis Infection
J. Immunol., November 1, 2006; 177(9): 6361 - 6369.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
S. D'Souza, M. Romano, J. Korf, X.-M. Wang, P.-Y. Adnet, and K. Huygen
Partial Reconstitution of the CD4+-T-Cell Compartment in CD4 Gene Knockout Mice Restores Responses to Tuberculosis DNA Vaccines.
Infect. Immun., May 1, 2006; 74(5): 2751 - 2759.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
D. Ordway, M. Harton, M. Henao-Tamayo, R. Montoya, I. M. Orme, and M. Gonzalez-Juarrero
Enhanced Macrophage Activity in Granulomatous Lesions of Immune Mice Challenged with Mycobacterium tuberculosis.
J. Immunol., April 15, 2006; 176(8): 4931 - 4939.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
D. Sud, C. Bigbee, J. L. Flynn, and D. E. Kirschner
Contribution of CD8+ T Cells to Control of Mycobacterium tuberculosis Infection
J. Immunol., April 1, 2006; 176(7): 4296 - 4314.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
C. Carranza, E. Juarez, M. Torres, J. J. Ellner, E. Sada, and S. K. Schwander
Mycobacterium tuberculosis Growth Control by Lung Macrophages and CD8 Cells from Patient Contacts
Am. J. Respir. Crit. Care Med., January 15, 2006; 173(2): 238 - 245.
[Abstract] [Full Text] [PDF]

Home page
 NEJMHome page
G. Kaplan
Rational vaccine development--a new trend in tuberculosis control.
N. Engl. J. Med., October 13, 2005; 353(15): 1624 - 1625.
[Full Text] [PDF]

Home page
 J. Immunol.Home page
M. Buettner, C. Meinken, M. Bastian, R. Bhat, E. Stossel, G. Faller, G. Cianciolo, J. Ficker, M. Wagner, M. Rollinghoff, et al.
Inverse Correlation of Maturity and Antibacterial Activity in Human Dendritic Cells
J. Immunol., April 1, 2005; 174(7): 4203 - 4209.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
C. Holscher, A. Holscher, D. Ruckerl, T. Yoshimoto, H. Yoshida, T. Mak, C. Saris, and S. Ehlers
The IL-27 Receptor Chain WSX-1 Differentially Regulates Antibacterial Immunity and Survival during Experimental Tuberculosis
J. Immunol., March 15, 2005; 174(6): 3534 - 3544.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
K. M. Patton, T. C. McGuire, D. G. Fraser, and S. A. Hines
Rhodococcus equi-Infected Macrophages Are Recognized and Killed by CD8+ T Lymphocytes in a Major Histocompatibility Complex Class I-Unrestricted Fashion
Infect. Immun., December 1, 2004; 72(12): 7073 - 7083.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
A. L. Gervassi, K. H. Grabstein, P. Probst, B. Hess, M. R. Alderson, and S. P. Fling
Human CD8+ T Cells Recognize the 60-kDa Cysteine-Rich Outer Membrane Protein from Chlamydia trachomatis
J. Immunol., December 1, 2004; 173(11): 6905 - 6913.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
H. M. Scott Algood and J. L. Flynn
CCR5-Deficient Mice Control Mycobacterium tuberculosis Infection despite Increased Pulmonary Lymphocytic Infiltration
J. Immunol., September 1, 2004; 173(5): 3287 - 3296.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
H. Shams, P. Klucar, S. E. Weis, A. Lalvani, P. K. Moonan, H. Safi, B. Wizel, K. Ewer, G. T. Nepom, D. M. Lewinsohn, et al.
Characterization of a Mycobacterium tuberculosis Peptide That Is Recognized by Human CD4+ and CD8+ T Cells in the Context of Multiple HLA Alleles
J. Immunol., August 1, 2004; 173(3): 1966 - 1977.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
S. Marino, S. Pawar, C. L. Fuller, T. A. Reinhart, J. L. Flynn, and D. E. Kirschner
Dendritic Cell Trafficking and Antigen Presentation in the Human Immune Response to Mycobacterium tuberculosis
J. Immunol., July 1, 2004; 173(1): 494 - 506.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
H. M. S. Algood, P. L. Lin, D. Yankura, A. Jones, J. Chan, and J. L. Flynn
TNF Influences Chemokine Expression of Macrophages In Vitro and That of CD11b+ Cells In Vivo during Mycobacterium tuberculosis Infection
J. Immunol., June 1, 2004; 172(11): 6846 - 6857.
[Abstract] [Full Text] [PDF]

Home page
 J. Exp. Med.Home page
M. Gilleron, S. Stenger, Z. Mazorra, F. Wittke, S. Mariotti, G. Bohmer, J. Prandi, L. Mori, G. Puzo, and G. De Libero
Diacylated Sulfoglycolipids Are Novel Mycobacterial Antigens Stimulating CD1-restricted T Cells during Infection with Mycobacterium tuberculosis
J. Exp. Med., March 1, 2004; 199(5): 649 - 659.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
D. A. Lewinsohn, A. S. Heinzel, J. M. Gardner, L. Zhu, M. R. Alderson, and D. M. Lewinsohn
Mycobacterium tuberculosis-specific CD8+ T Cells Preferentially Recognize Heavily Infected Cells
Am. J. Respir. Crit. Care Med., December 1, 2003; 168(11): 1346 - 1352.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
M. J. Tobin
Writing a Review Article for AJRCCM
Am. J. Respir. Crit. Care Med., October 1, 2003; 168(7): 732 - 734.
[Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
J. L. Johnson, E. Ssekasanvu, A. Okwera, H. Mayanja, C. S. Hirsch, J. G. Nakibali, D. D. Jankus, K. D. Eisenach, W. H. Boom, J. J. Ellner, et al.
Randomized Trial of Adjunctive Interleukin-2 in Adults with Pulmonary Tuberculosis
Am. J. Respir. Crit. Care Med., July 15, 2003; 168(2): 185 - 191.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
M. J. Tobin
Tuberculosis, Lung Infections, Interstitial Lung Disease, and Journalology in AJRCCM 2002
Am. J. Respir. Crit. Care Med., February 1, 2003; 167(3): 345 - 355.
[Full Text] [PDF]

This Article
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lazarevic, V.
Right arrow Articles by Flynn, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lazarevic, V.
Right arrow Articles by Flynn, J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Proc. Am. Thorac. Soc. Am. J. Respir. Cell Mol. Biol.
Copyright © 2002 American Thoracic Society