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Laboratory of Vaccinology and Mucosal Immunity, Hpital Erasme, Université Libre de Bruxelles, Brussels, Belgium
Institut National de la Santé et de la Recherche Médicale U, Institut Pasteur de Lille, Lille, France
Background.
Most individuals infected with Mycobacterium tuberculosis do not develop tuberculosis (TB) and can be regarded as being protected by an appropriate immune response to the infection. The characterization of the immune responses of individuals with latent TB may thus be helpful in the definition of correlates of protection and the development of new vaccine strategies. The highly protective antigen heparin-binding hemagglutinin (HBHA) induces strong interferon (IFN) responses during latent, but not active, TB. Because of the recently recognized importance of CD8+ T lymphocytes in anti-TB immunity, we characterized the CD8+ T lymphocyte responses to HBHA in subjects with latent TB.
Results.
HBHA-specific CD8+ T lymphocytes expressed memory cell markers and synthesized HBHA-specific IFN-. They also restricted mycobacterial growth and expressed cytotoxicity by a granule-dependent mechanism. This activity was associated with the intracellular expression of HBHA-induced perforin. Surprisingly, the perforin-producing CD8+ T lymphocytes were distinct from the IFN-producing CD8+ T lymphocytes.
Conclusion.
During latent TB, the HBHA-specific CD8+ T lymphocyte population expresses all 3 effector functions associated with CD8+ T lymphocytemediated protective immune mechanisms, which supports the notion that HBHA may be protective in humans and suggests that markers of HBHA-specific CD8+ T lymphocyte responses may be useful in the monitoring of protection.
Eight million new cases of tuberculosis (TB), as well as 23 million TB-related deaths, are recorded each year, and it is estimated that one-third of the world's population is presently infected with Mycobacterium tuberculosis [1]. Fortunately, not all individuals infected with M. tuberculosis will develop overt disease, and 90%95% of infected individuals will stay healthy throughout their lives. In contrast to individuals with active TB, the vast majority of individuals with latent TB can thus be considered to be protected by an appropriate, naturally acquired immune response to M. tuberculosis antigens. The characterization of the antiM. tuberculosis immune response in individuals with latent TB, compared with that in individuals with active TB, may thus be useful in the understanding of protective immune effector mechanisms and, ultimately, in the development of new vaccines against TB.
The only vaccine available at present, the bacille Calmette-Guérin (BCG), displays variable efficacy, ranging from 0% to 80% protection [24]. Improved vaccines are urgently needed, but their development is hampered by the still incomplete understanding of the mechanisms of protective immunity to TB. Cellular immune responses mediated by interferon (IFN)-producing CD4+ T lymphocytes have long been established as an essential component of protective immunity against M. tuberculosis, both in animal models [57] and in humans [8, 9]. Much less is known about the contribution of CD8+ T lymphocytes, although recent evidence also points to an important role for these cells in protection. 2m- or transporter associated with antigen processing1deficient mice rapidly die of M. tuberculosis infection [10, 11], and the maintenance of M. tuberculosis in a quiescent stage is controlled by CD8+ T lymphocytes in murine models [12, 13]. In humans, M. tuberculosisspecific cytolytic and IFN-producing CD8+ T lymphocytes are induced during active TB [14, 15]. Although these cells display a variety of effector functions in vitro, their involvement in protective immunity against TB remains unclear, because they recognize M. tuberculosis peptides in individuals with active TB, who, by definition, are not protected by their immune response. Virtually nothing is known about the induction of CD8+ T lymphocytes and their effector functions in healthy individuals with latent TB.
Heparin-binding hemagglutinin (HBHA) is a recently identified surface-exposed and surface-secreted antigen [16] that is involved in extrapulmonary dissemination [17] and that induces strong IFN- production by circulating CD4+ and CD8+ T lymphocytes in healthy individuals with latent TB but not in individuals with active TB [18, 19]. This antigen is thus able to discriminate between these 2 groups of individuals infected with M. tuberculosis, which suggests that a proper immune response to HBHA contributes to protective immunity. In murine models, immunization with HBHA induces IFN-producing, as well as cytotoxic and microbicidal, T lymphocytes and provides protection at levels equivalent to those induced by vaccination with BCG [19, 20]. In view of the importance of antigen-specific CD8+ T lymphocyte responses in protection against TB, we sought, in the present study, to characterize the effector functions of HBHA-specific CD8+ T lymphocytes during latent TB.
SUBJECTS, MATERIALS, AND METHODS
Subjects.
Venous blood samples were collected from 12 healthy subjects infected with M. tuberculosis who were selected on the basis of positive tuberculin skin test results (>18 mm of induration), normal chest radiographic findings, and absence of clinical signs of TB. The study was approved by the local ethical committee, and informed consent was obtained from all subjects included in the study.
Antigen preparation.
HBHA was purified from M. bovis BCG by heparin-Sepharose chromatography [16] and then by reverse-phase high-pressure liquid chromatography [18].
Preparation of peripheral blood mononuclear cells (PBMCs).
PBMCs were obtained as described elsewhere [18] and were resuspended at a concentration of 2 × 106 cells/mL in RPMI 1640 (BioWhittaker) supplemented with 40 g/mL gentamicin, 50 mol/L 2-mercaptoethanol, 1× nonessential amino acids (Invitrogen), 1× sodium pyruvate (Invitrogen), 2 mmol/L glutamin (Invitrogen), and 10% fetal calf serum (FCS), unless otherwise indicated.
Flow-cytometric analysis.
The antigen-specific IFN-, perforin-, and granzyme Aproducing cells were identified by 4-color immunofluorescent staining and flow-cytometric analysis. All reagents were purchased from BD Biosciences and were used in accordance with the manufacturer's instructions. Briefly, 2 × 106 PBMCs/mL were incubated in the absence or presence of 10 g/mL HBHA and a cocktail of anti-CD28/CD49d monoclonal antibodies (MAbs; 1 g/mL, L293/L253) overnight at 37°C in 5% CO2. Brefeldin A (10 g/mL) was then added for an incubation of an additional 4 h, and then EDTA (at a final concentration of 1 mmol/L) was added for an incubation of an additional 15 min. After leukocyte fixation, the cells were permeabilized, washed, and incubated with labeled anti-CD3, anti-CD4, anti-CD8, anti-CD16, anti-CD56, anti-CD57, or anti-CD45RO MAbs. AntiIFN-, anti-perforin, and antigranzyme A MAbs were used to stain intracellular mediators. All MAbs were directly conjugated to fluorescein isothiocyanate, phycoerythrin, peridinin chlorophyll protein, or allophycocyanin. The samples were analyzed using a FACSCalibur flow cytometer and CellQuest software (version 3.3; BD Biosciences) by first gating lymphocytes (forward vs. side scatter) and then gating the various cell subsets. Values for spontaneous production were subtracted from the values obtained after HBHA stimulation.
Generation of monocyte-derived macrophages (MDMs).
Isolated PBMCs were plated at a concentration of 1 × 107 cells/well in 6-well culture plates during a 2-h period at 37°C in 5% CO2. After washing, adherent monocytes were resuspended in supplemented RPMI 1640 containing 800 U/mL granulocyte-macrophage colony-stimulating factor (GM-CSF; Peprotech) and were cultured for 6 days. After 4 days of culture, a fresh solution of 800 U/mL GM-CSF was added.
51Cr-release assays.
Isolated PBMCs were cultured with 2 g/mL HBHA at a concentration of 2 × 106 cells/mL in supplemented RPMI 1640 containing 5% autologous serum instead of FCS in 96-well round-bottom culture plates for 1 week. HBHA-specific CD8+ T lymphocytes were then positively selected using an anti-CD8 MAb (SK1; BDBioscences) and the CELLection Pan Mouse IgG Kit (Dynal Biotech). Briefly, expanded PBMCs were incubated with the anti-CD8 MAb (1 g/1 × 106 target cells) in PBS/0.5% bovine serum albumin for 25 min at 4°C. After washing, dynabeads were added (bead : target cell ratio, 5 : 1), and the solution was incubated for 30 min at 4°C. Rosetted cells were separated using a magnetic device and then were incubated with a DNAse-containing buffer for 15 min. The purity of the isolated CD8+ T lymphocytes was >99%. Autologous MDMs were cultured in the presence or absence of 20 g/mL HBHA for 2 h, were pulsed with 75 Ci of 51Cr (>9.25 GBq/mg; Amershan Pharmacia Biotech) for 1 h, were washed, and were seeded at 5000 cells/well in 96-well V-bottom plates. The purified HBHA-specific CD8+ T lymphocytes were then added at various effector : target (E : T) cell ratios (100 : 1, 30 : 1, 10 : 1, and 3 : 1) in a final volume of 200 L/well and were incubated for 16 h at 37°C. To analyze the lytic mechanisms, an anti-Fas (ZB4; Dako) or an isotype-matched control MAb (IgG1, DAK-GO1; Dako) was added at a final concentration of 2 g/mL. To inhibit granule exocytosis, purified CD8+ T lymphocytes were preincubated with strontium (20 mmol/L; Sigma) for 3 h. Supernatants were harvested, and the release of 51Cr was measured using a gamma counter (Topcount; Packard Instrument). Maximum lysis was obtained by adding saponin (final concentration, 5%) onto target cells, and spontaneous release was measured in wells containing the target cells alone. The percentage of specific release was calculated using the following formula: percentage of isotope release = (counts per minute supernatant-counts per minute minimum lysis) × 100/(counts per minute maximum lysis-counts per minute minimum lysis).
Microbicidal assay with HBHA-specific T lymphocytes.
Autologous MDMs were infected with exponentially growing M. bovis BCG (strain 1173P2; World Health Organization) at an MOI of 5 bacilli/cell for 24 h. Preliminary experiments had indicated that this ratio was optimal to infect a high proportion of the macrophages while limiting the BCG-induced macrophage death. After washing to remove extracellular mycobacteria, HBHA-stimulated CD8+ T lymphocytes were positively selected and were cocultured with infected target cells at E : T cell ratios of 25 : 1 and 12.5 : 1 for 5 days. Unstimulated PBMCs were used to assess the specificity of the mycobactericidal activity. The MDMs were then lysed with saponin (final concentration, 0.3%), and 100-fold dilutions of the cell lysates were plated in triplicate onto 7H10 Middlebrook agar. Colony-forming units were enumerated after 3 weeks of incubation at 37°C. Results are expressed as percentages of BCG growth inhibition, which was calculated as being equal to 100-[100 × (colony-forming units in cultures containing antigen-expanded cells)/(colony-forming units in cultures containing only antigen-presenting cells)].
Statistical analysis.
The nonparametric Wilcoxon matched pairs test was used to determine statistical significance.
RESULTS
IFN- production by HBHA-specific CD8+ T lymphocytes.
HBHA has been shown to induce IFN- synthesis by PBMCs from healthy subjects with latent TB [18, 19]. The characterization of the phenotype of the IFN-producing cells by flow cytometry indicated that, for the 9 subjects studied here, a median of 0.30% (range, 0.04%3.88%) and 0.54% (0.08%2.25%) of the CD8+ and CD4+ T lymphocytes, respectively, produced increased amounts of IFN- on overnight stimulation with HBHA (figure 1). A more detailed phenotype analysis for 4 of these subjects indicated that most of the HBHA-specific IFN-producing CD8+ T lymphocytes expressed CD45RO (median, 99%; range, 49%100%), whereas only a minority of them expressed CD57 (median, 5%; range, 0%9%), and this latter expression was faint. These results indicate that the HBHA-specific CD8+ T lymphocytes were activated in an antigen-specific manner and had expanded from memory T cells. In addition to the major contribution by the CD8+ and CD4+ T lymphocytes to the HBHA-induced IFN- synthesis, a median of 1.43% (range, 0.01%5.28%) of CD3-CD16+CD56+ NK cells also produced IFN- on overnight stimulation with HBHA (data not shown), but the overall contribution of these cells was negligible because the proportion of circulating NK cells was low.
Cytotoxic mechanisms of HBHA-specific CD8+ T lymphocytes.
Two major mechanisms for CD8+ T lymphocytemediated cytotoxicity have been described, both of which result in the apoptosis of the target cells. They involve either the release of cytolytic mediators, such as perforin and granzyme protease, or a surface receptorligand interaction between the effector and target cells that is mediated by Fas-FasL.
The intracellular expression of perforin was analyzed in the lymphocytes from 8 healthy subjects with latent TB after overnight incubation of their PBMCs with HBHA. The median proportions of CD4+ and CD8+ T lymphocytes that expressed HBHA-induced perforin were 0.39% (range, 0%2.65%) and 4.41% (range, 0.56%12.43%), respectively (figure 3A). In contrast to the HBHA-specific IFN- production, primarily produced by the CD4+ T lymphocytes, the CD8+ T lymphocytes appeared to be the major perforin-producing cells in response to HBHA. Similar results were obtained for granzyme A expression (data not shown), which suggests that the HBHA-mediated cytotoxicity involves cytolytic mediators contained within granules.
Colabeling of intracellular perforin and IFN- in HBHA-stimulated CD8+ T lymphocytes indicated that >90% of the IFN-producing CD8+ T lymphocytes did not express perforin and, conversely, that 99% of the perforin-expressing CD8+ T lymphocytes were negative for the cytokine (figure 3C). These observations indicate that distinct HBHA-specific CD8+ T lymphocyte subsets are involved in IFN- production and in the synthesis of cytolytic mediators. Furthermore, we found no correlation between the percentage of HBHA-induced IFN-producing CD8+ T lymphocytes and that of HBHA-induced perforin-producing CD8+ T lymphocytes (data not shown), and the percentage of HBHA-induced perforin-producing cells was not influenced by the addition of up to 10 g/mL recombinant IFN- (data not shown).
Although cytotoxic effector functions in human CD8+ T lymphocytes have been reported to closely correlate with CD56 surface expression [21], the majority of HBHA-induced perforin-producing CD8+ T lymphocytes did not express CD56 at their surface. The HBHA-induced IFN- synthesis within CD8+ T lymphocytes was also not associated with the CD56+ cell subset (data not shown).
DISCUSSION
Because the vast majority of individuals infected with M. tuberculosis will not develop active TB during their lives, they may be considered to be protected by acquired immunity. A better understanding of the immune responses induced by natural infection in individuals who stay healthy may thus be of great benefit when novel vaccines against TB are being designed. We have reported elsewhere that these individuals produce high levels of HBHA-specific IFN-, whereas individuals with active TB do not [18, 19]. This finding suggests that HBHA-specific T lymphocyte responses may be protective in humans. The protective potential of HBHA has been shown against intravenous and aerosol challenge with M. tuberculosis in murine models [19, 20]. Although the contribution that CD4+ T lymphocytes make in protection against M. tuberculosis has long been recognized in both mice and humans [59], more recent evidence also points to a protective role for CD8+ T lymphocytes, because, in addition to producing antigen-specific IFN-, these cells may have cytolytic and microbicidal activity [14, 15, 2225]. In murine models, CD8+ T lymphocytes, in particular, have been shown to be involved in the control of latent M. tuberculosis infection by limiting its reactivation [12, 13].
In the present study, we therefore focused on the characterization of the HBHA-specific CD8+ T lymphocyte response in healthy subjects with latent TB as a potential correlate of protection. As was evidenced by flow cytometry, HBHA-stimulated CD8+ T lymphocytes from healthy subjects with latent TB produced IFN- in addition to its synthesis by HBHA-stimulated CD4+ T lymphocytes and a minor contribution from NK cells. The HBHA-specific IFN- production by the CD8+ T lymphocytes and NK cells was dependent on the presence of CD4+ T lymphocytes, as we found in CD4+ T lymphocytedepletion experiments (data not shown). The majority of the HBHA-specific CD8+ T lymphocytes expressed CD45RO and no or very low levels of CD57, which confirms that they were not naive cells but were activated in an antigen-specific manner and were expanded from memory T cells. These observations indicate that, during natural infection, HBHA is processed and presented to the CD8+ T lymphocytes, and these findings are consistent with the rapid in vitro expansion of these cells.
In addition to producing IFN-, the CD8+ T lymphocytes from healthy subjects with latent TB were also able to produce perforin and had cytolytic and mycobactericidal activity. Although antigen-specific production of IFN- is clearly crucial for protective immunity against mycobacterial infections [5, 9], recent evidence suggest that it is not sufficient [26] and that other effector functions, including cytolytic and mycobactericidal ones, are also needed. In fact, they may actually provide better surrogate markers of protective immunity to M. tuberculosis than does IFN- production [27]. The HBHA-specific CD8+ T lymphocytes from healthy subjects with latent TB induced the lysis of HBHA-loaded MDMs via the release of cytolytic mediators, such as perforin and granzyme, and the inhibition of granule release by strontium abolished the cytolytic functions, whereas the addition of anti-Fas antibodies had no effect. Thus, the Fas-FasL cytolytic pathway appears not to be involved in HBHA-specific CD8+ T lymphocytemediated cytotoxicity. In contrast, a direct relationship between the antigen-specific cytolytic activity and the up-regulation of perforin expression was found. In vivo, expression of the cytolytic activity of CD8+ T lymphocytes likely results in the release of mycobacteria, which can then be taken up by recruited and freshly activated macrophages able to kill them. In addition, CD8+ T lymphocytemediated lysis of infected cells may enhance the presentation of the mycobacterial antigens by major histocompatibility complex class I through cross-priming [28]. Interestingly, HBHA-specific IFN- and HBHA-specific perforin were produced by 2 distinct CD8+ T lymphocyte subsets, as was shown by flow-cytometric analysis. In contrast to what has been reported in other models [21], none of the subsets was associated with CD56 surface overexpression. The HBHA-specific CD8+ T lymphocytes were also able to restrict mycobacterial viability at levels similar to those reported by Cho et al. for isolated CD8+ T lymphocytes specific for mycobacterial peptides [15]. Although we have not directly investigated the mechanism of the mycobacterial killing, it is likely to be mediated by the granular enzymes also, because granulysin, colocalized with perforin in granules of lymphocytes, has been shown to be able to directly kill intracellular M. tuberculosis [22].
In conclusion, in the present study, we characterized HBHA-specific CD8+ T lymphocytes from healthy subjects with latent TB who were likely to be protected by their immune response to the infection. These CD8+ T lymphocytes produced IFN- and expressed effector functions, such as cytotoxicity and mycobactericidal activity. Thus, they present all the features known to be important for protection against TB, in agreement with the hypothesis that HBHA may be a protective antigen for humans, as has been shown in mice [19, 20]. HBHA should therefore be considered as a candidate in the development of new, acellular anti-TB vaccines. Furthermore, the cytolytic activities were correlated with HBHA-stimulated perforin expression, rather than with IFN- production, which suggests that perforin induction within CD8+ T lymphocytes on stimulation with HBHA may be a useful surrogate marker of protective immunity that could be used in the monitoring of individuals after infection or vaccination.
Acknowledgments
We thank A. Drowart, for her help in collecting the blood samples from healthy subjects with latent tuberculosis, as well as all the individuals who agreed to give blood for use in this study.
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