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,
Departments of
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Molecular Genetics and Biochemistry and
Medicine, and
Division of Rheumatology and Clinical Immunology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The role that CD8+ T cells play in the protective
immune response against M. tuberculosis is
controversial. CD8+ T cells may function as a
source of type 1 cytokines such as IFN-
and TNF-
, similar to
CD4+ T cells. It has also been suggested that the
protective effect of CD8+ T cells is dependent
upon the ability to lyse infected macrophages (M
) within the
tissues. In Chlamydia trachomatis infection, another
intracellular pathogen, CD8+ T cells induced
protection by IFN- production (7), while
CD8+ T cells mediated protection in a
Listeria monocytogenes model in an IFN--independent
manner (8).
Cytokine production by Ag-specific CD8+ T
cells upon in vitro restimulation has been demonstrated in human and
murine studies (9, 10, 11, 12, 13, 14). Partial protection of athymic mice
from M. tuberculosis infection by
CD8+ T cells required IFN- production
(15), suggesting that cytokine secretion by these cells
was induced in vivo. However, it is not clear whether
CD8+ T cells contribute significantly to the
cytokine pool during the normal course of infection. We showed
previously that CD8+ T cells freshly harvested
from infected lung were capable of IFN- and TNF- production,
although it appeared that only a small subset actively produced
cytokine at the site of infection (14). Human
CD8+ T cells stimulated with live M.
tuberculosis bacilli or mycobacterial Ags produced fewer cytokine
molecules per cell than did CD4+ T cells
(12).
In tuberculosis studies, mycobacteria-specific cytolytic
CD8+ T cells have been generated in mice and
isolated from humans (10, 16, 17, 18, 19, 20, 21, 22, 23). In addition to lysis of
infected M, human CD8+ T cells can directly
kill mycobacteria by granulysin, a granule-associated protein of
CD8+ T cells (24). However, the
existence of CD8+ CTL in mice infected with
virulent M. tuberculosis that are capable of lysing
M infected with live M. tuberculosis has not been
demonstrated. Moreover, no studies have addressed the presence of
CD8+ CTL in M.
tuberculosis-infected lungs. The participation of cytotoxic
CD8+ T cells in the protective immune response
against M. tuberculosis has been questioned by the
findings that deficiencies in perforin, granzyme B, or FAS-receptor
molecules did not affect short-term survival of M.
tuberculosis-infected mice (25, 26). Although this
may be due to compensatory mechanisms since doubly deficient mice were
not tested in those studies, these results demand a more thorough
investigation of the function of CD8+ T cells
induced during M. tuberculosis infection.
In this study, we demonstrate that mycobacteria-specific
CD8+ CTL are present in the lungs and
lung-draining lymph nodes of M. tuberculosis-infected
mice. These CD8+ T cells specifically lysed
M. tuberculosis-infected M mice in a
perforin-dependent manner. Recognition of mycobacterial Ags by the CTL
was ß2m dependent, MHC class I dependent, and
CD1 independent. We also detected expression of perforin by lymphocytes
in the organs of infected mice, further supporting a role for the
cytotoxic function of CD8+ T cells in the
protective immune response against M. tuberculosis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Eight- to 10-wk-old female C57BL/6 (The Jackson Laboratory, Bar Harbor, ME), BALB/c (The Jackson Laboratory), ß2m-/- (The Jackson Laboratory), MHC class II-/-, and perforin-deficient (P-/-) mice were used. MHC class II-/- and P-/- mice were bred in the pathogen-free facility at the University of Pittsburgh School of Medicine (Pittsburgh, PA). All mice were maintained in specific pathogen-free biosafety level 3 facilities. All experimental and animal handling procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Pittsburgh School of Medicine.
Bacteria and infections
M. tuberculosis (Erdman strain; Trudeau
Institute, Saranac Lake, NY) was passed through mice, grown in culture
once, and frozen in aliquots. Before infection, an aliquot was thawed,
diluted in PBS containing 0.05% Tween-80, and sonicated for 10 s
in a cup horn sonicator. Mice were infected i.v. via tail vein with
2 x 105 live bacilli in 100 µl or by
aerosol with 
100 live bacilli, as determined by viable counts on
7H10 agar plates (Difco Laboratories, Detroit, MI). For aerosol
infections, 107 CFU/ml were placed in nebulizer
and mice were exposed for 20 min, followed by 5 min of air only,
using an InTox aerosol unit (Albuquerque, NM).
Culture and infection of DC and M
Dendritic cells (DCs) and M were grown from murine bone
marrow precursors and cultured for 5 days using methods previously
described (14). For M infection, adherent cells were
washed twice with ice-cold PBS (Life Technologies, Grand Island, NY)
and infection media was added containing DMEM, 10% certified FBS, 1 mM
sodium pyruvate, and 2 mM L-glutamine (Life Technologies).
For DC infection, nonadherent cells were harvested, adjusted to
0.5 x 106 cells/ml in DC media containing
recombinant murine GM-CSF, and dispersed into
25-cm2 culture flasks (Costar, Cambridge, MA) for
infection.
For infection of APC, frozen aliquots were used to start cultures at a
concentration of 2.5 x 106/ml in liquid
medium (7H9 Middlebrook; Difco, Detroit, MI); bacteria were grown in
5% CO2 at 37°C. Four- to six-day-old cultures
were used to infect cells. Bacteria were washed, resuspended in DMEM
medium (Life Technologies), and sonicated for 15 s before
infection of cell cultures. Cells were infected for 16–18 h at
multiplicity of infection of 3–5. Extracellular bacteria were
separated from cells by low speed centrifugation (DCs) or by washing
adherent cells twice with PBS. Cells were incubated for 10 min on ice,
and harvested by forceful pipetting. In some experiments, infected and
uninfected M were cultured in fresh medium for an additional 24
h (see text) before use in CTL assays. For DCs and M, the percentage
of infection was estimated in each experiment by staining aliquots of
cells by the Kinyoun method for acid-fast bacteria (Difco). Routinely,
40–55% of DCs and 60–85% of M were infected.
FACS analysis of cell surface markers
Lung and lymph node cells were obtained from mice infected for various periods of time by crushing the organs in cell strainers (Becton Dickinson Labware, Lincoln Park, NJ) to obtain single cell suspensions. RBC were lysed with NH4Cl/Tris solution, and cells were washed twice. Cells were stained for cell surface markers using Abs against CD8 (anti-CD8 CyChrome Ab, clone 53-6.7), CD4 (anti-CD4 CyChrome Ab, clone H129.19), CD44 (anti-CD44 FITC Ab, clone IM7), CD45RB (anti-CD45RB FITC Ab, clone 16A), and CD69 (anti-CD69 FITC Ab, clone H1.2F3) in PBS containing 20% mouse serum, 0.1% BSA, and 0.1% sodium azide for 30 min at 4°C. All Abs were used at 0.2 µg/106 cells and obtained from PharMingen (San Diego, CA). Cells were fixed with 4% paraformaldehyde for 4–15 h and analyzed by FACS using CellQuest software (Becton Dickinson Immunocytometry Systems, San Jose, CA). Cells were gated on the lymphocyte population by size.
Intracellular staining
Single cell suspensions of lungs at various times postinfection were prepared, as described above. Staining for intracellular cytokines was performed as described previously (14). Briefly, cells were either stimulated with anti-CD3 (clone 145-2C11, 0.1 µg/ml) and anti-CD28 (clone 37.51, 1 µg/ml) Abs (PharMingen, San Diego, CA) or left unstimulated for 5–6 h in the presence of 3 µM monensin (Sigma, St. Louis, MO). At the end of stimulation period, cells were stained for CD4 and CD8, fixed, permeabilized, and stained for intracellular cytokines. Intracellular perforin staining was performed as previously described (27). Briefly, cells were fixed in 2% paraformaldehyde for 20 min on ice, washed, and permeabilized with 0.1% saponin. Cells were stained with anti-perforin Ab diluted 1/200 (clone KM 585; Kamiya Biomedical, Seattle, WA) in staining buffer containing 0.1% BSA, 0.1% sodium azide, 0.3% saponin, and 20% mouse serum at 4°C for 30 min, followed by staining with anti-rat IgG (anti-rat IgG2a FITC Ab, clone RG7/1.30; PharMingen). Cells were washed with 0.1% saponin, stained with anti-CD8 Ab or anti-CD4 Ab, and fixed in 4% paraformaldehyde for 1 h before analysis.
Culture of lung and lymph node cells
Lung and lymph node cells from mice infected for 2–5 wk were obtained as described above and plated in 96-well U-bottom plates (Corning, Corning, NY) in DMEM supplemented with 10% certified FBS, 1 mM sodium pyruvate, 2 mM L-glutamine, 25 mM HEPES (Life Technologies), 50 µM 2-ME (Sigma, St. Louis, MO), 30 µg/ml gentamicin (Life Technologies, Gaithersburg, MD), 15–20 U/ml recombinant murine IL-2 (Boehringer Mannheim, Indianapolis, IN), and 1 mM aminoguanidine (Sigma) at 2 x 105 cells/well. MHC class II-/- DCs infected for 18–24 h, as described above, were added to the cell cultures at 6.5–7 x 103 viable cells/well. After 2–3 days of culture, 100 µl of media was removed from each well and replaced with fresh media containing IL-2. Cells were cultured for additional 3–4 days before FACS analysis and CTL assays.
Perforin immunohistochemistry
Organs were fixed in 2% paraformaldehyde before paraffin embedment and sectioning. For immunohistochemistry, polyclonal anti-mouse perforin Ab was generated in rabbits, as described previously (28, 29). Sections were deparaffinized and stained using methods previously described (29). Briefly, sections were stained using rabbit anti-perforin Ab diluted 1/300, followed by biotinylated anti-rabbit IgG (Vector, Burlingame, CA). Perforin Ab was visualized using ABC kit (Vector) and 3-amino-9-ethyl-carbazole substrate (Sigma). Immunoreactivity was indicated by the appearance of the red-brown color.
Cytotoxicity assays
Lymphocytes harvested from 5–7-day stimulation cultures were
tested in a 4-h 51Cr release assay. Where
indicated, 15 mM strontium chloride (Aldrich Chemical, Milwaukee, WI)
was added to the T cell cultures for the final 12 h of culture. No
cytotoxicity associated with strontium treatment was observed. To
prepare targets, M uninfected or infected for indicated periods of
time were harvested as described above, and 1–2 x
106 cells were labeled with 100 µl of
Na51CrO4 (Amersham) in
Teflon jars (Savillex, Minnetonka, MN) for 1 h at 37°C. Cells
were washed three times with DMEM, added to wells of 96-well U-bottom
plates (Corning, Corning, NY) at 4 x 103
cells/well, and allowed to adhere for 20 min before addition of T
cells. Cultured cells were added at various E:T ratio in a total volume
of 0.1 ml in DMEM supplemented with10% certified FBS, 1 mM sodium
pyruvate, 2 mM L-glutamine, 25 mM HEPES, and 50 µM 2-ME,
and assay was conducted for 4 h. Where indicated, targets were
incubated for 2 h with increasing doses of perforin purified, as
described previously (28). Where indicated, anti-MHC
class I supernatant
(anti-KbDb, clone
28-8-6, 1/2 dilution) or anti-CD1d Ab (clone 19G11, 20 µg/ml) was
included in the CTL culture. After 2–4 h, 85 µl of supernatant was
removed from each well without disturbing the cells and counted in
gamma counter. Spontaneous release was determined by culturing target
cells in medium alone, and total release was determined by adding 0.1%
Triton-X to target cells. Percent of specific lysis was calculated by
the formula: 100 x [(experimental cpm - spontaneous
cpm)/(total cpm - spontaneous cpm)].
Statistics
The paired Student’s test was used to compare groups.
Statistical analysis was performed using StatView (Abacus Concepts,
Berkeley, CA). A p value of 
0.05 was considered
significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Lung-draining lymph nodes of mice infected i.v. with M.
tuberculosis were examined for the presence of activated effector
CD8+ T cells. The changes in the T cell
composition of lung-draining lymph nodes during the infection are
summarized in Fig. 1
, A and
B. As early as 1 wk postinfection, the number of
CD8+ T cells in the lymph nodes of infected mice
doubled (Fig. 1A), and the number of
CD69+ CD8+ T cells
increased 3–4-fold (Fig. 1B), indicating that
CD8+ T cells were recruited to the lymph nodes
and were undergoing priming. CD69 expression is frequently used as an
indication that T cells have encountered APC. This molecule is rapidly
up-regulated upon triggering of TCR, reaching a peak at 24–48 h
(30, 31, 32). By 4 wk postinfection, the total number of
CD8+ T cells in the lymph nodes was increased
5-fold as compared with uninfected controls, and total numbers of
CD69+ CD8+ T cells were
increased 10-fold (Fig. 1, A and B). However,
a significant proportion of CD8+ T cells found in
the lymph nodes retained a naive unactivated phenotype (data not
shown). The activation profiles of CD8+ T cells
in the lungs were similar to that of CD4+ T cells
(data not shown), indicating that both T cell subsets were efficiently
primed in the lymph nodes upon mycobacterial infection.
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C), indicating establishment of the effector/memory
phenotype. CD4+ T cells in the lungs undergo
similar changes with >90% T cells acquiring the
CD44highCD45RBlow phenotype
(data not shown). Thus, the number of lung CD8+ T
cells displaying a true naive phenotype in the lungs of infected mice
was negligible as early as 2 wk postinfection. In the lungs, a
substantial increase (8–10-fold) in the percentage of
CD69+ CD8+ T cells, as
compared with uninfected control, was observed by 2 wk postinfection;
by 4 wk postinfection, 50% of CD8+ T cells
were CD69+ (Fig. 1D). Starting from 2
wk postinfection, the percentage of CD8+ T cells
in the lungs expressing CD69 was equal to that of
CD4+ T cells (data not shown), supporting the
hypothesis that both T cell subsets are functioning during
M. tuberculosis infection. These data suggest that
effector CD8+ T cells are not only recruited to
the lung during infection, but also actively recognize mycobacterial
Ags and engage in the ongoing immune response at this site. Overall, the data presented in this study indicate that lungs and lung-draining lymph nodes contain large numbers of activated effector CD8+ T cells and are a likely source for mycobacteria-specific CTLs. To enrich for Ag-specific CD8+ T cells, lung and lymph node cells were cultured with murine bone marrow-derived M. tuberculosis-infected MHC class II-/- DCs in the presence of IL-2. Because these DCs are unable to present Ags to CD4+ T cells, by day 5 the resultant lung and lymph node T cell cultures were comprised of 70–85% CD8+ T cells (data not shown). Therefore, 3–5-fold enrichment in CD8+ T cells was observed in the in vitro cultures as compared with the infected lungs in which CD8+ T cells did not exceed 20–25% of total lymphocyte population (14). We were not able to grow CD8+ T cells from the lungs of uninfected mice, which indicates that culture conditions were not optimal for priming of naive CD8+ T cells and that only CD8+ T cells previously primed for mycobacterial Ags were expanded during the culture period (data not shown).
Cytotoxic activity of CD8+ T cells cultured from lungs and lung-draining lymph nodes of infected mice
Cytotoxic activity of CD8+ lung and lymph
node T cells harvested from mice 4 wk postinfection was tested after 5
days of coculture with M. tuberculosis-infected MHC
class II-/- DCs. Bone
marrow-derived M uninfected or infected with live M.
tuberculosis for 18 h before CTL assay were used as targets
in a 4-h 51Cr release assay. Both lung and lymph
node CD8+ T cells lysed infected M, albeit
with low efficiency (Fig. 2A). Similar results were obtained
with lung and lymph node cells harvested from mice 2, 3, 5, and 6 wk
postinfection (data not shown). We routinely observed low nonspecific
lysis of uninfected M by lung CTLs and somewhat higher nonspecific
lysis by lymph node CTLs. This might be due to the priming of naive
lymph node cells to foreign Ags, such as FBS Ags, during the short
stimulation with DCs. Since the majority of lung T cells had an
activated/effector phenotype and presumably defined Ag specificity
before ex vivo stimulation, priming of naive cells during short-term
culture was negligible.
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. We sought to improve the target cells by allowing more time for
Ag processing and presentation. Cytotoxic activity of lung
CD8+ T cells was tested using as targets M
infected for 18–20 or 42–44 h. In several independent experiments,
the longer interaction between M and M. tuberculosis
bacilli led to a substantial increase in the specific lysis by lung
CD8+ T cells (Fig 2B and data not
shown). There was no significant increase in MHC class I expression on
M at 42 h as compared with 18 h postinfection (data not
shown). The improvement in lysis efficiency by
CD8+ CTLs was most likely due to an increase in
the pool of available Ags or more efficient presentation of those Ags
with the longer infection period.
The possibility existed that M. tuberculosis-infected
M differ from uninfected M in their susceptibility to lysis by
CTLs. Because this infection is cytotoxic to some degree, infected M
might be lysed more readily, which would resemble low level specific
lysis. To exclude this possibility, dose-dependent lysis of uninfected
and infected M by purified perforin was assessed in a 2-h
51Cr release assay. No significant difference was
observed between purified perforin-mediated lysis of uninfected and
infected M (Fig. 2C),
indicating that infection does not lead to a more easily lysed
M.
CD8+ CTLs are present in the lungs and lymph nodes of mice infected via aerosol route
In the experiments described above, mice were infected with
M. tuberculosis i.v. Because infection with
M. tuberculosis naturally occurs via the respiratory
route, we tested whether the CTLs were also present in the lungs and
lung-draining lymph nodes of mice infected via aerosol. When mice were
infected via aerosol, a delay in the development of
CD8+ T cell-mediated immune response in the lungs
was observed, as compared with the immune response following i.v.
infection. However, by 6 wk postinfection, the activation parameters of
CD8+ T cells in the lungs of aerosol-infected
mice were similar to those of CD8+ T cells from
the lungs of i.v. infected mice (data not shown). Lung and lymph node
cells harvested from aerosol-infected mice were stimulated for 5 days,
as described above, and tested for cytotoxic activity against M
infected with M. tuberculosis for 42 h (Fig. 3). The lysis of infected M targets by
these CD8+ CTLs was similar to the lysis by CTLs
from i.v. infected mice, indicating that priming of
CD8+ CTLs specific for mycobacterial Ags and
their subsequent migration to and function in the lungs occur
regardless of infection route.
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In the murine model, the protective CD8+ T
cell-mediated immune response is believed to result from the
recognition of Ags presented in the context of MHC class I molecules
(5, 6). To examine the nature of Ag presentation in our
system, ß2m-deficient
(ß2m-/-)
M were used as targets. Specific lysis of M.
tuberculosis-infected
ß2m-/-
M by lung and lymph node CTLs was reduced 75–100%, compared with
wild-type M, in several independent experiments (Fig. 4, A and B). The
small degree of lysis observed with infected
ß2m-/-
targets was most likely due to residual levels of MHC class I molecules
detected in
ß2m-/-
mice. Thus, lysis was dependent on ß2m.
However, ß2m is a component of both MHC class I
and CD1 Ag-presenting complexes. In our experiments, M routinely
expressed high levels of MHC class I molecules and only marginal levels
of CD1d molecules (data not shown). Genes for type 1 CD1 molecules
(CD1a-c) are not present in the mouse system. In three independent
experiments, anti-MHC class I Ab cocktail inhibited specific lysis
by lung CD8+ CTLs by 63%, 53%, and 59% (Fig. 4C). MHC-mismatched M were also used as target cells.
Bone marrow M from BALB/c mice (H-2d) were used as targets for
CD8+ T cells derived from C57BL/6 (H-2b) mice.
Lysis of H-2d targets was reduced 66% compared with H-2b targets (Fig. 4D). Conversely, addition of anti-CD1d Ab had no effect
on the lysis by lung CTLs (Fig. 4E). This does not exclude
CD1d as a possible restricting element for CD8 T cell-mediated
recognition of mycobacterial Ags, because the very weak CD1d expression
observed on infected M might not have been enough to trigger TCR
stimulation. However, the majority of cells recovered from the lungs in
these studies appear to be MHC class I restricted.
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F), indicating that
lysis of M. tuberculosis-infected M by lung CTL
occurs via a granule-dependent pathway. Perforin expression in the organs of infected mice
Because lysis of infected M by cultured
CD8+ CTLs was perforin dependent, we sought to
confirm that perforin was expressed in the organs during infection.
Immunohistochemistry was performed to assess perforin expression in the
tissues of mice infected for 3 wk with M. tuberculosis.
Lung and liver sections of uninfected mice contained very few
lymphocytes, and perforin expression was not detected in these organs
(data not shown). Spleen sections from uninfected mice did not stain
positively for perforin either, even though this organ contains large
numbers of lymphocytes (data not shown). In contrast, perforin
expression was readily detected in the organs of infected mice. In the
lungs, perforin-expressing cells were distributed throughout the
lymphocytic infiltrates (Fig. 5,
A and B). In the liver sections,
perforin-expressing lymphocytes were clearly observed within the
granulomas, in the lymphocytic rim surrounding the M core (Fig. 5, C and D). Perforin staining was also evident in
the spleen sections of infected mice, albeit at lower frequency (data
not shown). That fewer lymphocytes expressed perforin in the spleen
correlates well with our observations that at least 45% of splenic
CD8+ T cells maintain an unactivated phenotype
during acute infection (unpublished data). The majority of perforin
expression was in small lymphocytic cells and was concentrated
unipolarly in the cells, suggesting that they were activated
lymphocytes (Fig. 5, B and D). A number of cells
expressed only few granules in the cytoplasm, but cells were also
detected that expressed large perforin aggregates.
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A). In contrast, no perforin
staining was observed in the CD4+ T cell subset
(Fig. 6B).
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Previously, it was demonstrated that single deficiencies in
perforin, granzyme B, or FAS receptor molecules did not affect
short-term survival of M. tuberculosis-infected mice
(25, 26). The interpretation of these results was that the
cytotoxic activity of CD8+ T cells does not
contribute to protective immunity during tuberculosis. Recently,
perforin deficiency was shown to have a profound effect on the
development of the CD8+ T cell-mediated immune
response during lymphocytic choriomeningitis virus infection;
CD8+ T cells were in a heightened state of
activation in P-/- mice
as compared with the wild-type mice (35). We examined
whether perforin deficiency affected development of
CD8+ T cells during the course of M.
tuberculosis infection. By 4 wk postinfection,
P-/- mice had increased
numbers of lymphocytes in the lungs, attributable to a doubling in the
numbers of CD8+ T cells (Fig. 7A).
Percentages of CD8+ CD69+
and CD8+ CD25+ T cells were
also increased in the lungs of infected
P-/- mice compared with
wild-type mice (Fig. 7B),
suggesting that CD8+ T cells at the site of
infection were in a state of hyperactivation. Additionally, mean
fluorescent intensity of CD25 expression on the surface of
P-/-
CD8+ T cells was increased 2-fold (data not
shown), suggesting enhancement in the activation state of individual
cells. No changes were observed in the numbers or activation state of
CD4+ T cells in
P-/- mice as compared
with the wild-type mice (data not shown).
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and TNF-. The total number of IFN--secreting
CD8+ T cells in the lungs of
P-/- mice was increased
4–4.5-fold as compared with the wild-type mice (Fig. 7C),
in accordance with data published previously (26). A
significant increase was also observed in the numbers of
TNF--secreting CD8+ T cells (data not shown).
To assess cytokine production by T cells at the site of infection, we
cultured fresh lung cells in the presence of monensin for 5 h
without ex vivo Ab stimulation. As described previously
(14), in the absence of stimulation, 5–8% of wild-type
CD8+ T cells secreted IFN-. In contrast,
P-/-
CD8+ T cells readily secreted IFN- without
stimulation, and the total number of IFN-+
CD8+ T cells in these cultures was increased
4-fold (Fig. 7C). Cytokine production by
CD4+ T cells from
P-/- mice with or without
Ab stimulation was unchanged as compared with the wild-type control
(data not shown). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in tissues. In this
study, we present evidence that M.
tuberculosis-specific CD8+ CTL are present
in the lungs and lung-draining lymph nodes of mice infected by either
the aerosol or i.v. route with virulent M.
tuberculosis. These cells express perforin in vivo and lyse
M. tuberculosis-infected M in an MHC class
I-dependent manner.
Studies of the cytotoxic function of mycobacteria-reactive
CD8+ T cells have been hampered by difficulties
in demonstrating the presence of CD8+ CTLs in the
organs of M. tuberculosis-infected mice. Various groups
have reported the generation of CD8+ T cells by
immunization strategies involving killed or attenuated mycobacteria, or
mycobacterial Ags (17, 19, 20, 36, 37, 38, 39). These cells were
demonstrated to recognize mycobacterial Ags in a MHC class I or
MHC-independent manner. Following infection with live M.
tuberculosis, CD8+ T cells specific for
epitopes of the 38-kDa glycolipoprotein were isolated (20, 36). However, with the exception of hsp65-specific CTLs
generated by immunization with a cell line expressing this Ag
(19), no other reports document the ability of
CD8+ CTLs to lyse M infected with live
M. tuberculosis, which is an important component of
protection by CTLs. Ags recognized by CD8+ T
cells could be generated during infection in a variety of ways,
including by infected M or DCs, or extracellular bacterial proteins
or apoptotic fragments of infected M taken up and processed by DCs.
It is important to demonstrate that the CD8+ T
cells specific for mycobacterial Ags can actually recognize infected
M in a functional manner, because these are the cells that must be
attacked by the CD8+ T cells to provide
protection. This study is the first to report the isolation of
CD8+ CTL from the lungs of mice infected with
virulent M. tuberculosis that specifically lyse M
infected with live M. tuberculosis.
Several possible explanations exist as to why M.
tuberculosis-specific CD8+ CTLs have been so
difficult to detect in infected mice. One hypothesis was that
CD8+ T cells were not primed during infection. A
number of recent studies providing evidence that activated
CD8+ T cells that produce cytokine in response to
mycobacterial Ags appear in the lungs following infection argue against
this hypothesis (13, 14). Most studies on CD8 CTL
responses in tuberculosis mouse models focused on the immune responses
in the spleen. We focused on CD8+ T cells present
in the lungs and lung-draining lymph nodes of infected mice. In the
lungs of infected mice, 
90% of CD8+ T cells
acquired an effector/memory phenotype by 14 days postinfection. By 4 wk
postinfection, 50% of CD8+ T cells in the
lungs were expressing CD69, suggesting active participation of this
cell subset in the ongoing immune response. Similar changes were also
observed in the lung-draining lymph nodes, with a
significant enrichment in the CD8+ T cells and
CD69+ CD8+ T cells,
indicating that CD8+ T cells were undergoing
priming to mycobacterial Ags. The phenotype of the
CD8+ T cells in infected lungs and draining lymph
nodes suggested that these organs were highly enriched for
effector CD8+ T cells and were most likely to
contain M. tuberculosis-specific CTL.
The choice of infected DCs for brief in vitro restimulation of the
CD8+ T cells also contributed to the success in
detecting mycobacteria-specific CD8 CTL. The number of mycobacterial
Ags recognized by CD8+ T cells is unknown. We
reasoned that restimulation with DCs infected with live M.
tuberculosis rather than cells transfected with the gene for one
potential Ag would allow expansion of a more complete pool of specific
CD8+ T cells. In other studies, bone marrow or
peritoneal derived M infected with live or killed M.
tuberculosis or transfected with mycobacterial genes were used as
stimulators in CD8+ T cells cultures. However,
infected M might not be well suited for this role because they
can be suppressive (40, 41). In our hands, bone
marrow-derived M. tuberculosis-infected M were poor
at stimulating mycobacteria-specific proliferation of
splenocytes4 or
IFN- production from lung CD4 or CD8+ T cells
(14) from infected mice. In human studies, DCs were
successfully used to expand or prime mycobacteria-specific
CD8+ T cells (11, 22). Infection of
murine DCs with bacteria enhanced biosynthesis, stabilization, and
surface expression of MHC class I molecules, suggesting effective
presentation of intracellular bacterial Ags to
CD8+ T cells (42). We reported that
M. tuberculosis infection of murine and human DCs
resulted in maturation of the cells and enhanced stimulation of
mycobacteria-specific T cell responses (14, 43).4 Using DCs deficient in MHC class II
molecules, we obtained lung and lymph node T cell cultures highly
enriched for CD8+ T cells (70%) after 5–7
days of stimulation. The brevity of the culture period was intended to
minimize priming of naive CD8+ T cells that might
be present in the lungs and lymph nodes and to reduce the potential
effects of IL-2.
In human studies, low level cytotoxic activity of
CD8+ T cells from bronchoalveolar lavage of
purified protein derivative-positive individuals against M.
tuberculosis-infected monocytes was reported (21). In
our murine studies, M infected with M. tuberculosis
for 42–44 h were much better targets for CTL than those infected
12–18 h, suggesting that processing and presentation of mycobacterial
Ags by M are not efficient. At present, it is not clear how
mycobacterial Ags are processed and presented by MHC class I molecules
during infection. Presentation of soluble OVA in the context of MHC
class I molecules was facilitated by virulent M.
tuberculosis (44), and it has been suggested that
M are capable of transferring Ags from phagosomal compartments into
the cytosol (45). Presentation of this type might require
a longer time for generation of Ags presented to
CD8+ T cells than is required during infection
with a cytosolic pathogen. Previously, we demonstrated that M
infected for 24 or 48 h did not stimulate the secretion of
significant levels of IFN- by CD8+ and
CD4+ T cells (14). The fact that
specific CD8-mediated lysis of M, albeit low, was observed after a
short (18-h) infection period suggests that, in vivo, cytotoxicity
might be the earliest elicited T cell response.
Although lysis of M. tuberculosis-infected M by
cultured CD8+ T cells demonstrated that
mycobacteria-specific CD8+ CTLs are present in
the lungs and lymph nodes of infected mice, it does not prove that
cytotoxic activity is exerted by CD8+ T cells in
vivo. However, perforin molecules were readily detected in cells in
lungs, livers, and spleens of infected mice and not in the organs of
uninfected mice; perforin+ cells were also
CD8+. It is not surprising that perforin
expression was detected in relatively few lymphocytes in lung sections.
CD8+ T cells in the lungs comprise only 20–25%
of the total lymphocyte population (14), and it is
possible that not all activated CD8+ T cells
express perforin. Also, some CD8+ T cells might
be depleted of perforin after recent encounter with infected M. Once
CD8+ CTL recognize the antigenic determinant
presented by infected M, the release of perforin- and
granzyme-containing granules is triggered, resulting in target lysis.
Having demonstrated that CD8+ T cells from lungs
of infected mice are capable of recognizing Ags presented by
M. tuberculosis-infected M and lysing the infected
M in a perforin-dependent manner, the finding that these cells
express perforin in vivo strongly implicates participation of
CD8+ CTLs in the immune response.
In human studies, alternative MHC class I presentation to
CD8+ T cells (12) and presentation
through CD1 or other nonpolymorphic MHC class Ib (11, 22, 24, 46) have been reported. In the murine model, the protective
CD8+ T cell-mediated immune response is
ß2m and TAP dependent (5, 6). The
data presented in this study indicate that CD8+
CTLs from lungs recognized mycobacterial Ags presented by MHC class I
molecules, and this recognition resulted in lysis of infected M.
Addition of anti-CD1d Ab did not affect lysis by CTLs. However, we
cannot rule out the possibility that CD1-restricted cells were present
in our cultures because expression of CD1d molecule by infected M
was difficult to detect. In any case, type I CD1 molecules are not
present in mice, so the contribution of CD1a-c presentation cannot be
tested in this system.
The involvement of cytotoxic CD8+ T cells in the
protective immune response to M. tuberculosis has been
challenged by findings that mice with single deficiencies in perforin-,
granzyme B-, and FAS receptor-deficient mice were shown to be resistant
to the infection (25, 26). However, it is becoming clear
that other lytic pathways can successfully compensate a deficiency in
any single cytotoxic mechanism. Clearance of influenza virus mediated
either by perforin- or FAS-dependent mechanisms was reported
(47). Additionally, partial protection of mice from
Listeria monocytogenes by single Ag-specific
P-/-
CD8+ T cells was demonstrated to require TNF-
production by these cells (48). Thus, the fact that
P-/- mice do not succumb to infection does not
necessarily rule out the involvement of cytotoxic perforin-mediated
responses during the normal course of infection. Recently, perforin has
been suggested to down-modulate the CD8+ T cell
responses during persistent viral infection (35). We
observed a substantial increase in CD8+ T cells
in the lungs of P-/- mice
following M. tuberculosis infection, and cytokine
secretion by CD8+ T cells in the lungs was also
enhanced (4-fold, compared with wild-type mice). These results
suggested that perforin plays a role in regulating CD8 T cell
proliferation and function during M. tuberculosis
infection as well. Failure of CD8+ T cells to
quickly lyse infected targets might result in the prolonged interaction
between these cells and infected M, inducing cytokine production by
CD8+ T cells. In addition, these data suggest
that lack of perforin-mediated killing by CD8+ T
cells may be compensated for by enhanced cytokine production by this
cell subset. The enhanced cytokine responses of
CD8+ T cells might be protective during the acute
stage of disease, but they also might induce pathology at the chronic
stage of disease. Recent data indicate that P-/- mice do
not survive as long as wild-type mice (49). Our data also
suggest that perforin-mediated cytotoxicity is exerted during the
normal course of M. tuberculosis infection and is at
least partially responsible for reduced cytokine responses of
CD8+ T cells. This down-modulating effect of
perforin is specific for the CD8+ T cell
subset.
In summary, we have demonstrated for the first time that the lungs of
mice infected with M. tuberculosis via either the i.v.
or aerosol route contain CD8+ CTLs with
specificity for mycobacterial Ags. These CD8+ T
cells lysed M infected with live M. tuberculosis in
an MHC class I- and perforin-dependent fashion and expressed
perforin in vivo. Our data provide evidence that cytotoxic
CD8+ T cells responses are operative during the
course of M. tuberculosis infection. In light of these
and other recent data, the importance of CD8+ T
cells in the protective response against this pathogen cannot be
neglected with respect to vaccine design. For a vaccine to be effective
against tuberculosis, it appears that specific
CD8+ T cells, in addition to other cell subsets,
must be induced. Whether IFN- production and cytotoxic activity of
CD8+ T cells are both important functions of
CD8+ T cells during infection remains to be
determined.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. JoAnne L. Flynn, Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA 15206.
3 Abbreviations used in this paper: ß2m, ß2-microglobulin; DC, dendritic cell; M, macrophage; P-/-, perforin deficient.
Received for publication January 11, 2000. Accepted for publication April 13, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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but requires TNF-. J. Immunol. 160:898.This article has been cited by other articles:
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J. Turner, C. D. D'Souza, J. E. Pearl, P. Marietta, M. Noel, A. A. Frank, R. Appelberg, I. M. Orme, and A. M. Cooper CD8- and CD95/95L-Dependent Mechanisms of Resistance in Mice with Chronic Pulmonary Tuberculosis Am. J. Respir. Cell Mol. Biol., February 1, 2001; 24(2): 203 - 209. [Abstract] [Full Text]
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J. E. Wigginton and D. Kirschner A Model to Predict Cell-Mediated Immune Regulatory Mechanisms During Human Infection with Mycobacterium tuberculosis J. Immunol., February 1, 2001; 166(3): 1951 - 1967. [Abstract] [Full Text] [PDF]
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 S. Cho, V. Mehra, S. Thoma-Uszynski, S. Stenger, N. Serbina, R. J. Mazzaccaro, J. L. Flynn, P. F. Barnes, S. Southwood, E. Celis, et al. Antimicrobial activity of MHC class I-restricted CD8+ T cells in human tuberculosis PNAS, October 12, 2000; (2000) 210391497. [Abstract] [Full Text]
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C. A. Scanga, V.P. Mohan, K. Yu, H. Joseph, K. Tanaka, J. Chan, and J. L. Flynn 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., August 7, 2000; 192(3): 347 - 358. [Abstract] [Full Text] [PDF]
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S. Cho, V. Mehra, S. Thoma-Uszynski, S. Stenger, N. Serbina, R. J. Mazzaccaro, J. L. Flynn, P. F. Barnes, S. Southwood, E. Celis, et al. Antimicrobial activity of MHC class I-restricted CD8+ T cells in human tuberculosis PNAS, October 24, 2000; 97(22): 12210 - 12215. [Abstract] [Full Text] [PDF]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) and M
) or infected with
M. tuberculosis for 44 h (
) were incubated
with increasing concentrations of purified perforin, and lysis was
assessed after 2 h. Error bars represent SE.
) were
determined by trypan blue exclusion. Cells were stained for CD8 and
analyzed by flow cytometry, and total numbers of CD8+ T
cells in the lymphocyte gate were determined (
). B,
Cells were stained for CD8, CD69, and CD25; fixed in paraformaldehyde;
and subjected to two-color flow cytometry. Cells were gated on CD8, and
expression of CD69 (
) and CD25 ([
) within the gate was analyzed.
Each bar represents 8–15 mice. Error bars represent SE.
C, Cells were stimulated with anti-CD3 and
anti-CD28 Abs (