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, Yet Succumb to Tuberculosis1
*
Department of Molecular Genetics and Biochemistry, and
Central Animal Facilities, University of Pittsburgh School of Medicine, Pittsburgh, PA 15206; and
Howard Hughes Medical Institute, Albert Einstein College of Medicine, Bronx, NY 10461
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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production in the lungs of
infected mice revealed that both mutant mouse strains were only
transiently impaired in their ability to produce IFN- following
infection. At 2 wk postinfection, IFN- production, assessed by
RT-PCR and intracellular cytokine staining, in the mutant mice was
reduced by >50% compared with that in wild-type mice. However, by 4
wk postinfection, both mutant and wild-type mice had similar levels of
IFN- mRNA and protein production. In CD4 T cell-deficient mice,
IFN- production was due to CD8 T cells. Thus, the importance of
IFN- production by CD4 T cells appears to be early in infection,
lending support to the hypothesis that early events in M.
tuberculosis infection are crucial determinants of the course
of infection. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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M. tuberculosis replicates within host macrophages, and
activated macrophages are essential to limiting the infection.
Macrophages activated by IFN- and either TNF-
or bacterial
products such as LPS or lipoarabinomannan produce reactive nitrogen
intermediates (RNI)4 and kill
intracellular mycobacteria (5, 6, 7). RNI produced by activated murine
macrophages are required in vivo (8, 9, 10) and in vitro (6, 7) to control
M. tuberculosis infection. We and others have demonstrated
an absolute requirement for IFN- (11, 12) and TNF- (13) in the
control of murine tuberculosis, which is in part related to the
requirement for these cytokines in early RNI production in vivo.
CD4 T cells are thought to be the major source of IFN- during
M. tuberculosis infection, and it is generally believed that
the primary role of CD4 T cells in controlling tuberculosis is
production of this cytokine. Previous studies have shown that both
murine and human M. tuberculosis-specific CD4 T cells
produce IFN- (14, 15, 16, 17) and can activate macrophages to kill M.
tuberculosis in vitro (18). Mycobacterium-specific human CD8 T
cells can also produce IFN- and lyse infected cells (19, 20, 21).
Previous murine studies relied on Ab depletion in vivo or adoptive
transfer of T cell subsets to show that CD4 T cells were involved in
protection against M. tuberculosis (22, 23, 24). More recently,
MHC class II-deficient mice were shown to have increased susceptibility
to M. tuberculosis infection (25). Here, we have compared
transgenic mice with defects in the expression of either MHC class II
(26) or CD4 (27) molecules to test the requirement for CD4 T cells in
M. tuberculosis infection and to assess the mechanism by
which these CD4 T cells are protective against tuberculosis. We
demonstrate that IFN- production by both CD4 T cell-deficient mouse
strains was only transiently reduced, yet the mice succumbed to the
infection. These mice may serve as a useful model for AIDS and
tuberculosis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All mice were bred in the specific pathogen-free facility at the University of Pittsburgh School of Medicine (Pittsburgh, PA). Breeding pairs for MHC class II-/- mice (26) were obtained from Dr. Diane Mathis (Strasbourg, France) and backcrossed four times onto the C57BL/6 background, and mice were bred as heterozygotes (+/- x -/- or +/- x +/-). To identify mice for use as breeders, mice were genotyped by Southern blot as previously described (26). Before use in experiments, each mouse was phenotyped by staining PBL with Ab against CD4 and analyzing by flow cytometry; mice lacking MHC class II molecules had very low levels of CD4 T cells compared with wild-type mice. Mice heterozygous or homozygous for the wild-type MHC class II gene (+/+ or +/-) were indistinguishable in response to M. tuberculosis (data not shown), so littermates (usually +/-) were used as control mice. CD4-/- breeding pairs were obtained from Dr. Tak Mak, backcrossed twice onto the C57BL/6 background, and bred as homozygotes. As controls, +/+ littermates were bred. Mice were phenotyped by staining PBL with anti-CD4 Ab and analyzing by flow cytometry to assess the presence or the absence of the CD4 molecule.
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 injection into mice, 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 the tail vein with 2–10 x 105 (depending on the experiment) live bacilli in 100 µl, as determined by viable counts on 7H10 agar plates (Difco, Detroit, MI). For immunization, Calmette-Guérin bacillus (BCG; Pasteur strain, Trudeau Institute; 1 x 105 live bacilli i.v.) and M. tuberculosis (1 x 103 live bacilli i.v.) were administered to mice. In some experiments mice were treated 4 wk after immunization with isoniazid (Sigma, St. Louis, MO; 1 mg/ml in drinking water) for 4 wk. Ten days following the end of antibiotic treatment, the mice were challenged with virulent M. tuberculosis as described above.
CFU determination
Organs retrieved from infected mice were homogenized in PBS/Tween-80 (0.05%) in plastic bags using a Stomacher homogenizer (Tekmar, Cincinnati, OH), and dilutions were plated on supplemented 7H10 agar plates. Colonies were counted after 21-day incubation at 37°C in 5% CO2.
Histology
Organs were fixed in 10% formalin, sectioned, and stained with hematoxylin and eosin or with Kinyoun’s stain for acid-fast bacilli. The numbers of granulomas in liver sections were counted in 5–10 x10 fields from each slide, using hematoxylin- and eosin-stained sections (three or four mice per group). For immunohistochemistry, paraffin-embedded sections were deparaffinized, and Ag retrieval was performed using a microwave technique as previously described (8). Anti-NOS2 Ab (Transduction Laboratories, Cincinnati, OH) was used to stain tissues, with biotinylated anti-rabbit IgG as a secondary Ab. The ABC method (Vector, Burlingame, CA) was used with diaminobenzidene as a substrate to visualize Ab binding. The proportion of NOS2+ granulomas was assessed by counting total granulomas versus NOS2+ granulomas in 5–10 x10 fields of liver sections from each mouse.
FACS analysis of cell surface markers
Spleen cells were obtained from infected mice at various time points postinfection by crushing the organ in mesh bags to obtain single cell suspensions. RBC were lysed with Tris/NH4Cl solution. Cells were stained for cell surface molecules using Abs against CD3 (anti-CD3-phycoerythrin), CD4 (anti-CD4-FITC), and CD8 (anti-CD8-CyChrome). All Abs were used at 0.2 µg/106 cells and were obtained from PharMingen (San Diego, CA). Cells were fixed with 2% paraformaldehyde overnight and analyzed by FACS (Becton Dickinson, Mountain View, CA). Two or three mice per group were used for each time point.
Spleen cell proliferation assays
Single cell suspensions were obtained by crushing spleens in mesh bags. RBC were lysed with Tris/NH4Cl, and the cells were washed extensively. Following resuspension in medium (RPMI 1640, 10% FBS, glutamine, and 2-ME), 5 x 105 cells/well of 96-well round-bottom plates were stimulated with medium alone, Con A (5 µg/ml), or PPD (10 µg/ml). In some experiments 1 x 104 peritoneal exudate macrophages from C57BL/6 mice were added to the wells before addition of spleen cells. Cells were pulsed with [3H]thymidine (1 µCi/well) after 60 h of culture and were harvested 12–18 h later. Incorporation of [3H]thymidine was measured by counting cell lysates on filters in a scintillation counter. The stimulation index was determined by dividing sample counts by background counts for each sample and is presented as an average for two mice per group. The experiment was repeated three times.
ELISA
IFN- production by cultured spleen cells was assessed by
sandwich ELISA using Abs R4-A62 and XMG1.2 (biotinylated; PharMingen),
according to the manufacturer’s protocol. Recombinant murine IFN-,
used to generate a standard curve, was a gift from Genentech (South San
Francisco, CA).
Semiquantitative competitive RT-PCR
Cytokine and NOS2 mRNA levels in lung and spleen were assessed using the QC-RT-PCR method as previously described (8, 28). PCR products were electrophoresed on 2% agarose gels followed by staining with Sybr Green (Molecular Probes, Eugene, OR). Bands were quantitated using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). The concentration of cDNA in each sample was standardized by determining the ratio of lung cDNA to competitor plasmid DNA for hypoxanthine phosphoribosyltransferase (HPRT), using a fixed concentration of competitor plasmid. A correction value for each sample for standardization was obtained. The ratio of lung cDNA to competitor plasmid DNA for each cytokine was obtained and was corrected using the HPRT values for each sample. Relative levels of cytokine mRNA are reported (lung cDNA/plasmid DNA), and a value of 1 is equivalent to HPRT mRNA level. Time zero indicates uninfected mice. The data presented are an average of three or four mice per group per time point.
In vivo intracellular cytokine staining
Single cell suspensions of lung and spleen cells at various time
points postinfection were prepared as described above. Cells were
stimulated for 6 h with anti-CD3 (clone 145-2C11; 0.1 and
anti-CD28 (clone 37.51; 1 µg/ml) Abs in the presence of 3 µM
monensin (Sigma) to halt egress of cytokines from the cells. Following
washing, cells were stained for cell surface molecules CD4 (0.2
µg/106 cells anti-CD4-CyChrome Ab, clone H129.19) and
CD8 (0.2 µg/106 cells anti-CD8-FITC Ab, clone 53-6.7)
in 20% mouse serum/1% FBS for 30 min at 4°C, washed, and fixed in
1% paraformaldehyde at 4°C overnight. Cells were permeabilized with
saponin (0.1% in PBS containing 1% FBS/0.1% sodium azide) and were
stained for IFN- or IL-4 (0.4 µg/106 cells,
anti-IFN--PE Ab, clone XMG1.2, or anti-IL-4-PE Ab, clone
11B11) in 20% mouse serum for 30 min at 4°C, washed, and analyzed by
FACS (Becton Dickinson). Isotype controls for each Ab were used, and an
uninfected control mouse was tested in each experiment. All Abs were
obtained from PharMingen. Cells could not be stained with anti-CD3
Ab for analysis, as anti-CD3 Ab was used to stimulate the cells.
Statistics
Student’s paired test was used to compare groups. Statistical analysis was performed using StatView (Abacus Concepts, Berkeley, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mice deficient in MHC class II molecules (26) were infected with
virulent M. tuberculosis (5 x 105 bacilli
i.v.). These mice succumbed to the infection with a mean survival time
of 39 ± 1 days, while control mice (homozygous +/+ or
heterozygous +/- littermates) survived for the length of the
experiment (>120 days; p < 0.0001; Fig. 1
). The MHC class
II-/- mice were deficient in their ability to
control the infection in lungs, liver, and spleen compared with the
control mice (Fig. 2). Whereas wild-type
or heterozygous mice began to control the infection in lungs at 3 wk
postinfection, MHC class II-/- mice did not,
and at 40 days postinfection there was a 50-fold increase in number of
bacteria in the MHC class II-/- mice compared
with the controls. In spleen and liver, mutant mice had >100-fold
higher bacterial numbers compared with control mouse organs. Thus, the
absence of MHC class II molecules prevented the mice from controlling
M. tuberculosis infection.
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). As a second
model, mice in which the gene CD4 molecule was disrupted
(CD4-/-) (27) were tested in our acute
tuberculosis model. The mean survival time for
CD4-/- mice infected with M.
tuberculosis (5 x 105 bacilli i.v.) was 60
± 7 days, which is significantly longer than that for MHC class
II-/- mice (Fig. 1
; p = 0.02).
Again, controls (+/+ littermates) survived the length of the experiment
(>120 days). Bacterial numbers in lung, liver, and spleen were at
least 100-fold greater in CD4-/- mice than in
control mice by 35 days postinfection (Fig. 2).
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Prior BCG immunization of immunocompetent mice does not prevent
infection with a virulent M. tuberculosis challenge, but the
bacterial numbers in the organs of the immunized and challenged mice
are reduced 10- to 100-fold compared with those in unimmunized mice.
Prior studies have shown that BCG immunization of
ß2m-/- mice (which lack MHC class
I molecules and, consequently, are deficient in CD8 T cells) (29) or
TNF-p55 receptor-/- mice (13) increased
survival time following M. tuberculosis challenge, although
the mice still succumbed to the infection. In contrast, BCG
immunization of MHC class II-/- mice for 3 mo
did not increase mean survival time following virulent M.
tuberculosis challenge (Table I).
Curiously, no adverse effects of BCG infection were observed despite
the absence of CD4 T cells in these mice. As an alternative
immunization strategy, in case BCG immunization was not adequate to
induce protective CD8 T cell responses, MHC class
II-/- mice were immunized with virulent
M. tuberculosis (1 x 103 i.v.) or BCG
(1 x 105 i.v.) for 4 wk, then treated with the
antibiotic isoniazid for 4 wk. Ten days after cessation of antibiotic
treatment, mice were challenged with virulent M.
tuberculosis (5 x 105 i.v.). Bacterial numbers
in lungs, liver, and spleen of BCG- or M.
tuberculosis-immunized mice were 100- to 1000-fold higher than
those in wild-type immunized mice at 3 and 6 wk postinfection and were
similar to those in unimmunized MHC class II-/-
mice (data not shown). The mean survival time of these mice was also
unchanged compared with that of unimmunized control mice (Table I);
there was no difference in protection between mice immunized with BCG
and those immunized with M. tuberculosis
(p = 0.61). Thus, immunization was not
effective in protecting against subsequent virulent M.
tuberculosis challenge in the absence of MHC class II Ag
presentation.
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In immunocompetent mice, the response to M.
tuberculosis includes the formation and maintenance of granulomas,
composed of CD4 and CD8 T cells surrounding epithelioid macrophages
(29). Granuloma formation was assessed in tissue sections from
wild-type, MHC class II-/-, and
CD4-/- mice at various times postinfection. At
1 wk postinfection liver sections from CD4-/-
and MHC class II-/- mice contained only
20–25% as many granulomas as liver tissue from wild-type control mice
(Table II). However, by 2 wk
postinfection the numbers of granulomas in liver sections from the
wild-type and CD4-deficient mice were equivalent and remained so over
the course of infection (Table II). A similar pattern was observed in
the lungs (data not shown).
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, A–C). Organized granulomas
consisting of an outer rim of mononuclear cells and central aggregates
of large, epithelioid histiocytes were more commonly seen in wild-type
animals. Well-organized granulomas were, however, also present in the
mutant mice. Lung tissue from wild-type,
CD4-/-, and MHC class
II-/- mice were evaluated and scored
histopathologically for the imflammatory processes present. These
included the infiltration of mixed, but primarily lymphohistiocytic,
cells throughout interstitial regions (interstitial pneumonitis), the
presence of circumscribed foci of granulomatous inflammation,
inflammatory cell infiltration into alveolar air spaces (alveolitis),
and histomorphological evidence of necrosis (Fig. 3, D–F).
Generally, wild-type mice demonstrated less severe interstitial
pneumonitis, necrosis, and alveolitis than their transgenic
counterparts, especially in the later stages of infection. Consistent,
significant differences between the histology of
CD4-/- and MHC class
II-/- tissues were not recognized within these
scoring categories.
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). By 3 wk postinfection the percentage of liver granulomas staining
positively for NOS2 protein was similar among wild-type and
CD4-deficient mice (Table II). NOS2 staining in lung followed a similar
pattern (data not shown). Thus, expression of NOS2 by macrophages was
delayed, but not absent, in the CD4 T cell-deficient mouse strains. The
early production of RNI by macrophages may be essential to control of
the infection. T cell subsets
It was previously reported that CD4-/-
mice had significant numbers of double-negative T cells
(CD4-8-), and that such cells were capable of
proliferating and producing IFN- in an MHC class II-restricted
manner in response to leishmanial Ag (30). This phenomenon was not
observed in MHC class II-/- mice. In those
studies CD4-/- mice were resistant to
Leishmania major challenge, while MHC class
II-/- mice were susceptible. In our acute
tuberculosis model, CD4-/- mice had a longer
mean survival time than MHC class II-/- mice,
although both strains of mice were much more susceptible than wild-type
littermate controls (Fig. 1). Therefore, we examined the presence and
function of the double-negative T cell population in
CD4-/- and MHC class
II-/- mice following infection with M.
tuberculosis. The T cell phenotype of spleen cells at 0, 14, and
24 days postinfection was assessed by Ab staining of cell surface
markers and flow cytometry. M. tuberculosis infection caused
a slight increase in double-negative CD3+ cells in
wild-type mice (Table III). As described
previously (26, 27), MHC class II-/- and
CD4-/- mice had higher numbers of CD8 T cells
compared with wild-type mice due to the deficiency in CD4 T cells.
Compared with wild-type mice, a much higher proportion of spleen cells
from both MHC class II-/- and
CD4-/- mice was CD3+
CD4-8- cells even before infection (Table III), and little increase in the percentage of
CD4-8- T cells was observed following
infection. In contrast to the reported findings in the L.
major system, we did not observe substantial differences between
the two CD4 T cell-deficient mouse strains in the numbers of
double-negative T cells following M. tuberculosis infection.
Thus, the increased survival of CD4-/- mice
compared with that of MHC class II-/- mice does
not appear to be due to an increase in the number of double-negative T
cells.
In vitro T cell proliferation
The ability of T cells from the CD4 T cell-deficient mice to
proliferate in response to mycobacterial Ags was examined by
stimulating spleen cells from mice at 2 and 4 wk post-M.
tuberculosis infection with Con A or PPD for 3 days and measuring
the incorporation of [3H]thymidine. Although T cells from
uninfected control (littermate) mice responded strongly to Con A, T
cells from infected wild-type mice had lower responses, suggesting a
suppressive effect of M. tuberculosis infection, which has
been described previously (31, 32). Spleen cells from infected
wild-type mice showed proliferation in response to PPD (Fig. 4A), although the stimulation
index was low. In contrast, there was little or no proliferation of
spleen cells from MHC class II-/- or
CD4-/- mice in response to PPD. Con A-induced
proliferation was lower in MHC class II-/-
spleen cells, but was similar to that in wild-type in
CD4-/- spleen cells (Fig. 4A).
Double-negative T cells present in the spleen cells of the mutant
strains did not appear to proliferate in response to MHC class II
presentation of Ag. Addition of wild-type macrophages (i.e., those with
MHC class II molecules) did not result in increased proliferation to
PPD by spleen cells from either CD4 T cell-deficient mouse strain (Fig. 4A). At later time points in the infection, increased
stimulation of the wild-type spleen cell response to PPD was observed
(our unpublished observations), but the limited life span of
infected CD4 T cell-deficient mice precluded comparisons of T cell
stimulation at the later time points.
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Alteration in cytokine production in the absence of CD4 T cells
was an obvious possibility to explain the inability of the MHC class
II-/- and CD4-/- mice
to control infection. We initially examined IFN- production by T
cells in response to PPD in vitro using ELISA. Spleen cells from
infected wild-type mice produced IFN- in response to PPD as well as
to the nonspecific stimulus of Con A (Fig. 4B). In contrast,
IFN- production in response to PPD was very low or undetectable in
spleen cell cultures from MHC class II-/- or
CD4-/- mice, although Con A induced IFN- in
these cultures (Fig. 4B). Addition of wild-type macrophages
(i.e., those with MHC class II molecules) as APCs to the cultures in
the presence of PPD did not increase the production of IFN- (data
not shown).
Cytokine mRNA expression in vivo following M. tuberculosis infection
In vitro proliferation assays may not be indicative of the actual
cytokine production by T cells in vivo, especially over the course of
infection, since it is a restimulation assay. In addition, this assay
primarily measures MHC class II-restricted proliferation, and the
contribution of MHC class I-restricted T cells (CD8+ or
CD4-8-) is not well represented. We were also
interested in the cytokine response in the lungs; bulk T cells from
lungs are difficult to obtain in sufficient numbers for in vitro
proliferation assays. To assess in vivo cytokine production, we used
QC-RT-PCR (28) to compare gene expression in the tissues of control and
knockout mice at various times postinfection. In the lungs of MHC class
II-/- mice, IFN- expression was
approximately 5-fold lower than that in wild-type lungs early in
infection, but expression levels were similar between the two groups at
3 wk postinfection (Fig. 5A).
Expression of NOS2 was similar between the mice in the lungs (Fig. 5A). IL-12 expression was also decreased in the lungs of MHC
class II-/- mice at 1 wk postinfection, but
IL-12 expression in the control mice decreased by 3 wk postinfection to
the level seen in the MHC class II-/- mice
(Fig. 5A). IL-10 expression in the lungs of all mice was
low, and IL-4 mRNA was not detected at any time point (data not shown).
Using our RT-PCR assay, we were unable to detect IL-2 mRNA in the lungs
or spleens of either wild-type or CD4-deficient mice, whether
uninfected or infected (data not shown).
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expression was reduced approximately 5-fold
compared with that in wild-type controls early in infection, but
returned to wild-type levels as the infection progressed (Fig. 5B). There was a more obvious decrease in NOS2 expression
early in infection in the CD4-/- mice compared
with wild-type controls, but it also returned to wild-type levels later
in infection (Fig. 5B). IL-12 expression was reduced in
CD4-/- mice at 2 wk postinfection compared with
that in wild-type mice (Fig. 5B). IL-4 and IL-2 mRNA were
undetectable at all time points (data not shown). In vivo intracellular cytokine staining
The data obtained by RT-PCR suggested that IFN- expression in
the lungs was lower in the absence of CD4 T cells at early time points
after infection, but quickly returned to wild-type levels as the
infection progressed. Using QC-RT-PCR on whole tissue RNA, it was only
possible to examine overall IFN- production, and no information
regarding the cells responsible for IFN- production could be
gathered. There was also variability among mice using the QC-RT-PCR
assay. For these reasons, we used intracellular cytokine staining of
lung and spleen cells directly from the infected mice to assess the
potential for each cell type to produce cytokines in vivo in response
to M. tuberculosis infection.
Lung and spleen cells from infected mice were obtained, stimulated with
anti-CD3 and anti-CD28 Abs, treated with monensin for only
6 h, stained for cell surface molecules, permeablized, and stained
for intracellular cytokine protein. Lung and spleen cells from
uninfected mice were used as controls; these cells produced little or
no IFN- or IL-4 after stimulation as described above (Fig. 6), indicating that only in vivo
activated T cells will produce cytokines upon short term (4–6 h)
stimulation with anti-CD3 and anti-CD28 Abs. Based on these
results, we chose this technique to compare cytokine production by
subsets of T cells without in vitro culture and obtain a clearer
picture of cytokine production in vivo during an infection. A
representative experiment is shown in Fig. 6, while Fig. 7 shows the combined results from at
least three experiments over the course of infection.
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Lung cells were used to analyze IFN- production following infection,
since the lung is the most relevant site of bacterial infection in
tuberculosis. In wild-type mice at 1 wk postinfection, IFN-
production by lung cells was very low (Fig. 7). By 2 wk postinfection,
IFN- production was increased, with approximately 11% of the lung
lymphocytes (gated by size) producing IFN- (Figs. 6 and 7). IFN-
was produced by both CD4 and CD8 T cells, with higher production by the
CD4 T cell subset. By 4 and 6 wk postinfection, IFN- production had
stabilized (
10% of lymphocytes), with contributions from both CD4
and CD8 T cells (Figs. 6 and 7). IL-4 was not detected in lung cells at
any time point (data not shown).
IFN- production in the lungs of MHC class
II-/- and CD4-/- at
various times postinfection was compared with that in wild-type mice
(Figs. 6 and 7). At 7 days postinfection, IFN- production was low in
all groups. At 14 days postinfection, IFN- production in MHC class
II-/- and CD4-/- mice
was only 44% of that in wild-type mice (p <
0.01). At 4 wk postinfection, total IFN- levels in the lungs of
wild-type and CD4-deficient mice were similar. In both MHC class
II-/- and CD4-/- lungs,
CD8 T cells were responsible for the majority of IFN- production,
with CD4-8- cells accounting for only a small
proportion of IFN- (Fig. 6). The apparent decrease in total IFN-
production in the MHC class II-/- lung cells at
the 6 wk point was probably due to the presence of necrosis and cells
in poor condition in the lungs, since the mean survival time is
approximately 6 wk. IL-4 was not detectable at any time point in the
wild-type or CD4-deficient mice (data not shown).
Spleen cells from these mice gave similar results as lung cells in
intracellular staining experiments, but the overall percentage of
IFN--producing T cells observed was lower (data not shown). This is
probably due to the large number of resident, nonspecific T cells in
the spleen. In the lung, 2- to 5-fold more T cells were isolated from
infected mice compared with uninfected mice (our unpublished
observations), suggesting that specific T cells migrate to or
proliferate at the site of infection in the lung. Uninfected mice have
a relatively small number of T cells. Thus, a higher percentage of the
T cells found in the lungs are likely to be mycobacterium-specific
IFN--producing T cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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is essential to limiting
replication of M. tuberculosis organisms (11, 12). This
cytokine is also likely to be important in human tuberculosis, as
humans deficient in receptors for IFN- or IL-12 are very susceptible
to mycobacterial infections (33, 34, 35, 36). Numerous studies report that CD4
T cells from M. tuberculosis-infected mice or humans
proliferate in response to mycobacterial Ags and produce IFN-. An
obvious conclusion from the previous studies was that the primary role
of CD4 T cells in protection against tuberculosis is the production of
IFN-. Here, we addressed how a loss of CD4 T cells affected the
ability of mice to control tuberculosis. Mice deficient in CD4 T cells
were strikingly more susceptible to M. tuberculosis
infection than were control mice. Surprisingly, the level of IFN-
production in the lungs was only transiently decreased early in
infection compared with that in wild-type mice. These data suggest that
early production of IFN- by CD4 T cells is essential to control of
this infection, and that IFN- production by other cells cannot
substitute for the CD4 T cell contribution. Our data do not rule out an
IFN--independent function of CD4 T cells in controlling
tuberculosis. In fact, it is likely that the immunodeficiency in
HIV+ patients that enhances susceptibility to tuberculosis
is complex and not simply due to decreased IFN- production by CD4 T
cells. MHC class II-/- and CD4-/- mice infected with M. tuberculosis had much higher bacterial numbers in the organs compared with control mice and succumbed to the infection. MHC class II-/- mice were shown previously to be susceptible to M. tuberculosis infection, but a transient plateau of CFU in the lung and spleens 20–60 days postinfection was reported, which was interpreted as the contribution of CD8 T cells to protection in this model (25). We did not observe such a plateau; however, bacterial numbers were calculated differently in our study: CFU per gram tissue were reported by Tascon et al., while we calculated the total number of CFU per organ. This may account for the apparent discrepancy in results, as the organs of MHC class II-/- and CD4-/- mice increase in size relative to those in wild-type mice as the infection progresses.
Although MHC class II-/- and
CD4-/- mice were more resistant than
IFN--deficient (11, 12) or TNF--deficient (13) mice, the mean
survival time was similar to that in ß2m-deficient mice
(29). It is simplistic to compare gene-disrupted mice to obtain a
meaningful picture of the relative protective effects of each immune
system component. However, it is clear that both CD4 and CD8 T cells
are important in protection against tuberculosis, and each subset is
incapable of substituting for the other in providing this protection.
Unlike mice deficient in MHC class I molecules (29), prior immunization
with BCG or M. tuberculosis did not extend the mean survival
time of the MHC class II-/- mice. These results
demonstrate that immunization depends on MHC class II Ag
presentation and confirm the central role of MHC class II molecules and
CD4 T cells in controlling tuberculosis. The data available to date
strongly indicate that CD4 and CD8 T cells are performing at least some
different functions during infection, and studies to define these
functions are essential to our understanding of this disease and the
most effective means for vaccination.
Spleen cells from M. tuberculosis-infected MHC class
II-/- and CD4-/- mice
were impaired in the ability to proliferate and produce IFN- in
response to PPD in vitro. However, in vitro proliferation assays under
the conditions used here favor the proliferation of CD4 T cells, rather
than CD8 T cells, and may give an inaccurate picture of the in vivo
potential of the T cells to produce IFN-. Thus, we turned to assays
that would allow assessment of in vivo production of IFN-.
IFN- mRNA levels in the lungs of CD4 T cell-deficient mice were
approximately 5-fold lower than those in wild-type mice early in
infection. However, by 3 wk postinfection there were no discernible
differences in levels of IFN- mRNA among CD4 T cell-deficient and
wild-type mice. IL-12 p40 mRNA, which is enhanced by IFN-
production, was also deficient only at early time points after
infection. Intracellular cytokine staining of lung cells directly
isolated from infected mice was used to assess the potential for
various cell types to produce IFN- during infection. This assay
identifies cells capable of IFN- production, but may overestimate
the percentage of cells actually producing IFN- at any one time
point during the infection. The results obtained were similar to those
obtained with RT-PCR in terms of kinetics of IFN- production in the
lungs. At 2 wk postinfection mice deficient in CD4 T cells produced
only 44% of the IFN- produced by wild-type mice. While IFN-
production by CD8 T cells was approximately the same in CD4 T
cell-deficient and wild-type mice at this time point, the contribution
of the CD4 T cells to IFN- was obviously absent in the mutant mice.
This suggests that IFN- production at the early time points of
infection must come from CD4 T cells to be effective, and that CD8 T
cell IFN- production is insufficient to control the infection.
Interestingly, by 4 wk postinfection, IFN- production by the CD8 T
cell subset in the CD4 T cell-deficient mice increased, so that total
IFN- production was similar to that in wild-type mice (Fig. 7),
consistent with mRNA data. However, this IFN- production was
apparently insufficient to control the infection in the absence of CD4
T cells.
CD4-/- mice had a longer mean survival time
than MHC class II-/- mice following M.
tuberculosis infection. A discrepancy in susceptibility between
these two mouse strains was also reported following L. major
infection, in which the protection of CD4-/-
mice was attributed to MHC class II-restricted IFN- production by
Leishmania-specific CD4-8- T cells
(30). No difference in the numbers of CD4-8-
cells between MHC class II-/- and
CD4-/- mice following M.
tuberculosis infection was observed, nor did these cells
proliferate or produce IFN- in response to mycobacterial Ag. In
vivo, these cells did not appear to contribute to IFN- production,
since most IFN- intracellular staining following stimulation of lung
cells was contributed by CD8 T cells in the mutant mice. The reasons
for the difference in susceptibility to tuberculosis between these two
strains is unclear, but appears not to be related to the direct
contribution of CD4-8- T cells.
Early IFN- production may not be the only role for CD4 T cells in
protection against tuberculosis. The effect of CD4 T cells on CD8 T
cell development and function must be taken into account, since CD8 T
cells are important in the control of murine tuberculosis (29, 37). The
number of CD8 T cells increases in the lungs following M.
tuberculosis infection in the presence or the absence of CD4 T
cells (our unpublished observations), and these cells are
capable of producing IFN-. However, another role for CD8 T cells in
protection against tuberculosis may be as CTL. The absence of CD4 T
cells in the mutant mice may prevent the CD8 T cells from becoming CTL,
which may contribute to the susceptibility of the mutant mice to
tuberculosis. However, effective CTL responses during viral
infections have been demonstrated in MHC class
II-/- and CD4-/- mice
(27, 38, 39, 40). In vitro, the requirement for CD4 T cells in the
generation of CD8 CTL can be bypassed by modulation of the APC by
either ligation of CD40 or viral infection (41, 42, 43). This suggests that
in vivo, infection with a pathogen such as M. tuberculosis
may also bypass the need for CD4 T cells in eliciting CD8 T cell
responses. Certainly, the data presented here indicate that CD4 T cells
are not necessary for eliciting CD8 T cells that produce IFN- during
mycobacterial infection; studies are currently underway to assess the
presence of mycobacteria-specific CTL in the infected mutant mice.
Another role for CD4 T cells in eliciting CD8 T cells is the production of IL-2. We were unable to detect IL-2 transcripts in the lungs or spleens of wild-type or mutant infected mice; this may be due to the time points chosen for analysis. However, the double-negative T cells (CD4-8-) in the mutant mice or even the CD8 T cells may be a source of sufficient IL-2 to drive T cell expansion. Previous studies have suggested that such cells may produce IL-2 in the absence of CD4 T cells (41).
Granulomas are believed to prevent the spread of infection throughout the organs. Both CD4 and CD8 T cells are present in the granulomas of wild-type mice, suggesting that each cell type contributes to the maintenance and function of the granuloma (29). Granuloma formation was delayed for about 1 wk in the CD4 T cell-deficient mice, suggesting that CD4 T cells are important in the initial formation of the granuloma and also for the elimination of bacilli within the granuloma. Although the numbers of granulomas present in the livers and lungs of CD4-deficient mice reached wild-type levels by 2 wk postinfection, the bacterial burden in these mice was not brought under control, indicating a loss of granuloma function. Indeed, in general, the granulomas present in the CD4 T cell-deficient mice were less organized than those in the tissues of wild-type mice.
NOS2 expression is dependent on IFN-. A delay in NOS2 gene and
protein expression was observed in the CD4 T cell-deficient mice
compared with that in control mice. By 3 wk postinfection, NOS2 protein
levels were similar among CD4 T cell-deficient and control mice, yet
the mutant mice were unable to control the infection. As we observed in
TNF receptor-deficient mice, later production of RNI is insufficient to
control an ongoing infection. These data lend support to the idea that
the early expression of antimycobacterial mechanisms of macrophages is
crucial to the control of tuberculosis.
In summary, we have shown that CD4 T cells are required for resistance
to tuberculosis, and that the absence of CD4 T cells results in a delay
in IFN- production as well as a delay in NOS2 expression and
granuloma formation. Thus, early production of IFN- is likely to be
a major role for CD4 T cells in protection against tuberculosis. The
contribution of CD8 T cells to IFN- production is obvious after 2 wk
of infection, but the CD8 T cell subset is insufficient to compensate
for the loss of CD4 T cells. It is clear that the early immunologic
events in M. tuberculosis have profound consequences for the
outcome of infection and disease. These findings have important
implications for tuberculosis and AIDS, and the murine models described
here may be useful to study the effects of tuberculosis on CD4 T
cell-deficient subjects as well as immunotherapies and drug regimens
that may be useful in an immunocompromised individual.
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Acknowledgments |
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Footnotes |
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2 Current address: School of Public Health, Harvard University, Boston, MA 02115.
3 Address correspondence and reprint requests to Dr. JoAnne L. Flynn, Department of Molecular Genetics and Biochemistry, E1240 Biomedical Science Tower, University of Pittsburgh School of Medicine, Pittsburgh, PA 15206. E-mail address:
4 Abbreviations used in this paper: RNI, reactive nitrogen intermediates; BCG, Calmette-Guérin bacillus; PPD, purified protein derivative; QC-RT-PCR, quantitative competitive RT-PCR; HPRT, hypoxanthine phosphoribosyltransferase.
Received for publication November 24, 1998. Accepted for publication February 8, 1999.
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H. M. Scott and J. L. Flynn Mycobacterium tuberculosis in Chemokine Receptor 2-Deficient Mice: Influence of Dose on Disease Progression Infect. Immun., November 1, 2002; 70(11): 5946 - 5954. [Abstract] [Full Text] [PDF]
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V. Lazarevic and J. Flynn CD8+ T Cells in Tuberculosis Am. J. Respir. Crit. Care Med., October 15, 2002; 166(8): 1116 - 1121. [Full Text] [PDF]
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 D. Kaushal, B. G. Schroeder, S. Tyagi, T. Yoshimatsu, C. Scott, C. Ko, L. Carpenter, J. Mehrotra, Y. C. Manabe, R. D. Fleischmann, et al. Reduced immunopathology and mortality despite tissue persistence in a Mycobacterium tuberculosis mutant lacking alternative sigma factor, SigH PNAS, June 11, 2002; 99(12): 8330 - 8335. [Abstract] [Full Text] [PDF]
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K. L. Elkins, A. Cooper, S. M. Colombini, S. C. Cowley, and T. L. Kieffer In Vivo Clearance of an Intracellular Bacterium, Francisella tularensis LVS, Is Dependent on the p40 Subunit of Interleukin-12 (IL-12) but Not on IL-12 p70 Infect. Immun., April 1, 2002; 70(4): 1936 - 1948. [Abstract] [Full Text] [PDF]
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A. L. Moreira, L. Tsenova, M. Haile Aman, L.-G. Bekker, S. Freeman, B. Mangaliso, U. Schroder, J. Jagirdar, W. N. Rom, M. G. Tovey, et al. Mycobacterial Antigens Exacerbate Disease Manifestations in Mycobacterium tuberculosis-Infected Mice Infect. Immun., April 1, 2002; 70(4): 2100 - 2107. [Abstract] [Full Text] [PDF]
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U. Palendira, A. G. D. Bean, C. G. Feng, and W. J. Britton Lymphocyte Recruitment and Protective Efficacy against Pulmonary Mycobacterial Infection Are Independent of the Route of Prior Mycobacterium bovis BCG Immunization Infect. Immun., March 1, 2002; 70(3): 1410 - 1416. [Abstract] [Full Text] [PDF]
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M. Panigada, T. Sturniolo, G. Besozzi, M. G. Boccieri, F. Sinigaglia, G. G. Grassi, and F. Grassi Identification of a Promiscuous T-Cell Epitope in Mycobacterium tuberculosis Mce Proteins Infect. Immun., January 1, 2002; 70(1): 79 - 85. [Abstract] [Full Text] [PDF]
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S. Ehlers, J. Benini, H.-D. Held, C. Roeck, G. Alber, and S. Uhlig {alpha}{beta} T Cell Receptor-positive Cells and Interferon-{gamma}, but not Inducible Nitric Oxide Synthase, Are Critical for Granuloma Necrosis in a Mouse Model of Mycobacteria-induced Pulmonary Immunopathology J. Exp. Med., December 17, 2001; 194(12): 1847 - 1859. [Abstract] [Full Text] [PDF]
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N. V. Serbina, V. Lazarevic, and J. L. Flynn CD4+ T Cells Are Required for the Development of Cytotoxic CD8+ T Cells During Mycobacterium tuberculosis Infection J. Immunol., December 15, 2001; 167(12): 6991 - 7000. [Abstract] [Full Text] [PDF]
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W. R. Waters, B. J. Nonnecke, T. E. Rahner, M. V. Palmer, D. L. Whipple, and R. L. Horst Modulation of Mycobacterium bovis-Specific Responses of Bovine Peripheral Blood Mononuclear Cells by 1,25-Dihydroxyvitamin D3 Clin. Vaccine Immunol., November 1, 2001; 8(6): 1204 - 1212. [Abstract] [Full Text] [PDF]
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A. A. Pathan, K. A. Wilkinson, P. Klenerman, H. McShane, R. N. Davidson, G. Pasvol, A. V. S. Hill, and A. Lalvani Direct Ex Vivo Analysis of Antigen-Specific IFN-{gamma}-Secreting CD4 T Cells in Mycobacterium tuberculosis-Infected Individuals: Associations with Clinical Disease State and Effect of Treatment J. Immunol., November 1, 2001; 167(9): 5217 - 5225. [Abstract] [Full Text] [PDF]
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J. L. Flynn and J. Chan Tuberculosis: Latency and Reactivation Infect. Immun., July 1, 2001; 69(7): 4195 - 4201. [Full Text] [PDF]
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N. V. Serbina and J. L. Flynn CD8+ T Cells Participate in the Memory Immune Response to Mycobacterium tuberculosis Infect. Immun., July 1, 2001; 69(7): 4320 - 4328. [Abstract] [Full Text] [PDF]
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C. G. Feng, U. Palendira, C. Demangel, J. M. Spratt, A. S. Malin, and W. J. Britton Priming by DNA Immunization Augments Protective Efficacy of Mycobacterium bovis Bacille Calmette-Guerin against Tuberculosis Infect. Immun., June 1, 2001; 69(6): 4174 - 4176. [Abstract] [Full Text] [PDF]
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N. Boechat, F. Bouchonnet, M. Bonay, A. Grodet, V. Pelicic, B. Gicquel, and A. J. Hance Culture at High Density Improves the Ability of Human Macrophages to Control Mycobacterial Growth J. Immunol., May 15, 2001; 166(10): 6203 - 6211. [Abstract] [Full Text] [PDF]
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C. G. Feng, C. Demangel, A. T. Kamath, M. Macdonald, and W. J. Britton Dendritic cells infected with Mycobacterium bovis bacillus Calmette Guerin activate CD8+ T cells with specificity for a novel mycobacterial epitope Int. Immunol., April 1, 2001; 13(4): 451 - 458. [Abstract] [Full Text] [PDF]
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J. C. Leemans, N. P. Juffermans, S. Florquin, N. van Rooijen, M. J. Vervoordeldonk, A. Verbon, S. J. H. van Deventer, and T. van der Poll Depletion of Alveolar Macrophages Exerts Protective Effects in Pulmonary Tuberculosis in Mice J. Immunol., April 1, 2001; 166(7): 4604 - 4611. [Abstract] [Full Text] [PDF]
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V. Nagabhushanam and C. Cheers Non-Major Histocompatibility Complex Control of Antibody Isotype and Th1 versus Th2 Cytokines during Experimental Infection of Mice with Mycobacterium avium Infect. Immun., March 1, 2001; 69(3): 1708 - 1713. [Abstract] [Full Text] [PDF]
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M. Gonzalez-Juarrero, O. C. Turner, J. Turner, P. Marietta, J. V. Brooks, and I. M. Orme Temporal and Spatial Arrangement of Lymphocytes within Lung Granulomas Induced by Aerosol Infection with Mycobacterium tuberculosis Infect. Immun., March 1, 2001; 69(3): 1722 - 1728. [Abstract] [Full Text] [PDF]
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T. Mogues, M. E. Goodrich, L. Ryan, R. LaCourse, and R. J. North The Relative Importance of T Cell Subsets in Immunity and Immunopathology of Airborne Mycobacterium tuberculosis Infection in Mice J. Exp. Med., February 5, 2001; 193(3): 271 - 280. [Abstract] [Full Text] [PDF]
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K. A. Bodnar, N. V. Serbina, and J. L. Flynn Fate of Mycobacterium tuberculosis within Murine Dendritic Cells Infect. Immun., February 1, 2001; 69(2): 800 - 809. [Abstract] [Full Text] [PDF]
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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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Y. A. W. Skeiky, P. J. Ovendale, S. Jen, M. R. Alderson, D. C. Dillon, S. Smith, C. B. Wilson, I. M. Orme, S. G. Reed, and A. Campos-Neto T Cell Expression Cloning of a Mycobacterium tuberculosis Gene Encoding a Protective Antigen Associated with the Early Control of Infection J. Immunol., December 15, 2000; 165(12): 7140 - 7149. [Abstract] [Full Text] [PDF]
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H.-J. Ullrich, W. L. Beatty, and D. G. Russell Interaction of Mycobacterium avium-Containing Phagosomes with the Antigen Presentation Pathway J. Immunol., December 1, 2000; 165(11): 6073 - 6080. [Abstract] [Full Text] [PDF]
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H. M. Dockrell, S. Brahmbhatt, B. D. Robertson, S. Britton, U. Fruth, N. Gebre, M. Hunegnaw, R. Hussain, R. Manandhar, L. Murillo, et al. A Postgenomic Approach to Identification of Mycobacterium leprae-Specific Peptides as T-Cell Reagents Infect. Immun., October 1, 2000; 68(10): 5846 - 5855. [Abstract] [Full Text] [PDF]
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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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N. V. Serbina, C.-C. Liu, C. A. Scanga, and J. L. Flynn CD8+ CTL from Lungs of Mycobacterium tuberculosis-Infected Mice Express Perforin In Vivo and Lyse Infected Macrophages J. Immunol., July 1, 2000; 165(1): 353 - 363. [Abstract] [Full Text] [PDF]
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C. M. Bosio, D. Gardner, and K. L. Elkins Infection of B Cell-Deficient Mice with CDC 1551, a Clinical Isolate of Mycobacterium tuberculosis: Delay in Dissemination and Development of Lung Pathology J. Immunol., June 15, 2000; 164(12): 6417 - 6425. [Abstract] [Full Text] [PDF]
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A. O. Sousa, R. J. Mazzaccaro, R. G. Russell, F. K. Lee, O. C. Turner, S. Hong, L. Van Kaer, and B. R. Bloom Relative contributions of distinct MHC class I-dependent cell populations in protection to tuberculosis infection in mice PNAS, April 11, 2000; 97(8): 4204 - 4208. [Abstract] [Full Text] [PDF]
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N. V. Serbina and J. L. Flynn Early Emergence of CD8+ T Cells Primed for Production of Type 1 Cytokines in the Lungs of Mycobacterium tuberculosis-Infected Mice Infect. Immun., August 1, 1999; 67(8): 3980 - 3988. [Abstract] [Full Text] [PDF]
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C. Manca, L. Tsenova, A. Bergtold, S. Freeman, M. Tovey, J. M. Musser, C. E. Barry III, V. H. Freedman, and G. Kaplan Virulence of a Mycobacterium tuberculosis clinical isolate in mice is determined by failure to induce Th1 type immunity and is associated with induction of IFN-alpha /beta PNAS, May 8, 2001; 98(10): 5752 - 5757. [Abstract] [Full Text] [PDF]
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),
CD4-/- (•), and wild-type littermate (
) mice were
infected i.v. with 5 x 105 viable M.
tuberculosis bacilli (strain Erdman). Mean survival times were
39 ± 1 days for MHC class II-/- mice, 60 ± 7
days for CD4-/- mice, and >120 days for wild-type mice.
At least eight mice per group were used. This is a representative
experiment of three performed.
) and wild-type (
), MHC class
II-/- (•), and CD4-/- (