| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
production by lung CD8+ T cells in
CD4-/- and wild-type mice was also comparable, suggesting
that emergence of IFN--producing mycobacteria-specific
CD8+ T cells in the lungs was independent of
CD4+ T cell help. In contrast, cytotoxic activity of
CD8+ T cells from lungs of M.
tuberculosis-infected mice was impaired in CD4-/-
mice. Expression of mRNA for IL-2 and IL-15, cytokines critical for the
development of cytotoxic effector cells, was diminished in the lungs of
M. tuberculosis-infected CD4-/- mice. As
tuberculosis is frequently associated with HIV infection and a
subsequent loss of CD4+ T cells, understanding the
interaction between CD4+ and CD8+ T cell
subsets during the immune response to M. tuberculosis is
imperative for the design of successful vaccination
strategies. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
CD8+ T cells contribute to immune protection
against certain intracellular noncytoplasmic pathogens, but the
interaction between CD4+ and
CD8+ T cell subsets during bacterial infections
has not been investigated. Protective immunity against
Mycobacterium tuberculosis involves
CD4+ and CD8+ T cells and
mice lacking either or both T cell subsets are more susceptible to
infection than the wild-type (WT) mice (15, 16, 17, 18, 19, 20).
Previously we reported that although IFN--secreting
CD8+ T cells emerge in the lungs of infected
CD4-/- mice, they are not sufficient for
protection (15). Chronically infected mice depleted of
CD4+ T cells were unable to prevent reactivation
of infection despite normal levels of IFN- and nitric oxide synthase
2 production, suggesting roles for CD4+ T cells
in addition to cytokine production and macrophage activation
(21). In the present study, we examined the effect of
CD4+ T cell deficiency on the development of
cytokine production and cytotoxic functions of
CD8+ T cells during acute tuberculosis. Our data
indicate that priming and amplification of mycobacteria-specific
CD8+ T cells occurred in the absence of
CD4+ T cells, as did migration of cytokine-secreting
CD8+ T cells to the lungs following M.
tuberculosis infection. However, development of cytotoxic
CD8+ effector cells in the lungs was
substantially diminished in the absence of CD4+ T
cells. These results suggest that CD4+ T cells
contribute to protective immunity not only by secretion of
antimicrobial cytokines such as IFN- but also by providing help for
maintaining optimal CD8+ T cell cytotoxic
responses.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Eight- to 10-wk-old C57BL/6 (WT; Charles River Breeding Laboratories, Rockland, MA, or The Jackson Laboratory, Bar Harbor, ME), CD4-/-, and MHC class II-/- mice were used. MHC class II-/- (22) and CD4-/- (8) breeding pairs were originally obtained from Dr. D. Mathis and Dr. T. Mak, respectively, and then from The Jackson Laboratory. These 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.
Bacteria and infections
M. tuberculosis (Erdman strain; Trudeau Institute,
Saranac Lake, NY) was passaged 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 by aerosol with 
50–100 live
bacilli as determined by viable counts on 7H10 agar plates (Difco,
Detroit, MI); 107 CFU/ml were placed in a
nebulizer, and mice were exposed for 20 min, followed by 5 min of air
only, using a nose-only unit (Intox, Albuquerque NM) as previously
described (23). For the CTL assay in which cells were
cultured before use in lysis assays, mice were injected i.v. via tail
vein with 2 x 105 live bacilli in 100
µl.
Culture and infection of DCs
Dendritic cells (DCs) were grown from murine bone marrow
precursors and cultured for 5 days using methods described previously
(24). For DC infection, nonadherent cells were harvested,
adjusted to 0.5 x 106 cells/ml in DC medium
containing recombinant murine GM-CSF, and dispersed into
25-cm2 culture flasks (Costar, Cambridge, MA) for
infection. Cells were infected for 16–18 h at multiplicity of
infection of 3–5. Extracellular bacteria were separated from cells by
low-speed centrifugation and fresh medium was added to the cells. For
FACS analyses, infected and uninfected DCs were cultured in fresh
medium for an additional 24 h. The percentage of infection was
estimated by staining aliquots of cells by the Kinyoun method for
acid-fast bacteria (Difco). Routinely, 50% of DCs were
infected.
FACS analyses of cell surface markers
Lung and mediastinal lymph node cells were obtained from mice infected for various periods of time as described previously (24). 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), CD69 (anti-CD69 FITC Ab, clone H1.2F3), and CD25 (anti-CD25 PE Ab, clone PC61). DCs were stained for surface markers using Abs against MHC class II (anti-I-Ab FITC Ab, clone AF6-120.1), MHC class I (anti-H2Db PE Ab, clone KH95), ICAM-1 (anti-CD54 FITC Ab, clone 3E2), B7.1 (anti-CD80 FITC Ab, clone 16-10A1), and B7.2 (anti-CD86 FITC Ab, clone GL-1). All staining procedures were performed in PBS containing 20% mouse serum, 0.1% BSA, and 0.1% sodium azide (FACS buffer) for 30 min at 4°C. All Abs were used at 0.2 µg/106 cells and obtained from BD PharMingen (San Diego, CA). Cells were fixed with 4% paraformaldehyde for at least 1 h and analyzed by flow cytometry using CellQuest software (BD Immunocytometry Systems, San Jose, CA). Cells were gated on the lymphocyte or monocyte population by forward and side scatter.
Intracellular staining
Single cell suspensions of lungs at various times postinfection were prepared and staining for intracellular cytokines was performed as described previously (24). 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 (BD PharMingen) or left unstimulated for 5–6 h in the presence of 3 µM monensin (Sigma-Aldrich, St. Louis, MO). At the end of the stimulation period, cells were stained for CD4 and CD8, fixed, permeabilized, and stained for intracellular cytokines.
Measurement of BrdU incorporation in vivo
Mice infected with M. tuberculosis for 4 wk were administered 1 mg of bromodeoxyuridine (BrdU) in PBS i.p. (catalog no. B5002; Sigma-Aldrich) 24 h before harvest of lungs. Single cell suspensions of lung tissue were stained with FITC-anti-BrdU Ab or FITC-IgG isotype Ab (catalog no. 36634K; BD PharMingen) according to the manufacturer’s instructions. Briefly, cells were stained for CD4 and CD8 cell surface Ags, permeabilized in ice-cold 70% ethanol, washed, and incubated in denaturing solution (2 M HCl, 0.5% BSA) for 20 min at room temperature. Cells were washed and pellet was resuspended in 0.1 M Na2B4O7, pH 8.5. Cells were incubated for 2 min at room temperature, washed, and stained with FITC-anti-BrdU or FITC-IgG isotype Abs for 30 min. Cells were washed and fixed with 4% paraformaldehyde and analyzed by flow cytometry.
IFN- ELISA
DCs uninfected or infected (multiplicity of infection 4) for
24 h as described were plated in 96-well U-bottom plates (Corning
Glass, Corning, NY) at 104 cells/well in
DMEM supplemented with 10% certified FBS, 1 mM sodium pyruvate, 2 mM
L-glutamine, 25 mM HEPES (Life Technologies, Grand Island,
NY), 50 µM 2-ME (Sigma-Aldrich), 30 µg/ml gentamicin (Life
Technologies, Gaithersburg, MD), 15–20 U/ml murine IL-2 (Boehringer
Mannheim, Indianapolis, IN), and 1 mM aminoguanidine (Sigma-Aldrich).
Lung cells were harvested from mice infected for 4 wk via aerosol, and
single cell suspensions were obtained as described (24).
Macrophages were depleted by adherence on plastic petri dishes for
2 h at 37°C. Lung cells were added at 105
cells/well and cultured with APC for 3 days. Culture of cells in medium
alone served as a baseline. As a positive control, cells were
stimulated with Con A (Boehringer Mannheim) at 5 µg/ml. Supernatants
were harvested after 3 days of culture and IFN- production was
measured by sandwich ELISA using Abs R4-A62 and XMG1.2 (biotinylated)
(BD PharMingen), according to the manufacturer’s protocol. Recombinant
murine IFN- used to generate a standard curve was a gift from
Genentech (San Francisco, CA).
Culture of lung and lymph node cells
Lung and lymph node cells from mice uninfected or infected for 2–5 wk were obtained as described above and plated in 96-well U-bottom plates (Corning Glass) 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-Aldrich), 30 µg/ml gentamicin (Life Technologies), 15–20 U/ml murine IL-2 (Boehringer Mannheim), and 1 mM aminoguanidine (Sigma-Aldrich) 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 medium were removed from each well and replaced with fresh medium containing IL-2. Cells were cultured for an additional 3–4 days before FACS analyses and CTL assays.
Cytotoxicity assays
Lymphocytes harvested from 5- to 7-day stimulation cultures were tested in a 4-h 51Cr release assay as previously described (23). Macrophages were infected for 48 h or left uninfected, labeled with 100 µl of Na51CrO4 (Amersham, Arlington Heights, IL) and used as targets. Target cells were added to wells of 96-well U-bottom plates (Corning) 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 ratios in a total volume of 0.1 ml in DMEM supplemented with 10% 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. After 4 h, 85 µl of supernatant was removed from each well without disturbing the cells and counted in a gamma counter. Spontaneous release was determined by culturing target cells in medium alone, and total release was determined by adding 0.1% Triton X-100 to target cells. Percent of specific lysis was calculated by the formula: 100 x (experimental counts per minute - spontaneous counts per minute/total counts per minute - spontaneous counts per minute).
Ab-dependent redirected target lysis assay
Lung cells harvested from WT mice treated with
anti-CD4 Ab (GK1.5, 0.5 mg/injection) i.p. 6 days before CTL assay
or CD4-/- mice were directly used as effectors
in a 4-h 51Cr release assay.
FcR+ P815 (H-2d) murine
mastocytoma tumor cells were labeled with 100 µl of
Na51CrO4 (Amersham) for
1 h, washed extensively, and added to wells of 96-well U-bottom
plates (Corning) at 4 x 103 cells/well.
Lung cells were added at various E:T ratios and the assay was conducted
in the presence or absence of anti-TCR
mAb (clone H57-597,
used at 10-4 µg/ml; BD PharMingen) for 4
h. Percentage of specific lysis was calculated as described above.
Ribonuclease protection assay
Mice were infected via aerosol with 100 viable bacilli, and
lungs were harvested at various times postinfection and flash-frozen.
Total RNA was isolated from frozen tissues using TRIzol (Life
Technologies) according to the manufacturer’s instructions, with an
additional phenol-chloroform extraction after the TRIzol extraction.
Expression of cytokine genes was examined using the RiboQuant
ribonuclease protection assay system (BD PharMingen) using mCK-1
ribonuclease protection assay template set. Intensities of the bands on
the autoradiographs were quantitated by densitometry (Personal
Densitometer SI; Molecular Dynamics, Sunnyvale, CA). The results are
expressed as the ratio of band intensities of genes of interest and
housekeeping gene, GAPDH or L32.
Statistical analysis
Data were analyzed by comparison of WT and CD4-/- mice at each time point, with 3–4 mice per time point (see text and figures); unpaired t tests were used. Prism 2.0 (GraphPad, San Diego, CA) was used for statistical analysis. Values of p < 0.05 were considered significant, and p values are not shown for nonsignificant differences. Each experiment was repeated at least once to ensure reproducibility (see text and figures).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
To address the requirement for CD4+ T cell
help in priming mycobacteria-specific CD8+ T
cells in vitro, we used DCs from MHC class
II-/- mice; the absence of MHC class II blocks
their ability to specifically stimulate CD4+ T
cell responses. It is believed that CD4+ T cells
function to condition APCs via CD40-CD40L interactions for improved T
cell priming interactions (1, 2, 3). Substantial increases in
cell surface expression of MHC class I, B7.1, and B7.2 were observed
when MHC class II-/- DCs were infected with
M. tuberculosis for 64–66 h (Fig. 1
A), suggesting that, as
demonstrated previously by our group and others (25, 26, 27),
infection with M. tuberculosis was by itself sufficient to
induce phenotypic maturation of DCs in the absence of
CD4+ T cells. Expression of MHC class II
molecules on the surface of these DCs was not detectable (data not
shown).
|
98%
CD8+, data not shown) was tested for cytotoxic
activity against uninfected or M. tuberculosis-infected
macrophages. Primed CD8+ T cells lysed infected
targets, but not uninfected targets (Fig. 1
B). Therefore,
priming, expansion, and acquisition of cytotoxic function by
mycobacteria-specific CD8+ T cells can occur in
the absence of CD4+ T cells in vitro. However,
IL-2 was supplied to these in vitro cultures during priming, which
would bypass an important function of CD4+ T
cells.
To address in vivo priming of M. tuberculosis-specific
CD8+ T cells in a CD4+ T
cell-deficient environment, the activation profiles of
CD8+ T lymphocytes in the mediastinal lymph nodes
of aerogenically infected CD4-/- and WT mice
were compared. CD8+ T cells in the lymph nodes of
uninfected mice express very low levels of the activation markers CD69
and CD25. Following infection, the numbers of
CD69+ and
CD25+CD8+ T cells in the
lymph node increase (24). Numbers and activation profiles
of CD8+ T cells in the lymph nodes of WT and
CD4-/- mice were comparable after aerogenic
infection with M. tuberculosis (Fig. 2, A and B). The
patterns of CD69 and CD25 expression in the lymph nodes from WT and
CD4+ T cell-deficient mice were comparable (Fig. 2, A and B). During the first 2 wk of infection,
higher percentages of CD8+ T cells expressed CD69
and CD25 in the lymph nodes of the knockout mice. Throughout the course
of infection, lymph node CD8+ T cells were
CD44lowCD45RBhigh in both
mouse strains; the expression of these markers was similar in the lymph
nodes from infected and naive mice (data not shown).
|
B).
CD8+ T cells from the lymph nodes of MHC class
II-/- mice yielded similar results (data not
shown). There were 6- to 7-fold more CD8+ T cells
in the lymph nodes of 4-wk-infected WT and
CD4-/- mice as compared with naive mice,
suggesting an increased numbers of CTL precursors in the lymph nodes of
both groups of mice following infection. Our experimental conditions
did not allow differentiation between in vitro priming during the 5- to
7-day culture period and restimulation of cells already primed in vivo.
However, the data demonstrate that lymph nodes of CD4-deficient mice
contained CD8+ T cells fully capable of
developing into cytotoxic effector cells. Acquisition of activated phenotype by CD8+ T cells in the absence of CD4+ T cell help
CD4+ T cells may be important for optimal
activation of effector CD8+ T cells, as suggested
in certain viral experimental systems. We previously reported that
migration of CD8+ T cells into the lungs
following i.v. infection was not diminished in the absence of
CD4+ T cells; in fact, percentages of
CD8+ T cells in the lungs of
CD4+ T cell-deficient mice were 2-fold higher
than in the lungs of the WT mice during acute infection
(15). In accordance with our previous data, the influx of
lymphocytes into the lungs was observed in both WT and
CD4-/- mice by the third week postaerosol
challenge (Fig. 3A) and the
numbers of CD8+ T cells in the lungs of
CD4+ T cell-deficient mice were 2-fold higher
than in the lungs of control mice up to 6 wk postinfection (Fig. 3B). It was not clear whether the increase in
CD8+ T cells observed in the lungs of infected
mice was due to the continuous migration of newly primed
CD8+ T cells into the lungs or to the
proliferation of CD8+ T cells at this site.
Because CD8+ T cells produce significantly less
IL-2 than CD4+ T cells, the absence of
CD4+ T cells might negatively affect
proliferation of CD8+ effectors in the lungs. To
address this issue, 4-wk-infected WT and CD4-/-
mice were administered BrdU, and 24 h later the lungs were
harvested. Incorporation of BrdU into T cells was assessed by flow
cytometry. In the lungs of WT mice, 30–40% of both
CD4+ and CD8+ T cells were
BrdU positive (data not shown). Similar percentages of BrdU-positive
CD8+ T cells were observed in the lungs of WT and
CD4-/- mice (Fig. 3C), suggesting
that absence of CD4+ T cells did not affect the
proliferation of CD8+ T cells in M.
tuberculosis-infected mice.
|
A). The up-regulation of CD44
expression by CD8+ T cells was concomitant with a
decrease in CD45RB expression in both WT and
CD4-/- mice, suggesting an effector/memory
phenotype (Fig. 4A). In the lungs of both WT and
CD4-/- mice,
CD69+CD8+ T cells were
detected as early as 1 wk postinfection and continued to increase
throughout the course of infection (Fig. 4, B and
C). The activation profiles of CD8+ T
cells from MHC class II-/- mice were comparable
to those of CD8+ T cells from
CD4-/- and WT mice (data not shown). These data
indicate that absence of CD4+ T cells had no
obvious effect on recruitment of primed CD8+ T
cells to the lungs and subsequent CD8+ T cell
activation during infection.
|
Previously, we demonstrated that IFN- production in the lungs
of mice devoid of CD4+ T cells was reduced by
>50% as compared with that in WT mice during the first 2 wk after
i.v. infection (15), indicating that early IFN-
production is primarily by CD4+ T cells. We
further examined the effect of a CD4+ T cell
deficiency on the development of cytokine-secreting
CD8+ T cells during acute M.
tuberculosis infection. In the lungs of aerogenically infected
mice, cytokine-producing T cells emerged by 2 wk postinfection, and the
percentages of IFN--secreting cells reached a plateau by wk 4
postinfection (data not shown). Cytokine production by
CD8+ T cells from the lungs of WT and
CD4-/- mice at 4 and 6 wk postaerosol infection
was assessed by intracellular cytokine staining. In these experiments,
brief (5–6 h) stimulation of lung cells with anti-CD3 and
anti-CD28 Abs followed by intracellular cytokine staining allowed
us to examine the total cytokine producing potential of the T cells.
Stimulation with anti-CD3/anti-CD28 Ab induced IFN- and
TNF- secretion by CD8+ T cells from both WT
and CD4-/- mice, and the percentage of
CD8+ T cells primed for cytokine production was
similar in both WT and CD4-/- mice (Fig. 5, A and B).
|
by ELISA.
WT mice were depleted of CD4+ T cells 4 days
before the lung harvest by administration of GK1.5 Ab i.p. (resulting
in 95% depletion, data not shown) so that similar populations of lung
cells could be compared. Low levels of IFN- were produced when cells
were cultured with uninfected DCs or in the medium alone (Fig. 5C). In contrast, stimulation with infected DCs induced
robust IFN- secretion by CD8+ T cells
comparable to that induced by Con A (Fig. 5D), indicating
that lungs of both WT and CD4-/- mice contained
M. tuberculosis-specific cytokine-producing
CD8+ T cells. Specific IFN- production was
also assessed by intracellular cytokine staining after short-term (12
h) culture of lung CD8+ T cells and infected DCs;
under these conditions, similar percentages of
CD8+ T cells from WT and
CD4-/- mice produced IFN- (data not
shown). We also examined ex vivo cytokine secretion by
CD8+ T cells in the absence of exogenous TCR
stimulation, a measure of T cells secreting cytokines in vivo at the
site of infection immediately before harvest. We observed that equal
percentages of unstimulated CD8+ T cells from
lungs of CD4-/- or WT mice secreted IFN-
(Fig. 5D). Similar data were obtained using MHC class
II-/- mice (data not shown). Previously we have
demonstrated that under conditions described above, IFN- cannot be
detected in lung T cells from uninfected mice (24),
suggesting that levels of nonspecific staining are minimal in our
assays. These data indicate that development of cytokine-producing
CD8+ T cells during M. tuberculosis
infection does not require CD4+ T cells. Cytotoxic activity of lung CD8+ T cells in the absence of CD4+ T cell help
The progressive loss of cytotoxic function of
CD8+ T cells during viral infections in the
absence of CD4+ T cells has been described.
Previously, we demonstrated that CD8+ T cells
from the lungs of M. tuberculosis-infected mice expressed
perforin in vivo and functioned as CTL after in vitro restimulation
(23). The in vitro cytotoxicity mediated by cultured
CD8+ T cells from lungs of infected mice depended
almost exclusively on perforin (23). We investigated
whether the absence of CD4+ T cells affected the
cytotoxic potential of mycobacteria-specific CD8+
T cells in the infected lungs. Lung cells harvested from either WT or
CD4-/- mice infected for 3–6 wk were cultured
with M. tuberculosis-infected MHC class
II-/- DCs for 5–7 days in the presence of
IL-2. At the end of the culture period, the cytotoxic activity of the
CD8-enriched populations was tested in a 4-h 51Cr
release assay using M. tuberculosis-infected macrophages as
targets. In several independent experiments, we observed substantial
reductions in the specific lysis mediated by CTLs from the lungs of
CD4-/- and MHC class
II-/- mice (Fig. 6A and data not shown),
suggesting that the cytotoxic activity of M.
tuberculosis-specific CTL was compromised in the absence of
CD4+ T cells.
|
60% CD8+ T cells and 20%
CD4-CD8- cells, and
contained no CD4+ T cells (data not shown). The
mean fluorescence intensity of TCR staining in the lungs
of WT and CD4-/- mice was 106 and 90,
respectively (data not shown). The cytotoxic activity of freshly
harvested lung cells from both WT and knockout mice was tested in the
4-h 51Cr release assay against
FcR+ P815 target cells in the presence or absence
of anti-TCR Ab. The Ab binding to the T cells triggers the
release of cytotoxic granules, and the Fc portion of the Ab binds to
the FcR on the target cells, bringing the T cells into close proximity
with the P815 cells. Only those cells primed to be cytotoxic will lyse
the targets in this assay. As shown in Fig. 6B, lung cells
from WT mice readily lysed target cells in the presence of anti-TCR
Ab. That no more than 30% of specific lysis was achieved in our assays
was not surprising, because only a subset of lymphocytes in the lungs
of M. tuberculosis-infected mice express perforin detectable
by immunohistochemistry in vivo (23). In contrast, T cells
from the lungs of CD4-/- mice failed to lyse
target cells in the presence of redirecting Ab (Fig. 6B),
confirming that the cytotoxic activity of CD8+ T
cells was compromised in the absence of CD4+ T
cells. In a separate ongoing study, we also observe that the frequency
of cytotoxic precursors (as measured by limiting dilution assay) in the
lungs of CD4-/- mice is reduced severalfold as
compared with that of WT CD8+ T cells (data not
shown). Production of IL-2 and IL-15 in the lungs of CD4-deficient mice
The above results suggested a defect in the development of
cytotoxic effectors in CD4-/- mice. IL-2 and
IL-15 are involved in development of cytotoxic functions of
CD8+ T cells, NK cells, and lymphokine-activated
killer cells (28, 29, 30, 31, 32). As
CD4+ T cells are the major source of IL-2, the
cytotoxic activity of CD8+ T cells might
crucially depend on the production of IL-2 by this cell subset. In
addition, CD4+ T cells may increase IL-15 secretion by
macrophages and DCs. WT and CD4-/- mice were
infected via aerosol, lungs were harvested 0, 3, and 4 wk
postinfection, and cytokine expression was measured using RNase
protection assays. Cytokine expression in the lungs of uninfected mice
was virtually undetectable (Fig. 7). At 3
and 4 wk postinfection, IL-2 levels were 2- and 4-fold lower,
respectively, in CD4-/- mice compared with WT
mice (Fig. 7A). IL-15 mRNA levels also were reduced in the
CD4-/- lungs at 3 and 4 wk postinfection,
compared with WT mice (Fig. 7B). In a separate experiment,
IL-2 and IL-15 were 6- and 2-fold lower, respectively, in
CD4-/- mice compared with WT mice at 2 wk
postinfection, with differences at 4 wk similar to the previous
experiment (data not shown); in that experiment, a 3-wk time point was
not obtained. We also observed that levels of IL-10 were higher in the
WT mice (Fig. 7C). One possible explanation for this
observation is that CD4+ T cells produce IL-10
during the infection. However, we have not been able to detect IL-10
production by CD4+ T cells stimulated ex vivo
(our unpublished data). Alternatively,
CD4+ T cell interaction with macrophages might
lead to the production of IL-10 by macrophages.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and TNF- (reviewed in Ref. 33). It is thought that the
major function of CD4+ T cells is production of
cytokines. Although the contribution of CD8+ T
cells to control of tuberculosis remains controversial (20, 33), these cells are capable of secreting cytokines (24, 34, 35, 36) as well as exerting cytotoxic activity (23, 37) during M. tuberculosis infection and following
rechallenge (38). Previously, we demonstrated that immune
responses during M. tuberculosis infection in mice lacking
CD4+ T cells are severely compromised despite the
increased numbers of IFN--producing CD8+ T
cells (15). Also, chronically infected mice depleted of
CD4+ T cells succumb rapidly to tuberculosis,
although overall IFN- production and inducible nitric oxide synthase
production by macrophages in the lungs remain unchanged
(21). These results suggested that IFN- production by
CD4+ T cells is not the only mechanism by which
these cells contribute to control of M. tuberculosis
infection. In some systems, the presence of regulatory CD4+ T cells is required for the optimal induction and maintenance of effector responses of CD8+ T cells (reviewed in Refs. 39 and 40). In this study, we examined the effects of CD4+ T cell deficiency on the development of cytotoxic and cytokine-producing effector functions of CD8+ T cells during M. tuberculosis infection. Priming and migration of CD8+ T cells to the lungs appeared normal in the absence of CD4+ T cells. However, the cytotoxic ability, but not cytokine production, of CD8+ T cells was significantly impaired in CD4-/- mice. A reduction in the levels of IL-2 and IL-15 gene expression in the lungs, cytokines essential for the development of cell-mediated cytotoxicity, may play a role in the deficient CTL activity. These results suggest that during M. tuberculosis infection an additional function of CD4+ T cells may be to optimize or maintain CD8+ T cell cytotoxic responses, possibly through regulation of the cytokine microenvironment.
In most viral systems studied, the mechanisms by which
CD4+ T cells regulate CD8+
T cell responses and function remain unclear. Development and
maturation of precursor CD8+ T cells into
cytotoxic and cytokine-producing effectors is a complex process
involving a number of regulatory steps. Activation of
CD8+ T cell effector functions via MHC class
I-TCR interactions depends on costimulation and the presence of a
variety of cytokines (41, 42). CD4+
T cells contribute to priming of CD8 T cells by "conditioning" the
APC via CD40-CD40L interaction for optimal presentation of Ag to
CD8+ T cells (1, 2, 3, 43). Indeed,
CD40L has been shown to be pivotal for control of some
(44, 45, 46) but not all (47) viral infections.
The CD4+ T cell-mediated "conditioning" is
thought to involve enhancement of costimulatory function of APCs
(48, 49) as well as induction of IL-12 secretion by APCs,
which induces IFN- production by T cells (49, 50, 51, 52, 53). In
this and previous studies (25, 27), we demonstrated that
infection with M. tuberculosis induced maturation of DCs in
the absence of T cells, apparently bypassing the need for interaction
of the DCs with T cells, and that infected DCs produce IL-12. DCs have
been reported to acquire CD8+ T cell priming
capacity after treatment with bacterial products such as LPS
(43). Mycobacterial lipopeptides were shown to induce
maturation of DCs via Toll-like receptor, suggesting at least one
mechanism by which activation of APCs can occur in the absence of
CD4+ T cell help (54). In a recent
study, DCs (CD11c+) isolated from murine lungs
matured and acquired Ag presenting ability upon ex vivo infection with
M. tuberculosis in the absence of T cells (55).
In accordance with the observed effect of M. tuberculosis
infection on the maturation of MHC class II-/-
DCs, the infected APCs primed naive CD8+ T cells
in vitro, suggesting that priming of precursor
CD8+ T cells to mycobacterial Ags can occur
independently of CD4+ T cells. The in vitro
primed CD8+ T cells recognized and lysed M.
tuberculosis-infected macrophages, supporting that the presence of
"conditioning" CD4+ T cells was not required
for generation of mycobacteria-specific CTLs. Our data are in agreement
with a report demonstrating that mice deficient in CD40L are fully
capable of mounting a protective immune response against M.
tuberculosis delivered i.v. (56). However,
CD8+ T cell function was not addressed in these
mice, and it remains to be determined whether
CD40L-/- mice are capable of controlling a
chronic M. tuberculosis infection.
The conditions of in vitro assays might not exactly reflect the in vivo environment in which priming of precursor CD8+ T cells occurs. For example, the APCs generated in vitro by culturing with recombinant cytokines might not be at the same stage of activation and maturation as APCs present in tissues in vivo. In addition, IL-2, which is primarily produced by CD4+ T cells, was added to the in vitro cultures but is reduced in vivo in the absence of CD4 T cells. Therefore, we examined the priming of CD8+ T cells in the lung-draining lymph nodes of M. tuberculosis-infected mice. Increased numbers of CD8+ T cells expressing CD69, an early activation marker, in the lymph nodes of both WT and CD4-/- mice were observed as early as 1 wk postinfection, suggesting that CD8+ T cell priming in the lymph nodes was independent of CD4+ T cells in vivo.
Several reports suggested that CD4+ T cells are
required not for priming but rather for survival and maintenance of
effector CD8+ T cells (reviewed in Ref.
40). In the absence of CD4+ T cell
help, CD8+ CTL were shown to be activated only
transiently, and these cells lost function during the chronic phase of
viral infection (14, 57). Exhaustion of
CD8+ T cell responses in chronic viral infection
was reported under conditions of CD4+ T cell
deficiency (10, 12), supporting the idea that absence of
CD4+ T cells may not only prevent the expansion
of CD8+ T cells but also induce their
elimination. In a gammaherpesvirus model, stimulation through CD40
reduced reactivation of a latent infection in
CD4+ T cell-deficient mice, although augmented
CD8+ T cell function could not be demonstrated
(58). In our experiments, infiltration of activated
(CD44highCD45low)
CD8+ T cells into the lungs was observed
throughout the course of infection in WT and CD4+
T cell-deficient mice. Substantial numbers of
CD8+ T cells also expressed CD69, suggesting
engagement of the T cells with APCs in the lungs; this effect was also
CD4+ T cell independent. The CD8+ T cells
proliferated equally in the presence or absence of CD4+ T
cells, although the BrdU assay performed does not allow one to
precisely conclude that the replication has occurred in the lungs. The
cells may have taken up BrdU at another site (e.g., the lymph node) and
trafficked to the lungs in the time between BrdU administration and
harvest of the lungs. Taken together, these results indicate that
CD4+ T cells are not required for migration,
expansion, and activation of CD8+ T cells in the
lymph nodes and lungs during M. tuberculosis infection.
Cytokine production by effector CD8+ T cells in
the lungs was also examined. We found that similar percentages of
CD8+ T cells in the lungs of both WT and
CD4-deficient mice secreted TNF- and IFN- in response to
anti-CD3/anti-CD28 Ab stimulation. Our data further indicated
that the observed cytokine secretion was at least partially due to the
activation of M. tuberculosis-specific cells.
CD8+ T cells also appeared to be actively
secreting IFN- in the lungs of both groups of mice, confirming that
priming of cytokine-producing effectors was not impaired in the absence
of helper cells.
Despite the highly activated phenotype of CD8+ T cells present in the lungs of CD4-deficient mice, they were impaired in their cytotoxic function. Cultured CD8+ T cells from lungs of mutant mice failed to lyse infected macrophages. It is possible that CD4+ T cells are required for in vitro culture of mycobacteria-specific CTLs. CD8+ T cells from lymph nodes of infected mice cultured under identical conditions had WT levels of lysis of infected macrophages, arguing that CD4 T cells are not necessary during the in vitro culture. However, it is not possible to distinguish in vitro priming of naive lymph node cells from restimulation of previously primed cells in the in vitro culture. The M. tuberculosis-specific CD8+ T cells from the lymph nodes may also not be cytotoxic following in vitro culture, and the lysis observed may be due to those CD8+ T cells primed in vitro, as we observed using uninfected mice. However, CD8+ T cells in the lungs of CD4-deficient mice do appear to be deficient in cytotoxic function. Because the antigenic repertoire of M. tuberculosis-specific CTLs is not known, it was not possible for us to test the cytotoxic activity of the full spectrum of Ag-specific CD8+ T cells directly ex vivo. To examine the cytotoxic potential of CD8+ T lymphocytes without in vitro cell culturing, we used a redirected target lysis assay. CD8+ T cells freshly harvested from lungs of infected CD4-/- mice failed to induce Ab-redirected target lysis. The failure of CD8+ T cells to exert cytotoxicity was not due to the general unresponsiveness of these cells, as they readily secreted cytokines upon TCR triggering. A similar situation was recently reported to occur during chronic LCMV infection in the absence of CD4+ T cells (59). In these studies, the presence of activated (CD69+CD44highCD62Llow) CD8+ T cells during infection was observed under conditions of CD4 T cell deficiency. Although these virus-specific CD8+ T cells expressed activation markers and proliferated in vivo, they were unable to exert effector functions. Recently, Moser et al. (60) described polyoma virus-specific CD8+ T cells that lacked cytotoxic activity despite intact perforin expression, suggesting that regulation of lytic activity in CD8+ T cells extends beyond the expression of perforin or granzyme molecules. Preliminary data from ongoing studies in our laboratory suggest that perforin levels are similar between CD8+ T cells from WT and CD4+ T cell-deficient mice (data not shown).
One possible explanation for the silencing of the effector responses of CD8+ T cells in the absence of CD4+ T cell help is the lack of appropriate costimulation by APCs. Our results indicate that absence of CD4+ T cells might not affect the quality of APC during priming of the immune response to M. tuberculosis. However, the effects of CD4+ T cells on APC function and stimulation of CD8+ T cell responses during ongoing infection in the lungs remain to be determined. In addition to "conditioning" the APCs during infection, CD4+ T cells likely function as a source of cytokines, such as IL-2. CD4+ T cell deficiency in mice is associated with lower IL-2 responses (8); this cytokine is involved in development of cytotoxic responses. We hypothesized that CD4+ T cells in the lymph nodes and lungs secrete IL-2 and possibly other cytokines that activate and maintain the cytotoxic function of CD8+ T cells. IL-2 gene expression was diminished in the lungs of CD4-deficient mice throughout the course of infection. Interestingly, the reduced IL-2 levels did not appear to affect the incorporation of BrdU by CD8+ T cells, suggesting that even low levels of this cytokine are sufficient to sustain proliferative responses in vivo. CD8+ T cells can make low levels of IL-2 under certain circumstances and may be responsible for the IL-2 expression in the absence of CD4+ T cells.
The levels of another CTL-activating cytokine, IL-15, were also reduced in the CD4-/- lungs as compared with the WT lungs. Although IL-15 production is attributed to nonlymphoid cells such as monocytes and DCs (61, 62), optimal IL-15 production might require interaction of these cells with CD4+ T cells, as was shown for IL-18 production by macrophages from tuberculosis patients (63). IL-15 has been shown to enhance and restore CD8+ T cell responses in Toxoplasma gondii-infected or immunized mice (31, 32). In addition, exogenous administration of IL-15 enhanced the ability of mice to control M. tuberculosis infection (64), although CD8+ T cell responses were not assessed. In our system, diminished CTL responses in the absence of CD4+ T cell help might be due to a deficient cytokine environment. This hypothesis is consistent with our observation that, in contrast to cytotoxic function, cytokine production by CD8+ T cells is unimpaired in CD4-deficient mice. Recently, HIV-specific clones were reported to be deficient in cytotoxic activity while still retaining the ability to secrete cytokines (65). Thus, the mechanism by which CD4+ T cells regulate CD8+ T cell function might be common to both viral and bacterial pathogens.
CD8+ T cell effector function in the absence of
CD4+ T cell help has been studied mostly in viral
systems, and the mechanisms underlying the dependence of
CD8+ T cells on CD4+ T cell
help are not completely clear. To our knowledge, this is the first
study that examines in detail the effect of CD4+
T cell deficiency on the effector functions of
CD8+ T cells during bacterial infection. In
summary, we have demonstrated that a CD4+ T cell
deficiency severely impaired the development of cytotoxic
CD8+ T cell responses during M.
tuberculosis infection while having no discernible effect on the
priming, migration, and activation of this cell subset in the lymph
nodes and lungs. In contrast to cytotoxic function, cytokine production
by CD8+ T cells was not obviously affected by the
absence of CD4+ T help, suggesting differential
regulation of the development of these effector functions. The lack of
CD4+ T cells resulted in diminished levels of
IL-2 and IL-15, which may contribute to reduced effector functions of
CD8+ T cells in the lungs. The absence of
CD4+ T cells in both acute and chronic
tuberculosis results in a fatal course of infection, even though there
are CD8+ T cells capable of producing high levels
of IFN-. This supports the hypothesis that
CD4+ T cells have important, nonredundant roles
in control of M. tuberculosis in addition to IFN-
production. The data presented in this work suggest that
CD4+ T cells provide help for the development of
cytotoxic CD8+ T cell populations and the
cytotoxicity exerted by effector CD8+ T cells
might be an important component of antimycobacterial immunity. These
findings are relevant with respect to the development or maintenance of
immune responses under conditions of immunodeficiency, such as HIV
infection, when CD4+ T cells are depleted. The
bacterial load and numbers of functional CD4+ T
cells may be important factors in determining whether successful
cytotoxic or cytokine-producing CD8+ T cells are
primed and maintained during a bacterial infection such as
tuberculosis. The effect of diminished CD4+ T
cells on the development or maintenance of a CD8+
T cell memory response in tuberculosis is also unclear. Because HIV
infection and the subsequent loss of CD4 T cells are major risk factors
for development of active tuberculosis, these questions are important
for vaccine and therapeutic approaches against tuberculosis.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Current address: Department of Medicine, Infectious Disease Service, Sloan-Kettering Institute, New York, NY 10021.
3 Address correspondence and reprint requests to Dr. JoAnne L. Flynn, Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, E1240 Biomedical Science Tower, Pittsburgh, PA 15261. E-mail address: joanne{at}pitt.edu
4 Abbreviations used in this paper: CD40L, CD40 ligand; LCMV, lymphocytic choriomeningitis virus; DC, dendritic cell; BrdU, bromodeoxyuridine; WT, wild type.
Received for publication June 19, 2001. Accepted for publication October 16, 2001.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, yet succumb to tuberculosis. J. Immunol. 162:5407. interferon. Infect. Immun. 66:830. and NOS2. J. Exp. Med. 192:347. interferon-secreting CD8+, as well as CD4+, T cells in lungs following aerosol infection with Mycobacterium tuberculosis. Infect. Immun. 67:3242.
T cells by a mechanism involving the - and c-chains of the IL-2 receptor. J. Invest. Dermatol. 105:837.[Medline]
This article has been cited by other articles:
|
|
 |
|
  D. J. Lee, P. A. Sieling, M. T. Ochoa, S. R. Krutzik, B. Guo, M. Hernandez, T. H. Rea, G. Cheng, M. Colonna, and R. L. Modlin LILRA2 Activation Inhibits Dendritic Cell Differentiation and Antigen Presentation to T Cells J. Immunol., December 15, 2007; 179(12): 8128 - 8136. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Andersson, A. Samarina, J. Fink, S. Rahman, and S. Grundstrom Impaired Expression of Perforin and Granulysin in CD8+ T Cells at the Site of Infection in Human Chronic Pulmonary Tuberculosis Infect. Immun., November 1, 2007; 75(11): 5210 - 5222. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. V. Guidry, R. L. Hunter Jr, and J. K. Actor Mycobacterial glycolipid trehalose 6,6'-dimycolate-induced hypersensitive granulomas: contribution of CD4+ lymphocytes Microbiology, October 1, 2007; 153(10): 3360 - 3369. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-S. Yu, J. W. Peacock, W. R. Jacobs Jr., R. Frothingham, N. L. Letvin, H.-X. Liao, and B. F. Haynes Recombinant Mycobacterium bovis Bacillus Calmette-Guerin Elicits Human Immunodeficiency Virus Type 1 Envelope-Specific T Lymphocytes at Mucosal Sites Clin. Vaccine Immunol., July 1, 2007; 14(7): 886 - 893. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Ngai, S. McCormick, C. Small, X. Zhang, A. Zganiacz, N. Aoki, and Z. Xing Gamma Interferon Responses of CD4 and CD8 T-Cell Subsets Are Quantitatively Different and Independent of Each Other during Pulmonary Mycobacterium bovis BCG Infection Infect. Immun., May 1, 2007; 75(5): 2244 - 2252. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A.-H. Hovav, M. J. Cayabyab, M. W. Panas, S. Santra, J. Greenland, R. Geiben, B. F. Haynes, W. R. Jacobs Jr., and N. L. Letvin Rapid Memory CD8+ T-Lymphocyte Induction through Priming with Recombinant Mycobacterium smegmatis J. Virol., January 1, 2007; 81(1): 74 - 83. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Kamath, J. S.M. Woodworth, and S. M. Behar Antigen-Specific CD8+ T Cells and the Development of Central Memory during Mycobacterium tuberculosis Infection J. Immunol., November 1, 2006; 177(9): 6361 - 6369. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. A. Murray, N. Mansoor, R. Harbacheuski, J. Soler, V. Davids, A. Soares, A. Hawkridge, G. D. Hussey, H. Maecker, G. Kaplan, et al. Bacillus Calmette Guerin Vaccination of Human Newborns Induces a Specific, Functional CD8+ T Cell Response J. Immunol., October 15, 2006; 177(8): 5647 - 5651. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Lutjen, S. Soltek, S. Virna, M. Deckert, and D. Schluter Organ- and Disease-Stage-Specific Regulation of Toxoplasma gondii-Specific CD8-T-Cell Responses by CD4 T Cells. Infect. Immun., October 1, 2006; 74(10): 5790 - 5801. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Spies, V. Eggers, G. Szabo, A. Lau, V. von Dossow, H. Schoenfeld, H. Althoff, K. Hegenscheid, B. Bohm, T. Schroeder, et al. Intervention at the Level of the Neuroendocrine-Immune Axis and Postoperative Pneumonia Rate in Long-term Alcoholics Am. J. Respir. Crit. Care Med., August 15, 2006; 174(4): 408 - 414. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. L. Lin, S. Pawar, A. Myers, A. Pegu, C. Fuhrman, T. A. Reinhart, S. V. Capuano, E. Klein, and J. L. Flynn Early Events in Mycobacterium tuberculosis Infection in Cynomolgus Macaques Infect. Immun., July 1, 2006; 74(7): 3790 - 3803. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. D'Souza, M. Romano, J. Korf, X.-M. Wang, P.-Y. Adnet, and K. Huygen Partial Reconstitution of the CD4+-T-Cell Compartment in CD4 Gene Knockout Mice Restores Responses to Tuberculosis DNA Vaccines. Infect. Immun., May 1, 2006; 74(5): 2751 - 2759. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Sud, C. Bigbee, J. L. Flynn, and D. E. Kirschner Contribution of CD8+ T Cells to Control of Mycobacterium tuberculosis Infection J. Immunol., April 1, 2006; 176(7): 4296 - 4314. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Carranza, E. Juarez, M. Torres, J. J. Ellner, E. Sada, and S. K. Schwander Mycobacterium tuberculosis Growth Control by Lung Macrophages and CD8 Cells from Patient Contacts Am. J. Respir. Crit. Care Med., January 15, 2006; 173(2): 238 - 245. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. M. Sullivan, O. Jobe, V. Lazarevic, K. Vasquez, R. Bronson, L. H. Glimcher, and I. Kramnik Increased Susceptibility of Mice Lacking T-bet to Infection with Mycobacterium tuberculosis Correlates with Increased IL-10 and Decreased IFN-{gamma} Production J. Immunol., October 1, 2005; 175(7): 4593 - 4602. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. M. Lindell, T. A. Moore, R. A. McDonald, G. B. Toews, and G. B. Huffnagle Generation of Antifungal Effector CD8+ T Cells in the Absence of CD4+ T Cells during Cryptococcus neoformans Infection J. Immunol., June 15, 2005; 174(12): 7920 - 7928. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Santosuosso, X. Zhang, S. McCormick, J. Wang, M. Hitt, and Z. Xing Mechanisms of Mucosal and Parenteral Tuberculosis Vaccinations: Adenoviral-Based Mucosal Immunization Preferentially Elicits Sustained Accumulation of Immune Protective CD4 and CD8 T Cells within the Airway Lumen J. Immunol., June 15, 2005; 174(12): 7986 - 7994. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Wang, M. Santosuosso, P. Ngai, A. Zganiacz, and Z. Xing Activation of CD8 T Cells by Mycobacterial Vaccination Protects against Pulmonary Tuberculosis in the Absence of CD4 T Cells J. Immunol., October 1, 2004; 173(7): 4590 - 4597. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. L. Pearce, D. J. Shedlock, and H. Shen Functional Characterization of MHC Class II-Restricted CD8+CD4- and CD8-CD4- T Cell Responses to Infection in CD4-/- Mice J. Immunol., August 15, 2004; 173(4): 2494 - 2499. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. J. Gehring, K. M. Dobos, J. T. Belisle, C. V. Harding, and W. H. Boom Mycobacterium tuberculosis LprG (Rv1411c): A Novel TLR-2 Ligand That Inhibits Human Macrophage Class II MHC Antigen Processing J. Immunol., August 15, 2004; 173(4): 2660 - 2668. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. L. Marzo, V. Vezys, K. D. Klonowski, S.-J. Lee, G. Muralimohan, M. Moore, D. F. Tough, and L. Lefrancois Fully Functional Memory CD8 T Cells in the Absence of CD4 T Cells J. Immunol., July 15, 2004; 173(2): 969 - 975. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. C. Derrick, C. Repique, P. Snoy, A. L. Yang, and S. Morris Immunization with a DNA Vaccine Cocktail Protects Mice Lacking CD4 Cells against an Aerogenic Infection with Mycobacterium tuberculosis Infect. Immun., March 1, 2004; 72(3): 1685 - 1692. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Ranjbar, N. Ly, S. Thim, J.-M. Reynes, and A. E. Goldfeld Mycobacterium tuberculosis Recall Antigens Suppress HIV-1 Replication in Anergic Donor Cells via CD8+ T Cell Expansion and Increased IL-10 Levels J. Immunol., February 1, 2004; 172(3): 1953 - 1959. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. P. Junqueira-Kipnis, A. Kipnis, A. Jamieson, M. G. Juarrero, A. Diefenbach, D. H. Raulet, J. Turner, and I. M. Orme NK Cells Respond to Pulmonary Infection with Mycobacterium tuberculosis, but Play a Minimal Role in Protection J. Immunol., December 1, 2003; 171(11): 6039 - 6045. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Botha and B. Ryffel Reactivation of Latent Tuberculosis Infection in TNF-Deficient Mice J. Immunol., September 15, 2003; 171(6): 3110 - 3118. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. A. R. Tobian, N. S. Potter, L. Ramachandra, R. K. Pai, M. Convery, W. H. Boom, and C. V. Harding Alternate Class I MHC Antigen Processing Is Inhibited by Toll-Like Receptor Signaling Pathogen-Associated Molecular Patterns: Mycobacterium tuberculosis 19-kDa Lipoprotein, CpG DNA, and Lipopolysaccharide J. Immunol., August 1, 2003; 171(3): 1413 - 1422. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Al-Attiyah, F. A. Shaban, H. G. Wiker, F. Oftung, and A. S. Mustafa Synthetic Peptides Identify Promiscuous Human Th1 Cell Epitopes of the Secreted Mycobacterial Antigen MPB70 Infect. Immun., April 1, 2003; 71(4): 1953 - 1960. [Abstract] [Full Text]
|
|
|
|
|
|
D. J. Shedlock, J. K. Whitmire, J. Tan, A. S. MacDonald, R. Ahmed, and H. Shen Role of CD4 T Cell Help and Costimulation in CD8 T Cell Responses During Listeria monocytogenes Infection J. Immunol., February 15, 2003; 170(4): 2053 - 2063. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. R. Waters, T. E. Rahner, M. V. Palmer, D. Cheng, B. J. Nonnecke, and D. L. Whipple Expression of L-Selectin (CD62L), CD44, and CD25 on Activated Bovine T Cells Infect. Immun., January 1, 2003; 71(1): 317 - 326. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
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]
|
|
|
|
|
|
R. Dudani, Y. Chapdelaine, H. v. Faassen, D. K. Smith, H. Shen, L. Krishnan, and S. Sad Multiple Mechanisms Compensate to Enhance Tumor-Protective CD8+ T Cell Response in the Long-Term Despite Poor CD8+ T Cell Priming Initially: Comparison Between an Acute Versus a Chronic Intracellular Bacterium Expressing a Model Antigen J. Immunol., June 1, 2002; 168(11): 5737 - 5745. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) and macrophages infected for 42 h
with M. tuberculosis (•). Error bars represent
SE.
)
and CD4-/- (
) mice were infected with M.
tuberculosis bacilli via aerosol, and lung-draining lymph nodes
were harvested 0, 1, 2, 3, and 4 wk postinfection. Numbers of viable
cells in lymph nodes were counted by trypan blue exclusion. Cells were
stained for CD8 and CD69, gated on lymphocyte population by size, and
analyzed by flow cytometry. The total numbers of CD8+ T
cells were calculated based on percentages of the CD8+ and
CD8+CD69+ cells in the lymphocyte gate.
C, Lymph node cells from WT and CD4-/-
mice infected for 4 wk were cultured for 5 days with M.
tuberculosis-infected MHC class II-/- DC and used
in a 4-h 51Cr release cytotoxicity assay. Targets:
uninfected bone marrow-derived macrophages (
