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Infect Immun, January 1998, p. 98-106, Vol. 66, No. 1
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Processing of Mycobacterium tuberculosis
Bacilli by Human Monocytes for CD4+ 
and 
T
Cells: Role of Particulate Antigen
Kithiganahalli N.
Balaji and
W. Henry
Boom*
Department of Medicine, Case Western Reserve
University and University Hospitals of Cleveland, Cleveland, Ohio
44106-4984
Received 26 June 1997/Returned for modification 18 July
1997/Accepted 14 October 1997
 |
ABSTRACT |
Mycobacterium tuberculosis readily activates both
CD4+ and V
2+ 
T cells. Despite
similarity in function, these T-cell subsets differ in the antigens
they recognize and the manners in which these antigens are presented by
M. tuberculosis-infected monocytes. We investigated
mechanisms of antigen processing of M. tuberculosis antigens to human CD4 and T cells by monocytes. Initial uptake of M. tuberculosis bacilli and subsequent processing were
required for efficient presentation not only to CD4 T cells but also to V2+ T cells. For T cells, recognition of
M. tuberculosis-infected monocytes was dependent on
V2+ T-cell-receptor expression. Recognition of M. tuberculosis antigens by CD4+ T cells was restricted
by the class II major histocompatibility complex molecule HLA-DR.
Processing of M. tuberculosis bacilli for
V2+ T cells was inhibitable by Brefeldin A,
whereas processing of soluble mycobacterial antigens for T cells
was not sensitive to Brefeldin A. Processing of M. tuberculosis bacilli for CD4+ T cells was unaffected
by Brefeldin A. Lysosomotropic agents such as chloroquine and ammonium
chloride did not affect the processing of M. tuberculosis
bacilli for CD4+ and T cells. In contrast, both
inhibitors blocked processing of soluble mycobacterial antigens for
CD4+ T cells. Chloroquine and ammonium chloride
insensitivity of processing of M. tuberculosis bacilli was
not dependent on the viability of the bacteria, since processing of
both formaldehyde-fixed dead bacteria and mycobacterial antigens
covalently coupled to latex beads was chloroquine insensitive. Thus,
the manner in which mycobacterial antigens were taken up by monocytes
(particulate versus soluble) influenced the antigen processing pathway
for CD4+ and T cells.
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INTRODUCTION |
Mycobacterium
tuberculosis, the etiologic agent of human tuberculosis, is spread
readily from person to person by inhalation of aerosolized mycobacteria
(8). A hallmark of M. tuberculosis infection is
the ability of most healthy individuals to control the infection by
mounting an acquired immune response, in which antigen-specific T cells
and mononuclear phagocytes arrest the growth of M. tuberculosis bacilli and maintain control over dormant bacilli
within granulomas (reviewed in reference 25). This
protective cellular immune response results in conversion of the
tuberculin skin test from negative to positive and probably in
increased resistance to reinfection with tubercle bacilli.
CD4+ 
-T-cell-receptor ( TCR)-bearing T cells
(CD4+ T cells) are readily activated by mycobacterial
antigens and have a dominant role in the protective immune response to
M. tuberculosis in humans (2, 34). These
CD4+ T cells not only secrete cytokines but also serve
directly as cytotoxic effector cells against M. tuberculosis-infected macrophages (6). In addition to
CD4+ T cells, M. tuberculosis antigens activate
other human T-cell subsets such as TCR+ T cells
( T cells) (15, 16, 18). V2+ and
V9+ T cells are particularly responsive to live
M. tuberculosis (15). A role for both and
CD4+ T cells in protective immunity to acute M. tuberculosis infection has been demonstrated in murine models
(20, 21, 26, 27). A recent study of humans suggests that
V9+ and V2+ T-cell numbers and
function are reduced in tuberculosis patients (23).
Functional comparisons of human CD4+ and T-cell
responses of healthy tuberculin-positive persons demonstrate that both
T-cell subsets have similar cytotoxic effector functions for M. tuberculosis-infected monocytes and produce large amounts of gamma
interferon (IFN-), with T cells being slightly more efficient
producers of IFN- than CD4+ T cells (37).
Despite similarities in function, these two T-cell subsets differ in
the mycobacterial antigens recognized by their TCRs and the manners in
which antigens are presented to them by M. tuberculosis-infected mononuclear phagocytes. CD4+ T
cells recognize a wide diversity of mycobacterial peptides in the
context of class II major histocompatibility complex (MHC) molecules,
which include secreted as well as somatic antigens (6, 13, 33,
37). In contrast, V9+ and V2+
T cells, the dominant TCR subsets activated by M. tuberculosis, recognize mycobacterial antigens in a
non-MHC-restricted manner and the repertoire of antigens includes small
phosphate-containing antigens such as TUBag's (5, 9, 19, 22, 29,
36).
Both blood monocytes and alveolar macrophages infected with M. tuberculosis are efficient antigen-presenting cells for
mycobacterial antigen-specific CD4+ and T cells
(1, 5). However, little is known about how M. tuberculosis-infected mononuclear phagocytes process antigens for
these two T-cell subsets. M. tuberculosis bacilli are taken up by mononuclear phagocytes through a variety of surface receptors, including complement receptor 4, mannose receptor, and complement receptor 3 (17, 31, 32). Within mononuclear phagocytes, the
mycobacteria reside within phagosomes and modulate the phagosome by
preventing fusion with acidic lysosomal compartments (7). Although the vacuolar membranes surrounding the phagosome acquire endosomal markers, the vesicular proton ATPase is actively excluded, resulting in an elevated pH of 6.3 to 6.5 compared to the normal lysosomal pH of 4.5 (7, 35). The elevated pH in the
phagosome does not appear to inhibit the ability of mycobacterial
antigens to be processed and presented to CD4+ and
V2+ T cells. This study was undertaken to gain
insight into the mechanisms used by monocytes infected with live
M. tuberculosis bacilli to process mycobacterial antigens
for presentation to both CD4+ and T cells.
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MATERIALS AND METHODS |
Chemical reagents and monoclonal antibodies.
Chloroquine,
ammonium chloride, cytochalasin D, and Brefeldin A were purchased from
Sigma (St. Louis, Mo.). Chloroquine and ammonium chloride were
dissolved in phosphate-buffered saline, and cytochalasin D and
Brefeldin A were dissolved in dimethyl sulfoxide. Phycoerythrin
(PE)-conjugated anti-interleukin 2 receptor alpha chain (IL-2R)
(CD25-PE; Becton Dickinson, San Jose, Calif.), fluorescein
isothiocyanate (FITC)-conjugated OKT-4 (CD4-FITC; Ortho Diagnostics,
Raritan, N.J.), and FITC-conjugated TCR-1 (-FITC; T Cell
Sciences, Cambridge, Mass.) were purchased and used according to the
manufacturers' instructions with FITC- and PE-conjugated isotypic
controls. Purified anti-human TCR-V2 antibody C448.15D was a gift
from Simon R. Carding, University of Pennsylvania, Philadelphia.
Purified L243 (anti-HLA-DR) was a gift from Cliff Harding, (Case
Western Reserve University, Cleveland, Ohio).
Bacteria and antigens.
M. tuberculosis H37Ra was
cultured in Middlebrook 7H9 with ADC enrichment, and frozen stocks were
prepared as described previously (5, 15). Bacterial counts
and viability were performed by light microscopy and by counting CFU on
7H10 medium. M. tuberculosis-H37Ra stocks were tested
periodically for viability and with an M. tuberculosis complex-specific DNA probe (AccuProbe; Gen-Probe, San Diego, Calif.) to
ensure purity of the M. tuberculosis stocks. Before use in T-cell assays, mycobacteria were washed three times in RPMI 1640 and
sonicated for 20 s to disrupt clumps. The viability was routinely more than 50%.
Formaldehyde-fixed M. tuberculosis was prepared by
suspending bacilli in 1 ml of RPMI 1640 (109/ml) containing
1.5% (vol/vol) formaldehyde as previously described (12).
In brief, mycobacteria were incubated at room temperature for 2 h
with constant mixing, pelleted, and washed three times. The cells were
resuspended in 1 ml of RPMI 1640 and kept at 4°C. Viability was less
than 1 CFU/ml.
Cytosolic antigens of
M. tuberculosis H37Ra were prepared as
previously described (
37). Cytosolic mycobacterial antigens
were coupled covalently to carboxylated latex beads by carbodiimide
linkage (Polysciences, Inc., Warrington, Pa.) as per the
manufacturer's
instructions. Briefly, 0.5 ml of a 2.5% suspension of
carboxylated
beads was washed with carbonate buffer followed by
phosphate buffer.
To the suspension of beads in phosphate buffer, an
equal volume
of 2% solution of carbodiimide was added, and the mixture
was
incubated for 4 h. Beads were washed with borate buffer,
before
500 µg of cytosolic mycobacterial antigens per ml was added,
and
incubated overnight on a rotary shaker. After incubation, beads
were pelleted and resuspended in 0.1 M ethanolamine and then
centrifuged
and blocked in bovine serum albumin before being stored in
phosphate-buffered
saline. Beads were tested with peripheral blood
mononuclear cells
(PBMC) from healthy tuberculin skin test-positive
donors to test
their ability to stimulate T-cell proliferation, with
equivalent
levels of proliferation to soluble mycobacterial antigens
being
observed at 10
6 to 10
7 beads/ml.
Isolation of PBMC and monocytes.
PBMC were isolated by
density centrifugation over sodium diatrizoate-Hypaque gradients, and
monocytes were obtained by adherence from PBMC as previously described
(37). Briefly, PBMC were incubated on plastic tissue culture
dishes precoated with pooled human serum, nonadherent cells were
removed, and plastic-adherent cells (
90% monocytes by Wright's,
peroxidase, and nonspecific esterase staining) were collected by
scraping the dishes with a plastic policeman. PBMC were isolated from
healthy tuberculin-positive persons (18 to 45 years old). They were
selected for consistency of resting T-cell expansion (20 to 50%
TCR+ T cells) after stimulation with live M. tuberculosis.
Generation of M. tuberculosis-specific
CD4+ and T-cell lines.
CD4+ and
T-cell lines specific for M. tuberculosis were
generated as described in previous studies (4, 6, 37). In brief, PBMC were stimulated with M. tuberculosis bacilli or
with soluble mycobacterial antigens for 7 to 10 days. Then,
CD4+ and T-cell subsets were enriched by negative
selection with magnetic beads coated with antibodies (Dynal, Great
Neck, N.Y.). For T-cell enrichment, cells were treated
simultaneously with anti-CD4- and anti-CD8-coated beads. For
CD4+ T-cell enrichment, cells were treated first with
TCR-1 and then with goat anti-mouse immunoglobulin G-coated beads
and anti-CD8-coated beads. Antibody-coated beads were used at a 10:1
bead-to-cell ratio, based on the estimated number of T cells from each
T-cell subset (CD4+, CD8+, and T cells)
present after 7 to 9 days of stimulation with live M. tuberculosis. Generally, one cycle of treatment was sufficient for
depletion of the T cells, although in some experiments two cycles were
performed. Purity of selected T-cell populations was assessed by
two-color cytometry.
CD4
+ and
T cell lines were maintained with biweekly
stimulation with mycobacterial antigens, irradiated PBMC, and
recombinant
IL-2. CD4
+ T cell lines were maintained on
autologous PBMC as APC, and generally
-T-cell lines were
stimulated with HLA-mismatched PBMC. Mycobacterial
antigen-specific
T-cell lines stimulated in vitro two to three
times and maintained for
8 weeks were considered short-term lines
and were derived from seven
donors (seven lines for CD4
+ and four lines for
T
cells). T-cell lines (
n = 4 for CD4 T
cells,
n = 2 for
T cells) maintained for more than 12 weeks
were considered long-term lines. Most experiments shown were
performed
with long-term T-cell lines; however, results were validated
with
both short-term and long-term lines. Purity of phenotype of T-cell
lines was monitored by flow cytometry.
Proliferation assays.
CD4 and T cells (2 × 104 to 2.5 × 104 per 200-µl well) were
cocultured with 5 × 104 monocytes per well as APC for
72 h in 96-well plates. Cells were pulsed with 1 µCi of
[3H]thymidine (ICN, Costa Mesa, Calif.) for 12 to 16 h before being harvested on glass fiber filters.
[3H]thymidine incorporation was measured by liquid
scintillation counting and expressed as counts per minute.
For antigen uptake and processing, monocytes were incubated with whole
M. tuberculosis (5:1 or 10:1 bacterium-to-cell ratio)
for
4 h. Then cells were washed and fixed with paraformaldehyde
as
previously described (
3). Briefly, cells were washed and
fixed with paraformaldehyde for 1 min, followed by neutralization
with
0.15 M glycine (pH 7.2) for 20 min at room temperature and
then washing
(four times). Cells were incubated in tissue culture
medium for 60 min
to remove residual paraformaldehyde before use
in proliferation assays.
Before the cells were pulsed with 1 µCi
of
[
3H]thymidine, 50 µl of supernatant was harvested to
measure the
levels of secreted IFN-
. The levels of IFN-
were
measured by
enzyme-linked immunosorbent assay (Endogen, Cambridge,
Mass.).
For cytochalasin D treatment, monocytes were pulsed with cytochalasin D
(10 µg/ml) for 30 min before addition of mycobacteria
(5:1
bacterium-to-cell ratio) and incubated for 2 h. Monocytes
then
were washed five times to remove unphagocytosed bacteria
and irradiated
before addition to the proliferation assay with
CD4 and
T cells.
For class II MHC blocking, irradiated monocytes were incubated with
anti-HLA-DR (L243) monoclonal antibody (MAb) (10 µg/ml)
for 120 min
on ice before addition of CD4 T cells in the proliferation
assay. For
anti-V
2 antibody blocking experiments,
T cells
were incubated
with C448.D15 (2 µg/ml) for 60 min on ice before
being added to
irradiated monocytes in the proliferation assay.
Cytotoxicity assay.
Monocytes were incubated for either 2 to
4 h or 10 to 12 h with live M. tuberculosis (5:1
or 10:1 bacterium-to-monocyte ratio), soluble mycobacterial antigens
(50 µg/ml), soluble antigens covalently coupled to latex beads (5:1
or 10:1 bead-to-monocyte ratio) or no antigen. After incubation,
monocytes were washed and labeled for 1 h at 37°C with 100 µCi
of 51Cr (New England Nuclear, Boston, Mass.) before being
used as targets in 4-h cytotoxicity assays with CD4+ and
T cells as previously described (37).
For inhibition of ongoing antigen processing, monocytes were
preincubated for 8 to 12 h with the antigens indicated in the
figures before addition of chloroquine (400 µM) or Brefeldin A
(5 µg/ml) according to published procedures (
3,
14,
30).
Antigen-pulsed monocytes were treated with inhibitors for 120
min, and
inhibitors were removed by washing. Monocytes were then
pulsed with
51Cr before being used as targets.
For inhibition of initial antigen uptake and processing, monocytes were
incubated with chloroquine or ammonium chloride (NH
4Cl,
10 and 50 mM) for 30 min before addition of
M. tuberculosis
bacilli,
soluble mycobacterial antigens, or latex beads. Antigen and
inhibitors
were coincubated for 120 min. After coincubation, monocytes
were
washed and labeled with
51Cr. The inhibitors did not
increase average spontaneous
51Cr release from the usual 15 to 20% for
M. tuberculosis-infected
monocytes.
Statistical analysis.
Statistical analysis was determined by
paired Student's t test, and a P of <0.05 was
considered significant.
| |
RESULTS |
Requirement for phagocytosis and processing of M. tuberculosis bacilli by monocytes for CD4+ and
T cells.
In earlier studies, we demonstrated that live M. tuberculosis bacilli readily activate peripheral blood T
cells from healthy tuberculin-positive persons. Activation of T cells
by live M. tuberculosis is not restricted to T cells
but also results in activation of CD4+ T cells. A
representative experiment is shown in Fig.
1, in which IL-2R (CD25) expression on
CD4+ and T cells was measured after stimulation of
PBMC with live M. tuberculosis bacilli. The activation of
these two T-cell subsets by M. tuberculosis antigens is
dependent on antigen-presenting cells, and blood monocytes are
efficient APC for both CD4+ and T cells. We have
used the activation of peripheral blood CD4+ and T
cells by M. tuberculosis bacilli as a means to derive M. tuberculosis antigen-specific CD4+ and
T-cell lines for the antigen-processing experiments described below.
Whereas TCR usage and mycobacterial antigen recognition by
CD4+ T cells are characterized by marked diversity,
T-cell activation by mycobacterial antigens is limited predominantly to
T cells expressing TCR consisting of V9 and V2 chains. As shown
in Fig. 2B, activation of T cells
was inhibited by MAb C448.15D, specific for the V2 chain of
TCR, indicating that activation of T cells by live-M.
tuberculosis-infected monocytes was dependent on V2 expression.
Recognition of M. tuberculosis by CD4 T cells was blocked by
anti-HLA-DR antibody (Fig. 2A); in contrast, recognition of M. tuberculosis by T cells is not restricted by MHC molecules (5).
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FIG. 1.
Upregulation of IL-2R (CD25) expression on peripheral
blood CD4+ and T cells by live M. tuberculosis. Shown are the results of an analysis by two-color
flow cytometry of CD25 expression on CD4+ and T
cells from peripheral blood T cells stimulated for 7 days with either
live M. tuberculosis (5 × 106 bacilli per
ml) (B and D) or no antigen (A and C). The y axis represents
PE fluorescence for CD25, and the x axis represents FITC
fluorescence for either CD4+ or T cells.
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FIG. 2.
(A) Restriction of CD4 T cells by class II MHC (HLA-DR)
expression on M. tuberculosis-infected monocytes. Monocytes
were pretreated with either L243 (10 µg/ml) or isotypic control MAb
(10 µg/ml) on ice for 120 min before the addition of CD4 T cells
(2.5 × 104/well) from a long-term T-cell line with
and without M. tuberculosis or soluble mycobacterial antigen
in a proliferation assay. The results are the means and standard
deviations of triplicate wells and are representative of four
experiments. (B) Dependence on V2 TCR expression by T cells
for activation by M. tuberculosis-infected monocytes.
T cells (2.5 × 104/well) were pretreated with either
C448.15D (15D) or isotypic control MAb (2 µg/ml) on ice for 60 min
before being added to irradiated heterologous monocytes (5 × 104/well) with and without M. tuberculosis
(5 × 106/ml) in a proliferation assay. The results
are the means and standard deviations of triplicate wells and are
representative of three experiments. Ag, antigen; MTB, M. tuberculosis.
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Next, we determined if phagocytosis and processing by monocytes of
M. tuberculosis bacilli were required for presentation
of
antigens to CD4
+ and
T-cell lines. Monocytes were
pretreated with cytochalasin
D, an inhibitor of phagocytosis by
inhibiting actin polymerization,
for 30 min before
M. tuberculosis bacilli were added. After 2
h of incubation,
infected monocytes were washed extensively to
remove nonphagocytosed
bacilli and cytochalasin D, before treated
monocytes were added as APC
to the T-cell proliferation assay.
As shown in Fig.
3, pretreatment of monocytes with
cytochalasin
D inhibited the proliferation of both CD4
+ and
T cells. Cytochalasin D pretreatment did not inhibit the
ability
of monocytes to present soluble mycobacterial antigens
to both T-cell
subsets, indicating that cytochalasin D treatment
did not affect APC
function of monocytes (41,921 cpm).
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FIG. 3.
Requirement for phagocytosis by monocytes of M. tuberculosis bacilli for activation of CD4+ and
T cells. Monocytes (MO) were incubated with M. tuberculosis
(MTB) in the presence (CCD-MO+MTB) or absence (MO+MTB) of cytochalasin
D (CCD) for 120 min, after which they were washed and irradiated before
coculture with CD4+ (A) and (B) T-cell lines in a
proliferation assay. Results are means and standard deviations of
triplicate wells and are representative of five experiments. Monocyte
targets were from the same donor as the CD4+ T-cell line,
and the T-cell line was derived from an unrelated donor.
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Fixation of monocytes with paraformaldehyde before exposure to
M. tuberculosis prevented processing and presentation of
mycobacterial
antigens to both CD4
+ and
T cells,
whereas monocytes fixed with paraformaldehyde
after infection with
M. tuberculosis were able to activate both
T-cell subsets
(Fig.
4). In addition, we measured
IFN-
release
in response to fixed APC by CD4 and
T-cell
lines. IFN-
results
were similar to proliferation results. Fixing
the monocytes before
pulsing them with bacteria did not stimulate CD4
and
T cells
to make detectable levels of IFN-
, while fixing
the monocytes
after infection with
M. tuberculosis induced
IFN-
production
by CD4 T cells (949 pg/ml) and
T cells (354 pg/ml). Consistent
with the results of the proliferation experiments,
IFN-
levels
in response to fixed APC were reduced 50% in comparison
with those
in response to unfixed APC.
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FIG. 4.
Requirement for uptake and processing of M. tuberculosis bacilli for presentation to both CD4+ and
T cells. Monocytes (MO) were fixed with paraformaldehyde either
before (MO FIXED) or after [(MO+MTB)FIXED] a 120-min incubation
with live M. tuberculosis (MTB). Fixed monocytes then were
added to a proliferation assay with CD4+ (A) and (B)
T cells. Results are means and standard deviations of triplicate wells
and are representative of four experiments.
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Inhibition by Brefeldin A of mycobacterial antigen processing for
but not CD4+ T cells.
The above-described
experiments demonstrated that initial uptake by phagocytosis and
antigen processing were required to present antigens from M. tuberculosis bacilli by monocytes to CD4+ and
T cells. To further characterize the antigen-processing pathways
of M. tuberculosis-infected monocytes, the effects of inhibitors of antigen processing were tested in cytotoxicity assays with CD4+ and T cells as cytotoxic T lymphocytes
(CTL). Processing of M. tuberculosis bacilli by monocytes
was inhibited by Brefeldin A for T cells. The results of two
representative experiments of five are shown in Fig.
5A and C. Processing for CD4+
T cells (Fig. 5B) was not inhibited by Brefeldin A in the CTL assay
(n = 4, P > 0.375). The same M. tuberculosis-infected monocytes were used in CTL assays with both
CD4+ and T cells (Fig. 5A and B), clearly
establishing the differential effects of Brefeldin A on processing of
M. tuberculosis bacilli for these two T-cell subsets
(inhibition for T cells was 53 to 60% in five experiments;
P < 0.025). Processing for T cells (and for
CD4+ T cells [data not shown]) of soluble cytosolic
antigens of M. tuberculosis by monocytes was not sensitive
to Brefeldin A treatment (5 µg/ml), in contrast to the findings with
intact bacilli (Fig. 5D) (n = 5, P > 0.4). In these experiments, monocytes were exposed first to either live
M. tuberculosis or soluble mycobacterial antigens and second
to treatment with Brefeldin A for 2 h and then washed extensively
before being tested in the CTL assay. No further antigens were added
during the CTL assay, indicating that the ligand(s) recognized by
T cells was stably expressed on the surfaces of antigen-pulsed
monocytes. Increasing the concentration of Brefeldin A to 20 µg/ml
did not change the differential effects on antigen processing for
and CD4+ T cells (data not shown). These
results indicated that in M. tuberculosis-infected
monocytes, Brefeldin A inhibited ongoing processing for T cells
of antigens originating from the bacilli but not for CD4+ T
cells.
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FIG. 5.
Differential effects of Brefeldin A on antigen
processing of M. tuberculosis bacilli for and
CD4+ T cells. Monocytes incubated for 12 h with either
M. tuberculosis (MTB) (A, B, and C) or cytosolic
mycobacterial antigens (Ag) (D) were treated with Brefeldin A (BFA) for
120 min before serving as targets in a CTL assay with either
T-cell lines (A, C, and D) or CD4+ T cell lines (B). In
experiment 1 (EXP 1), the same monocyte targets were used for both
CD4+ and T-cell lines, with the T-cell lines
being derived from an HLA-mismatched donor. Results are representative
of four experiments. E:T ratio, effector-to-target ratio.
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The V
9
+ and V
2
+ T-cell lines in
this study did respond to isopentenyl pyrophosphate (IPP; 25 µg/ml).
However, in contrast
to
M. tuberculosis bacilli or soluble
mycobacterial antigens,
IPP could not be stably pulsed onto monocytes
for use in CTL assays
but needed to be added and present continuously
during a 4-h CTL
assay (data not shown).
Inability of lysosomotropic agents to inhibit processing of
M. tuberculosis bacilli for CD4+ and T
cells.
Since M. tuberculosis bacilli reside largely
within the phagosomes of mononuclear phagocytes and are able to inhibit
phagosomal acidification, the effects of lysomotropic agents on antigen
processing of M. tuberculosis bacilli for and
CD4+ T cells were tested. As shown in Fig.
6A and C, treatment of M. tuberculosis-infected monocytes with chloroquine did not affect ongoing antigen processing for CD4+ T cells. In fact, in
three of six experiments chloroquine appeared to enhance processing of
M. tuberculosis, as was reflected in increased cytotoxicity
(Fig. 6A and 6C) (n = 6, P > 0.375).
Processing of M. tuberculosis bacilli for T cells was
not affected by chloroquine (data not shown).
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FIG. 6.
Effect of an inhibitor of lysosomal acidification on
antigen processing of M. tuberculosis bacilli by monocytes
for CD4+ T-cell lines. Monocytes incubated for 12 h
with either M. tuberculosis (MTB) (A and B) or cytosolic
antigens (Ag) (C and D) of M. tuberculosis were treated with
chloroquine (Chloro) for 120 min before serving as targets in a CTL
assay with CD4+ T cells. The long-term CD4+
T-cell line used in experiment 1 (EXP. 1) was generated against total
cytosolic proteins of M. tuberculosis, and the long-term
CD4+ T-cell line used in experiment 2 was generated against
purified 30-kDa (85B) antigen of M. tuberculosis. Results
are representative of six experiments. E:T ratio, effector-to-target
ratio.
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The lack of effect of chloroquine on
M. tuberculosis
processing was observed for concentrations ranging from 40 to 2,000 µM,
with 400 µM being used as the optimal concentration. The
insensitivity
to chloroquine was not dependent on the load of
M. tuberculosis bacilli when
M. tuberculosis-to-monocyte
ratios were varied from
1:1 to 30:1 (data not shown). Antigen
processing of soluble mycobacterial
antigens for CD4
+ T
cells was inhibited by chloroquine, serving as a control for
the effect
of chloroquine (Fig.
6B and D) (
n = 6,
P < 0.025).
The inability of chloroquine to inhibit
processing of live
M. tuberculosis for CD4
+ T
cells suggested that processing for class II MHC molecules
of
M. tuberculosis antigens from live bacilli differed from processing
of soluble mycobacterial antigen. However, in these experiments
chloroquine was added to monocytes already infected with
M. tuberculosis or pulsed with soluble antigens, and thus the effect
of chloroquine
was on ongoing antigen processing. Thus, we determined
the effect
of chloroquine treatment on monocytes as they initiate the
processing
of mycobacterial antigens for CD4
+ T cells. In
these experiments, monocytes were treated with chloroquine
during a 4-h
exposure to
M. tuberculosis bacilli. As shown in
the results
of a representative experiment in Fig.
7,
chloroquine
did not inhibit processing of antigens of live
M. tuberculosis bacilli (
n = 4,
P > 0.375) but did inhibit processing of soluble
antigens (data not shown).
To ensure that insensitivity of processing
of
M. tuberculosis bacilli for CD4
+ T cells and
T
cells was not restricted to chloroquine, additional
experiments with an
alternative lysomotropic agent, ammonium chloride
(NH
4Cl),
were performed. As shown in Fig.
8,
treatment of monocytes
with ammonium chloride at the time of initial
uptake did not inhibit
subsequent processing of
M. tuberculosis bacilli for CD4
+ T cells (Fig.
8A)
(
n = 3,
P > 0.375) but did inhibit
processing
of soluble antigen (Fig.
8B) (
n = 3,
P < 0.05), indicating that
processing of
M. tuberculosis bacilli was resistant to two different
lysomotropic
agents.
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FIG. 7.
Effect of chloroquine on the initial uptake and
processing of M. tuberculosis bacilli for CD4+ T
cells. Monocytes were preincubated with or without chloroquine (CHLORO)
for 30 min, and then M. tuberculosis bacilli (MTB) were
added for 120 min and chloroquine treatment was continued. Monocytes
were then tested in a CTL assay with a CD4+ T-cell line. In
this experiment, chloroquine treatment reduced soluble antigen (Ag)
presentation by monocytes by 40%. The long-term CD4+
T-cell line used in this assay is reactive to total cytosolic proteins
of M. tuberculosis. Results are representative of three
experiments. E:T ratio, effector-to-target ratio.
|
|
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|
FIG. 8.
Effect of ammonium chloride (NH4Cl) on the
initial uptake and processing of M. tuberculosis bacilli for
CD4+ T cells. Monocytes were preincubated with or without
ammonium chloride for 30 min, and then M. tuberculosis
bacilli (MTB) (A) or cytosolic mycobacterial antigens (CYTOSOL) (B)
were added for 120 min on initial uptake and processing of M. tuberculosis bacilli for CD4+ T cells and ammonium
chloride treatment was continued. Monocytes then were tested in a CTL
assay with a CD4+ T-cell line. Results are representative
of three experiments. Ag, antigen; E:T ratio, effector-to-target
ratio.
|
|
Chloroquine insensitivity of processing of particulate
mycobacterial antigens for CD4+ T cells.
To determine
if chloroquine insensitivity of antigen processing for CD4+
T cells was unique to live M. tuberculosis bacilli or due to the particulate nature of the antigen, experiments were performed with
cytosolic antigens of M. tuberculosis covalently linked to latex beads. As shown in Fig. 9A and C,
these particulate antigen preparations were also found to be
chloroquine insensitive (n = 3; P > 0.3), whereas processing of soluble cytosolic antigens for
CD4+ T cells remained inhibitable with chloroquine in the
same experiment (Fig. 9B) (P < 0.005). When
formaldehyde and dead M. tuberculosis bacilli were used,
again no inhibition by chloroquine was observed, whereas soluble
antigens were inhibited (data not shown). These findings suggested that
it is the particulate nature of mycobacterial antigens and not the
viability of the bacilli which determines the chloroquine insensitivity
of antigen processing for CD4+ T cells by human monocytes.
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[in a new window]
|
FIG. 9.
Chloroquine insensitivity of antigen processing of
cytosolic mycobacterial antigens coupled to latex microspheres for
CD4+ T cells. Monocytes were incubated with latex beads
coupled with cytosolic mycobacterial antigens (A and C) or with soluble
mycobacterial antigens (B) and then treated with chloroquine (Chloro)
before use in a CTL assay with a CD4+ T-cell line. Results
of two of four representative experiments are shown. Ag, antigen; E:T
ratio, effector-to-target ratio.
|
|
| |
DISCUSSION |
Monocytes infected with M. tuberculosis are efficient
antigen-presenting cells for mycobacterial antigen-specific
CD4+ and T cells from healthy tuberculin skin
test-positive persons (25). The results of our studies
indicate that there are a number of unique features to the mechanisms
used by human monocytes to process and present antigens originating
from M. tuberculosis-containing phagosomes to both of these
T-cells subsets. First, uptake of M. tuberculosis bacilli by
phagocytosis and subsequent processing were required for antigen
presentation to both CD4+ and T cells. Second, the
recognition of M. tuberculosis-infected monocytes by
T cells was dependent on V2+ TCR and by CD4+
T cells on class II MHC molecules. Processing of antigens for V2+ T cells was inhibitable by Brefeldin A. In
contrast, processing of M. tuberculosis for CD4+
T cells was unaffected by Brefeldin A. Third, lysomotropic agents such
as chloroquine and ammonium chloride did not affect the processing of
M. tuberculosis bacilli for CD4+ and T
cells. This was true both when inhibitors were added during initial
uptake and short-term processing of M. tuberculosis bacilli
and when lysomotropic agents were added to ongoing antigen processing.
In contrast, both inhibitors were able to block the processing of
soluble mycobacterial antigens for CD4+ T cells in both
situations. Fourth, the chloroquine and ammonium chloride
insensitivity of processing of M. tuberculosis bacilli was
not dependent on the viability of the bacteria, since both formaldehyde-fixed dead bacteria and mycobacterial antigens
covalently coupled to latex beads were chloroquine insensitive. Thus,
the particulate nature of mycobacterial antigens may have a role in determining insensitivity to agents.
In the last few years, increasing evidence suggests that M. tuberculosis-activated V2+ T cells are
predominantly activated by small phosphate-containing molecules. A
number of these antigens have been identified and include four TUBag's
isolated from M. tuberculosis, which are small (±500 Da)
phosphorylated molecules which also contain nucleotide (9).
Others have described IPP and related prenyl phosphates as possible
antigens for V2+ T cells (36). These
phosphate ligands are not secreted by mycobacteria belonging to the
M. tuberculosis group. Instead, they remain associated with
the bacterial cell mass (10). Studies with TUBag's and synthetic IPP determined that these small antigens could be presented by fixed APC without a requirement for processing (22, 24). However, these small antigens were not stably associated with APC since
antigen had to be present continuously during the assay and could not
be pulsed onto APC (10). These studies also determined that
T-cell-APC contact was required for activation of V2+
T cells.
In contrast, our studies with M. tuberculosis bacilli
indicated that antigens for V2+ T cells are
processed and remain stably associated on the surfaces of monocytes.
This conclusion was supported by three lines of evidence. First,
monocytes fixed before addition of M. tuberculosis bacilli
were unable to activate V2+ T cells whereas those
fixed after infection were able to present antigen. Second, monocytes
infected with M. tuberculosis or pulsed with soluble
mycobacterial antigens could be washed extensively and still present
antigen to V2+ T cells, indicating that antigens
for V2+ T cells remained stably associated with
monocyte surfaces. The -T-cell lines used in our study did react
to synthetic small phosphate-containing antigens such as IPP. However,
IPP was not stably associated on monocytes for recognition by T
cells and was readily removed by washing. In contrast, soluble
mycobacterial antigen and live M. tuberculosis bacilli were
stably pulsed onto monocytes and extensive washing did not remove
antigen for V2+ T cells. Third, Brefeldin A could
inhibit processing of M. tuberculosis antigens for
V2+ T cells. Brefeldin A inhibits transport from
the endoplasmic reticulum to the trans-Golgi network, thus
suggesting either that in M. tuberculosis-infected
monocytes, antigen(s) for T cells becomes associated with
molecules migrating from the endoplasmic reticulum to the
trans-Golgi network or that M. tuberculosis
antigens for T cells require transport through the endoplasmic
reticulum and trans-Golgi network to arrive on the surfaces
of monocytes. These results are consistent with a model in which small
phosphate antigens or epitopes of M. tuberculosis are
associated with a carrier molecule which requires processing and allows
phosphate antigens or epitopes to remain stably associated on the
surfaces of APC. Our observation that M. tuberculosis
bacilli contain a 10- to 14-kDa cytosolic antigen which stimulates
V2+ T cells is consistent with this model
(4). Further supporting evidence for a carrier molecule was
provided by the ability of cytosolic antigens covalently linked to
latex beads to stimulate V2+ T cells with
monocytes as APC (data not shown).
In contrast to the poorly understood processing of antigens for
T cells, the cellular pathways for processing of soluble antigens for
presentation by class II MHC molecules to CD4+ T cells are
much better characterized (reviewed in reference 11). Antigens are internalized by endocytosis or
phagocytosis and concentrated within endosomes. As endosomes mature and
fuse with lysosomes, proteases break down protein antigens into peptide fragments and the pH progressively decreases. Class II MHC molecules are present in late endosomal and early lysosomal compartments, where
antigen-derived peptides bind to class II MHC molecules. The processing
of particulate antigens (e.g., bacteria) is less-well-understood, and
the site where mycobacterial peptides are loaded on class II MHC
molecules in macrophages is unknown. After phagocytosis, M. tuberculosis bacilli generally remain within phagosomes and class
II MHC molecules can be found in these phagosomes (7). M. tuberculosis bacilli modulate the phagosome by preventing
fusion with acidic lysosomal compartments and excluding the vesicular proton ATPase, resulting in an elevated pH of 6.3 to 6.5 compared to
the normal lysosomal pH of 4.5 (35). Nevertheless, M. tuberculosis-infected monocytes readily process and present
antigens to CD4+ T cells.
Our finding that processing of M. tuberculosis bacilli is
resistant to lysomotropic agents is consistent with the possibility that mycobacterial antigens become associated with class II MHC molecules within phagosomes. Chloroquine-resistant processing of
M. tuberculosis, however, is not unique to live bacteria,
since processing of particulate mycobacterial antigens for
CD4+ T cells also is chloroquine resistant. Thus,
processing of particulate mycobacterial antigens by monocytes may not
depend on acidification of the phago-lysosomal compartment. Our studies
were performed with live M. tuberculosis, formaldehyde-fixed
bacteria, and cytosolic antigens of M. tuberculosis linked
to latex beads. These are complex antigen preparations and include not
only a large number of different proteins but also nonprotein
constituents such as lipoarabinomannan, phosphatidyl-myo-inositol
mannosides and other phospholipids, and complex carbohydrates, which
may influence antigen processing in endosomal compartments. Traffic of
lipoarabinomannan out of phagosomes has been demonstrated,
supporting the concept of dynamic interactions between M. tuberculosis phagosomes and endosomal compartments
(38). Chloroquine-resistant processing of particulate mycobacterial antigens may be unique to mycobacteria, since the original studies of inhibition of antigen processing for class II MHC
molecules by agents were performed with particulate heat-killed Listeria monocytogenes and murine peritoneal macrophages
(39). Alternatively, it is possible that human monocytes
differ from murine macrophages in how they process particulate antigens
for class II MHC molecules. Based on studies with human macrophages chronically infected with M. bovis BCG, it has been
suggested that mycobacteria can reside within a compartment outside the route of normal antigen processing for CD4+ T cells
(28). This result is consistent with our findings that particulate mycobacterial antigens may have unique antigen-processing requirements.
The antigen-processing mechanisms used by M. tuberculosis-infected mononuclear phagocytes are critical in the
recruitment of T-cell subsets and in the determination of the antigen
repertoire recognized by CD4+ and T cells. Our study
indicates that there are distinct pathways for the processing by human
monocytes of antigens emanating from M. tuberculosis-containing phagosomes for CD4+ and
T cells. In addition, the manner in which the antigen is present within
monocytes (particulate versus soluble) influences the
antigen-processing mechanisms. Further studies of the antigen processing of M. tuberculosis bacilli by macrophages is
necessary to understand the regulation of T-cell-subset activation in
the protective immune response to M. tuberculosis, an
understanding of which is necessary for the design of improved vaccines
and immunotherapies for tuberculosis.
| |
ACKNOWLEDGMENTS |
Special thanks go to Cliff Harding for many helpful suggestions
for experiments with inhibitors of antigen processing and for
critically reviewing the manuscript.
This work was supported by National Institutes of Health grant
AI-27243.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Division of
Infectious Diseases and Department of Medicine, Case Western Reserve
University School of Medicine and University Hospitals of Cleveland,
10900 Euclid Ave., Cleveland, OH 44106-4984. Phone: (216) 368-4847. Fax: (216) 368-2034. E-mail: whb{at}po.cwru.edu.
Editor: S. H. E. Kaufmann
| |
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Infect Immun, January 1998, p. 98-106, Vol. 66, No. 1
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Abstract
Introduction
