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ß TCR+ Cells by Mycobacterium tuberculosis Via an Alternate Class I MHC Antigen-Processing Pathway1
Departments of
*
Medicine and
Pathology, Case Western Reserve University and University Hospitals of Cleveland, Cleveland, OH 44106
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells, and the role of
CD8+ 
ß TCR+ T cells in this response is
poorly understood. Stimulation of T cells from healthy tuberculin skin
test-positive persons with live M. tuberculosis-H37Ra or
soluble M. tuberculosis Ags readily up-regulated
IL-2R (CD25) expression on CD8+ T cells. Purified
resting and activated CD8+ T cells produced IFN- and
proliferated to both M. tuberculosis bacilli and soluble
mycobacterial Ags with monocytes as APC. Precursor frequency of
mycobacterial Ag-specific CD8+ T cells by IFN-
enzyme-linked immunospot was 5–10-fold lower than the precursor
frequency of CD4+ T cells, and IFN- secretion by
CD8+ T cells was 50–100-fold lower. CD8+ T
cells secreted 
10-fold less IFN- per cell than CD4+ T
cells in response to mycobacterial Ags. CD8+ T cell
responses to M. tuberculosis bacilli were blocked by
anti-MHC class I antibody and required Ag processing. Processing of
M. tuberculosis bacilli by monocytes for presentation to
MHC class I-restricted CD8+ T cells was insensitive to
brefeldin A treatment, which blocks the conventional MHC class I
Ag-processing pathway. These results represent the first demonstration
that human cells can process pathogen Ags via an alternate
Ag-processing pathway for MHC class I and suggest a mechanism for
participation of IFN--secreting CD8+ T cells in the
human immune responses to M.
tuberculosis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß TCR+ (CD4+), cells,
both CD8+ ß TCR+ (CD8+) cells
and TCR+ T cells have a role in the cellular immune
response to mycobacteria.
The importance of CD4+ T cells is well defined in humans
and in animal models. CD4+ T cell deficiency induced by
HIV-1 infection is associated with increased susceptibility to M.
tuberculosis (3). In murine models, the importance of
CD4+ T cells was established in cell transfer experiments
and more recently in MHC class II-deficient animals (4). The function
of CD4+ T cells in response to M. tuberculosis
is both to secrete macrophage-activating cytokines such as IFN- and
to serve as cytotoxic effector cells (CTL) (5, 6, 7, 8).
The role of CD8+ T cells in the human immune response to M. tuberculosis remains poorly characterized. In murine models, CD8+ T cells from immune mice provide partial protection in irradiated T cell-deficient mice (9). ß2-Microglobulin gene-knockout mice, which lack MHC class I and functional CD8+ T cells, are more susceptible to M. bovis bacille Calmette Guérin and M. tuberculosis (4, 10). Functionally, murine CD8+ T cells proliferate to mycobacterial Ags and express CTL activity (11, 12). We and others have shown recently that mycobacterial Ags can activate human MHC class I-restricted CD8+ T cells in healthy tuberculin skin test-positive (PPD+)3 persons and that these CD8+ T cells can serve as cytotoxic T cells (13, 14). Others have isolated human CD8+ T cell clones that respond to mycobacterial Ags (12, 15). Little is known about the mycobacterial Ags recognized by CD8+ T cells and how these Ags are processed for MHC class I presentation by macrophages.
In general, MHC class I-restricted CD8+ T cells respond to microbial Ags that are present in the cytoplasm of APCs. These microbial Ags are processed via a conventional MHC class I Ag-processing pathway, in which cytoplasmic proteins are degraded into peptide fragments by proteasomes. These peptides then are transported by the transporter for Ag presentation into the endoplasmic reticulum (ER). Here, peptides bind to MHC class I molecules, and the resulting complexes are transported to the cell surface (16). This pathway is effectively blocked by brefeldin A, which inhibits anterograde ER-Golgi transport (17, 18).
M. tuberculosis bacilli are phagocytosed, remain within phagosomes, and do not enter the cytoplasm of macrophages (19, 20). How M. tuberculosis-derived peptides become loaded onto MHC class I molecules is unknown. In murine macrophages, Ags from phagosomal compartments can be presented to CD8+ T cells (16, 21, 22). There is also evidence that soluble Ags can be presented to CD8+ T cells (23, 24). This alternate mechanism of Ag processing for MHC class I does not require proteasomes or trafficking through the ER (16, 22, 25). This alternate MHC class I pathway has been demonstrated in several murine model Ag systems but has not been demonstrated in human APCs.
The current study was undertaken to further characterize the functional
responses of human CD8+ T cells to mycobacterial Ags and to
ascertain the Ag-processing pathways used by macrophages to present
M. tuberculosis Ags to human CD8+ T cells. We
have found that CD8+ T cells respond both to live M.
tuberculosis bacilli and to soluble mycobacterial Ags.
CD8+ T cells produced IFN-, albeit at much lower amounts
than CD4+ T cells. Furthermore, we demonstrate for the
first time evidence of an alternate MHC class I pathway in human APCs
for a microbial pathogen.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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M. tuberculosis bacilli (H37Ra; American Type Culture Collection (ATCC), Manassas, VA) were grown to log phase in Middlebrook 7H9 medium (Difco, Detroit, MI) with albumin, dextrose, and catalase enrichment (Difco); harvested; and frozen at -70°C as described (26). Bacterial counts and viability were determined by light microscopy and by counting CFUs on 7H10 medium. Before use, mycobacteria were washed once in RPMI 1640 (BioWhittaker, Walkersville, MD) and sonicated for 50 s to disrupt clumps. Late culture filtrate Ags of M. tuberculosis (CF) were prepared from M. tuberculosis bacilli (H37Ra) by growing bacilli in Proskauer Beck medium for 6 wk, as described (27). In brief, Ags from CF were precipitated in 60% ammonium sulfate, dialyzed against 0.1 M ammonium bicarbonate, lyophilized, reconstituted, and frozen at -70°C. Cytosolic Ags was prepared from M. tuberculosis bacilli (H37Ra) harvested after 4 wk of culture in 7H9 broth with albumin, dextrose, and catalase enrichment, as described (28). In brief, the mycobacterial pellet was sonicated on ice (30 min, three times), followed by passage through a French press (three times). Cytosolic Ags were harvested as supernatant after centrifugation of lysates for 2 h at 145,000 x g at 4°C. Cytosolic Ags were concentrated and stored at -70°C.
PBMC and monocyte preparation
Whole blood was obtained from healthy PPD+ volunteers. PBMC were isolated by density centrifugation over Ficoll/diatrizoate sodium (Ficoll-Paque, Pharmacia Biotech, Uppsala, Sweden). Monocytes were obtained by adherence purification on plastic plates (Falcon, Lincoln Park, NJ). Monocytes were washed extensively after 1 h of adherence and then scraped off with a cell scraper (Falcon). Monocytes were irradiated with 5000 rad by a 137Cs source in all experiments.
Expansion of M. tuberculosis-specific CD8+ T cells
For expansion of M. tuberculosis-specific T cells, PBMC (2 x 106 cells per 2-ml well) from healthy PPD+ persons were incubated in 24-well plates (Costar, Cambridge, MA) with live M. tuberculosis (H37Ra) (5 x 106 bacteria per ml). Culture medium consisted of RPMI 1640 supplemented with 10% pooled human serum, 20 mM HEPES, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. rIL-2 (20 U/ml; Chiron, Emeryville, CA) was added after 48–72 h of culture. After 10–14 days of culture, viable cells were harvested by density sedimentation over sodium diatrizoate/Hypaque gradients before isolating T cell subsets with Ab-coated beads as described below.
Isolation of CD8+ and CD4+ T cells
Starting cell populations were either resting primary PBMC or
PBMC stimulated in vitro with M. tuberculosis.
CD8+ T cells were purified with anti-CD8 magnetic beads
(Dynal, Oslo, Norway) by positive selection following the
manufacturer’s guidelines. Cells were incubated with beads at a 5:1
ratio for 30–60 min, nonbound cells were removed, and Detachabead
(Dynal) was applied to obtain CD8+ cells. This positively
selected CD8+ T cell population was then incubated with
TCR-1 (anti- Ab) as ascites at 1:200 dilution (TCR-1
was a gift of M. Brenner, Harvard University, Boston, MA). After
washing, cells were incubated with goat anti-mouse IgG-coated beads
(Dynal) and anti-CD4-conjugated beads (Dynal) to further purify by
negative selection the CD8+ T cell population.
CD4+ T cells were positively selected by anti-CD4
magnetic beads and detached by Detachabead. Further purification of
CD4+ T cells by negative selection was not necessary for
CD4+ T cells. Purity of the CD4+ and
CD8+ T cells was assessed by flow cytometry, as described
below.
Immunofluorescence analysis
Purity of CD8+ and CD4+ T cells was
assessed by two-color flow cytometry. Phycoerythrin-conjugated
anti-CD3 antibody was used with FITC-conjugated anti-CD4, CD8
(all from Becton Dickinson, San Jose, CA), anti-pan-TCR ß (T
Cell Diagnostics, Woburn, MA), or anti- Ab V
2 (T Cell
Diagnostics). Cells were analyzed by flow cytometry on a
FACScan (Becton Dickinson) using the LYSYSTMII software.
Primary and activated CD8+ T cells were 95–97%
CD3+, with <2% contamination by and
CD4+ T cells. CD4+ cells were at least 96%
CD3+ in all cases. T cells were confirmed to be ß
TCR+ (T Cell Diagnostics).
Phycoerythrin-conjugated anti-CD25 (Becton Dickinson) antibody was
used with FITC-conjugated anti-CD4, CD8, and Abs for CD25
expression on PBMC after 7 days of stimulation with M.
tuberculosis. Cells were gated on a two-parameter plot of 90° vs
forward-angle scatter. The gate for lymphocytes was set widely. The
position of the cutoff marker for positive and negative fluorescence
was set manually based on the distribution of cells stained with FITC-
and phycoerythrin-conjugated isotypic controls alone and was kept
constant for the experiment. The percentage reported for a given cell
surface marker represents the proportion of gated cells with a positive
signal less the percentage of cells staining positive with isotypic
control alone.
Proliferation assays
Purified CD8+ T cells (2.5–5 x 105/200 µl) were placed in a round-bottom 96-well plate with irradiated autologous monocytes (0.5–1 x 105). Cells were incubated with Ags for 5 days with primary CD8+ T cells and 3 days with in vitro-stimulated CD8+ T cells. [3H]Thymidine (1 µCi; ICN, Costa Mesa, CA) was added for overnight incubation. Plates were harvested onto a glass wool filter with a Filtermate-196 harvester (Packard, Meriden, CT). [3H]Thymidine incorporation was measured by liquid scintillation counting. In experiments with blocking Abs, monocytes were preincubated at 4°C with mAbs for 90 min before addition of T cells and Ags. Abs remained in the cultures during the entire 3–5-day assay. Anti-MHC class I Ab was W6/32 (ATCC) and was used at a 5 µg/ml final concentration and prepared by protein A-affinity chromatography of culture supernatants. Isotype control was IgG2a (Zymed, South San Francisco, CA) at 5 µg/ml final concentration.
IFN- production and enzyme-linked immunospot (ELISPOT)
CD8+ or CD4+ T cells (2 x
105/200 µl) were incubated with irradiated monocytes and
M. tuberculosis (1 x 106) or cytosol (10
µg/ml). Supernatants were harvested and assayed by standard capture
ELISA with IFN- matched-pair Abs M-700A and M-701B (Endogen,
Cambridge, MA).
For ELISPOT, resting or activated T cells were incubated overnight with
M. tuberculosis and monocytes. After overnight stimulation,
cells were washed and added in triplicate to anti-IFN- capture
Ab M-700A (Endogen)-coated nitrocellulose ELISPOT wells (T-SPOT Assay
Plates; Athersys, Cleveland, OH). After an overnight incubation, cells
were washed away from the ELISPOT plates with PBS/Tween 0.05%, and
biotin-anti-IFN- M-701B (Endogen) was added.
Streptavidin-horseradish peroxidase at 1:2000 dilution (Dako,
Carpinteria, CA) was added next, and IFN- spots were visualized
after the addition of 3-amino-9-ethylcarbazole peroxide
substrate. Spots were counted with a T-SPOT Image Analyzer (Athersys),
and triplicates were averaged for each dilution. Means of no-Ag
wells were subtracted for analysis and contained fewer than 10 IFN-
spots.
Immunoprecipitation of MHC class I
Monocytes (2.5 x 107) were incubated with or without brefeldin A (1 µg/ml; Sigma Chemical, St. Louis, MO) in leucine-deficient medium (Life Technologies, Grand Island, NY) with dialyzed FCS for 15 min, pulsed with [3H]leucine (230 µCi/ml, 221 Ci/mmol; ICN) for 90 min, washed, and chased for 30 min in leucine-containing medium. Cells were cooled to 4°C; incubated with purified W6/32 at 10 µg/ml for 20 min to ligate cell surface MHC class I molecules; washed extensively; and lysed with 2% Triton X-100 (Sigma Chemical), 0.2 mM PMSF (Sigma Chemical), 20 µg/ml leupeptin (Sigma Chemical), 2 µg/ml pepstatin A (Sigma Chemical), and 5 mM iodoactamide (Sigma Chemical). Triton-insoluble material was removed by centrifugation. W6/32-bound MHC class I molecules ("surface" MHC class I) were precipitated with 100 µl of protein-A Sepharose beads (Sigma Chemical). The remaining supernatant ("intracellular pool") was then incubated with W6/32 bound to protein A-coated Sepharose beads and precipitated. Precipitates were analyzed by SDS-PAGE (12% polyacrylamide) under reducing conditions. Gels were impregnated with EnHance (NEN, Boston, MA) and dried. Autoradiographs were prepared and analyzed by densitometry using Kodak 1D Image Analysis software (Rochester, NY).
Monocyte Ag presentation assay
Adherent monocytes were cultured with IFN- (100 U/ml; R&D
Systems, Minneapolis, MN) overnight in six-well plates (Falcon). Live
M. tuberculosis (20:1 ratio) was spun onto cells at 200
x g for 5 min. After a 4-h incubation at 37°C, monocytes
were harvested by scraping and washed in cold RPMI 1640. Monocytes were
then fixed in 0.5% parafomaldehyde at room temperature for 15 min,
washed, and incubated with 0.2 M lysine for 20 min at room temperature.
Cells were washed twice in RPMI 1640. For brefeldin A treatment, cells
were pretreated with brefeldin A at 1 µg/ml (Sigma Chemical) before
adding M. tuberculosis bacilli. Fixed monocytes (2–5
x 105 per well) were added to 0.5–1 x
105 in vitro-stimulated CD8+ T cells in
round-bottom 96-well plates. Recombinant IL-2 (20 U/ml) and
anti-CD28 Ab (clone 9.3 ascites at 1:2000 dilution; a gift of C.
King, Case Western Reserve University, Cleveland, OH) were added as
costimulators. After 3 days, supernatants were harvested and IFN-
was measured by ELISA.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In earlier studies, we demonstrated that live M.
tuberculosis bacilli and soluble mycobacterial Ag readily
activated peripheral CD4+ and + T cells
from healthy PPD+ persons (6). In addition, we have found
that alveolar T cells from PPD+ persons after expansion
with purified protein derivative of M. tuberculosis
and IL-2 contained CD8+ T cells with CTL activity against
mycobacterial Ag-pulsed macrophages (13). To further characterize human
CD8+ T cell responses to mycobacterial Ags, PBMC from 10
healthy PPD+ persons were stimulated with either soluble
cytosolic Ags or intact bacilli of M. tuberculosis. After 7
days, cells were analyzed by two-color flow cytometry for CD25
(IL-2R) expression on CD8+, CD4+, and V2
TCR+ T cells. As shown in Fig. 1
, stimulation of PBMC with both soluble
Ags and M. tuberculosis bacilli resulted in up-regulation of
CD25 expression on CD8+ T cells compared with unstimulated
CD8+ T cells. CD25 expression on CD8+ T cells
increased from 0.19% ± 0.45% (SD) with no Ag to 28.4% ± 14.7%
with live M. tuberculosis and 11.6% ± 6.4% with M.
tuberculosis cytosol (all p 
0.005). CD25
expression on CD8+ T cells in freshly isolated PBMC was
<4%. Stimulation of PBMC with M. tuberculosis was
associated with an increase in number of CD8+ T cells
compared with unstimulated cells (data not shown). Consistent with our
earlier findings, live M. tuberculosis bacilli were
particularly effective in activating V2+ T cells,
and soluble Ags in activating CD4+ T cells. Thus, both
M. tuberculosis cytosol and bacilli activated
CD8+ T cells, but to a lesser extent than CD4 and T
cells, when measured as a percentage of CD25-expressing T cells and as
total number of T cells.
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Experiments described in Fig. 1
did not distinguish between
bystander and direct activation of CD8+ T cells by
mycobacterial Ags, since the starting population consisted of whole
PBMC, which contained CD4+ T cells, a source of IL-2 and
helper activity for CD8+ T cells. To determine directly the
ability of CD8+ T cells to respond to mycobacterial Ags,
highly purified resting CD8+ T cells were isolated from
PBMC by positive selection with anti-CD8-coated magnetic beads,
followed by negative selection with anti-CD4 and anti-
Ab-coated beads. Purity of CD8+ cells was nearly 100%, and
94% were CD3+. Proliferative responses of resting
CD8+ T cells to mycobacterial Ags were measured with
irradiated monocytes as APC (Fig. 2A). The mean stimulation
index of four donors to M. tuberculosis was 11.6
(p < 0.01); to cytosolic Ags, 3.89
(p < 0.05); and to CF Ags, 2.64
(p < 0.1). Thus, resting CD8+ T
cells responded directly to M. tuberculosis bacilli and
cytosolic Ags with autologous monocytes as APC and with lesser
responses to CF Ags.
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+ T cells as
described above. Negative selection of T cells was particularly
important, since in some donors substantial contamination (up to 20%)
by CD8+ (bright) V2+ + T
cells was measured. After negative selection, purity of
CD8+CD4- TCR- T
cells was 95%.
As shown in Fig. 2B, purified M.
tuberculosis-activated CD8+ T cells proliferated to
both M. tuberculosis and cytosol with monocytes as APC. The
stimulation index was 15.2 to live M. tuberculosis
(p < 0.001) and 8.3 to M.
tuberculosis cytosol (p < 0.001).
M. tuberculosis-activated CD8+ T cells did not
respond to Candida Ags or hen egg lysozyme, confirming their
Ag specificity (data not shown). To assure that measured proliferation
was by CD8+ T cells and not by contamination by
CD4+ or T cells, purity of CD8+ T cells
was monitored by flow cytometry before and after stimulation with
M. tuberculosis. Purity of CD3+CD8+
T cells remained 95% CD3+CD8+.
To determine whether CD8+ T cell responses to mycobacterial
Ags were MHC class I restricted, autologous monocytes were incubated
with W6/32 (anti-MHC class I) and isotypic control mAbs for 90 min
at 4°C before adding CD8+ T cells. As shown in Fig. 3, W6/32 blocked proliferation of
activated CD8+ T cells to both live bacilli and soluble
M. tuberculosis cytosol Ag (p
0.01), demonstrating an MHC class I-restricted response.
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production by CD8+ T cells and comparison with
CD4+ T cells
In earlier studies, we demonstrated that CD8+ T cells
were cytolytic for M. tuberculosis-infected monocytes (13).
The other major effector function of T cells in the immune response to
M. tuberculosis is secretion of macrophage-activating
cytokines such as IFN-. In the next series of experiments, IFN-
responses of resting primary CD8+ T cells were measured and
compared with CD4+ T cells. As shown in a representative
experiment in Fig. 4A,
supernatants from mycobacterial Ag-activated resting CD8+ T
cells contained IFN-. Fig. 4B shows the difference in
IFN- production in this same donor between equal numbers of
CD8+ and CD4+ T cells in response to M.
tuberculosis. Table I demonstrates
that IFN- production by CD8+ T cells ranged from 187 to
2,179 pg/ml (n = 7) in response to live M.
tuberculosis. In response to cytosol (n = 4),
IFN- production ranged from 0 to 312 pg/ml (data not shown). When
CD8+ and CD4+ T cells were compared from the
same donors (n = 7), CD4+ T cells
consistently produced far greater amounts of IFN- in response to
M. tuberculosis than CD8+ T cells (2,954–93,600
pg/ml vs 187–2,179 pg/ml) (p 0.01). Mean
CD4+ T cell IFN- production was 78 ± 96
times greater than that of CD8+ T cells.
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between
CD4+ and CD8+ T cells were due to differences
in precursor frequencies of mycobacterial Ag-specific CD4 and CD8
cells, IFN- ELISPOT assays were performed. Purified resting primary
CD4+ and CD8+ T cells were stimulated overnight
with M. tuberculosis or medium and monocytes as APC and then
placed on ELISPOT filter plates coated with monoclonal IFN- capture
Ab for another 18 h. Spots were screened with an image analyzer
(T-SPOT image analyzer; Athersys), and the numbers were confirmed by
manual counting. Precursor frequency of IFN--producing
CD8+ T cells in response to M. tuberculosis
ranged from 1/845 to 1/3539. For CD4+ T cells, precursor
frequency of IFN--producing cells was 1/59 to 1/1705. The precursor
frequency of M. tuberculosis-responsive CD4+
cells was 7.8 ± 5.6 times higher than CD8+ T cells
(p 0.001). Based on differences in precursor
frequencies and secreted IFN- production, CD4+ T cells
were calculated to produce 11 times more IFN- per cell than
CD8+ T cells in response to M. tuberculosis.
This conclusion is also supported by the relative intensity of IFN-
ELISPOTS of CD4+ and CD8+ T cells. At a similar
spot number, CD4+ T cell IFN- spots (Fig. 5, left) were larger and more
intense than CD8+ T cell IFN- spots (Fig. 5, right). Activation of CD8+ T cells by M.
tuberculosis for 10 days resulted in a 3- to 43-fold increase in
precursor frequency of M. tuberculosis-responsive
CD8+ T cells, as shown in Table II.
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In Fig. 3, we demonstrated that CD8+ T cell responses
to M. tuberculosis bacilli were restricted by MHC class I
molecules. However, these studies did not determine whether Ag
processing was required. To exclude the possibility that small Ag
fragments present in the M. tuberculosis preparation were
presented without processing, experiments with fixed APCs were
performed. In preliminary experiments, we determined that proliferative
responses of CD8+ T cells were markedly reduced by 0.5%
paraformaldehyde fixation of APC. However, IFN- production was still
evident with fixed APC, and the addition of IL-2 and anti-CD28 Abs
as costimulators enhanced Ag-specific IFN- production by
CD8+ T cells. As shown in Fig. 6A, fixation of monocytes
after infection with M. tuberculosis bacilli resulted in
IFN- production by CD8+ T cells, whereas addition of
M. tuberculosis bacilli to fixed monocytes did not. Thus,
presentation of M. tuberculosis Ags to CD8+ T
cells required Ag processing and was not due to surface binding of free
peptides.
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(100 U/ml) overnight to optimize Ag
processing and presentation function and then infected with live
M. tuberculosis in the continued presence or absence of
brefeldin A (1 µg/ml). After a 4-h incubation, monocytes were fixed
with paraformaldehyde, washed, and then incubated with CD8+
T cells. As shown in Fig. 6B, brefeldin A did not inhibit
processing and presentation of M. tuberculosis Ags to
CD8+ T cells. To demonstrate directly that brefeldin A
inhibited surface expression of newly synthesized MHC class I,
immunoprecipitation of surface and intracellular MHC class I molecules
was performed. Fig. 6C demonstrates that brefeldin A reduced
newly synthesized MHC class I expression on monocytes by 94%. Thus, monocytes were able to process M. tuberculosis Ags for presentation by MHC class I in the presence of brefeldin A. Brefeldin A inhibited egress of newly synthesized MHC class I complexes from ER, an essential function for the conventional MHC class I Ag-processing pathway. These results indicate that human monocytes use an alternate pathway for processing of M. tuberculosis bacilli for presentation to MHC class I-restricted CD8+ T cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and respond to mycobacterial Ags.
Recognition of M. tuberculosis Ags on mononuclear phagocytes
by CD8+ T cells was dependent on presentation by MHC class
I molecules and required Ag processing. Chemical fixation of monocytes
before addition of M. tuberculosis bacilli did not activate
CD8+ T cells, whereas fixation of monocytes after infection
with M. tuberculosis resulted in Ag-specific IFN-
production. These studies excluded direct binding to surface MHC class
I by mycobacterial peptides present in or generated by M.
tuberculosis bacilli. This same preparation of M.
tuberculosis bacilli was used to determine whether processing of
bacilli for MHC class I in human monocytes required traffic through ER
and trans-Golgi complex. Brefeldin A resistance of Ag processing
of M. tuberculosis bacilli for CD8+ T cells
indicated use of an alternate processing pathway in human monocytes, in
which M. tuberculosis peptides are loaded onto MHC class I
molecules distal to the ER.
In general, MHC class I-restricted CD8+ T cells respond to microbial Ags that are present in the cytoplasm of APC. These microbial Ags are processed via the conventional MHC class I Ag-processing pathway, in which cytoplasmic proteins are degraded into peptide fragments by proteasomes. These peptides are transported by the transporter for Ag presentation into the ER, where peptides bind to MHC class I molecules, and peptide-MHC class I complexes are transported to the cell surface. This pathway is readily blocked by brefeldin A. M. tuberculosis bacilli, however, remain within phagosomes after phagocytosis and do not enter the cytoplasm of macrophages. In murine macrophages, Ags from the phagosomal compartment also can be presented to CD8+ T cells (16), and there is evidence that soluble Ags also can be presented to CD8+ T cells (23, 24). A number of studies support an alternate mechanism of Ag processing of these Ags for MHC class I that does not require trafficking of Ag through the ER (16, 22, 25). This alternate MHC class I pathway has never been demonstrated before in human APC. Mechanisms for MHC class I loading of mycobacterial peptides in M. tuberculosis-infected macrophages include direct loading in the phagosome or endosomes that communicate with the phagosome. Another possible mechanism involves peptide regurgitation, in which peptides are released from the cell and bind to MHC class I on the surface. Future experiments are necessary to further dissect the mechanism for processing of M. tuberculosis Ags for MHC class I presentation.
CD8+ T cells were activated both by live M.
tuberculosis bacilli and by soluble mycobacterial Ags.
CD8+ T cells recognized mycobacterial Ags in M.
tuberculosis cytosol more readily than Ags in CF. This pattern of
response to M. tuberculosis and soluble mycobacterial Ags
was similar to that observed for T cells. In contrast,
CD4+ T cells responded well to both cytosol and CF Ags.
CD8+ T cells produced IFN- in response to M.
tuberculosis-infected mononuclear phagocytes, but at much lower
efficiency than CD4+ T cells based on a comparison of
IFN- ELISPOT and supernatant IFN- measurements. Diminished
IFN- production also distinguishes M.
tuberculosis-specific CD8+ T cells from T cells
that produce more IFN- than CD4+ T cells.
Highly purified CD8+ ß TCR T cells were used for these
experiments. This was particularly important in light of the less
vigorous proliferative and IFN- responses of CD8+ T
cells compared with CD4+ and + T cells.
Besides confirmation of purity of the starting populations of resting
and activated CD8+ T cells, purity of CD8+ T
cells was checked by flow cytometry after stimulation with
mycobacterial Ags to assure that measured responses were from
CD8+ T cells and not due to expansion of small numbers of
contaminating CD4+ and/or T cells. In some donors,
before resorting to more rigorous purification methods,
V2+ T cells were found that expressed high levels
of CD8 (CD8-bright), which had expanded from positively selected
CD8+ T cells in response to M. tuberculosis.
Blocking of MHC class I inhibited proliferation and IFN- production
by CD8+ T cells, confirming that M.
tuberculosis-reactive CD8+ T cells were MHC class I
restricted and did not belong to the subset of CD1-restricted
CD8+ T cells.
The role of MHC class I-restricted CD8+ T cells in human
immune responses to M. tuberculosis and their relationship
to other T cell subsets activated by M. tuberculosis are not
well defined. CD8+ T cells are present in the affected lung
of tuberculosis patients, and memory M.
tuberculosis-specific CD8+ T cells are found in
alveolar spaces of healthy PPD+ persons (13, 29). Healthy
PPD+ persons are thought to be protected from exogenous
reinfection, suggesting that CD8+ T cells might have a role
in protective immunity to M. tuberculosis in humans. T cell
responses to M. tuberculosis are characterized by marked
diversity, with activation of multiple subsets including
CD4+, V2+, MHC class I-restricted
CD8+, and more recently CD1-restricted CD8+ T
cells (30, 31). The requirement for these different T cell populations
in the immune response to M. tuberculosis may be determined
by differences in function, Ag repertoire, or both. Evidence for major
differences in function is not strong. CD4+, , and
CD8+ T cells all serve as cytotoxic effector cells and
produce IFN-, albeit at differing amounts, in response to M.
tuberculosis. Furthermore, there is no evidence for a Th1-Th2-like
dichotomy among these T cell subsets in M. tuberculosis
infection.
Differences in IFN- production do not necessarily imply differences
in control of M. tuberculosis growth. The primary role of
IFN- may be to regulate Ag processing and presentation of
mycobacterial Ags to T cells rather than stimulate macrophage
activation for M. tuberculosis killing. IFN- is not a key
macrophage-activating cytokine for M. tuberculosis killing
in humans (32, 33). Macrophage activation that is dependent on T cell
contact may be as important for activation of mycobacterial killing as
the presence of soluble cytokines. Whether T cell contact-dependent
mechanisms are mediated through CTL activity or specific
receptor-ligand interactions is not known. ATP, signaling through
purinergic P2Z(P2Z7) receptors, may be a major mediator for
mycobacterial killing, whereas perforin- or FasL-mediated killing of
macrophages has minor effects on mycobacterial viability (34). Whether
CD8+ T cells deliver different cell contact-dependent
signals to macrophages for mycobacterial killing than CD4+
and T cells is unknown.
The requirement for multiple T cell subsets in the immune response to
M. tuberculosis is likely determined by the need to
recognize a broad repertoire of mycobacterial constituents.
CD8+ T cells differ from CD4+ and T
cells in the molecules they use to recognize mycobacterial Ags and the
mechanisms used to process Ags for these molecules. Little is known
about the repertoire of mycobacterial Ags recognized by MHC class
I-restricted CD8+ T cells. Studies with CD8+ T
cell clones described recognition of mycobacterial Ags of 14, 19, 65,
71, and 100 kDa (15), and a recent study suggests that ESAT 6 is
recognized by CD8+ T cells (35). Most of these Ags are also
recognized by CD4+ T cells. Whether CD8+ T
cells recognize the same broad mycobacterial Ag repertoire as
CD4+ T cells remains to be determined. Ag processing for
and expression of the appropriate MHC molecules may be critical in
regulating differential CD4+ and CD8+ T cell
responses. The alternate Ag-processing pathway for MHC class I
molecules facilitates presentation of M. tuberculosis Ags by
MHC class I. Uptake of soluble mycobacterial Ags such as cytosol by
macrophages for presentation by MHC class I molecules further
facilitates activation of CD8+ T cells. Whether other cells
within the pulmonary microenvironment are able to take up soluble Ags
for presentation by MHC class I remains to be determined. Recognition
by CD8+ T cells of mycobacterial Ags on cells that do not
normally express MHC class II molecules such as bronchial epithelial
cells or pneumocytes would expand greatly immune defenses to M.
tuberculosis and provide an explanation for a CD8+ T
cell requirement in the immune response to M. tuberculosis.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. David H. Canaday, Division of Infectious Disease, Case Western Reserve University, School of Medicine, BRB 10-8, 10900 Euclid Avenue, Cleveland, OH 44106-4984. E-mail address:
3 Abbreviations used in this paper: PPD+, tuberculin skin test (purified protein derivative) positive; ELISPOT, enzyme-linked immunospot; ER, endoplasmic reticulum; CF, culture filtrate Ags of M. tuberculosis.
Received for publication March 20, 1998. Accepted for publication September 15, 1998.
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