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T Cells: A Mechanism for the Loss of T Cells in Patients with Pulmonary Tuberculosis1
*
Department of Microbiology, University of Pennsylvania School of Medicine, and
Division of Pulmonary Medicine and Critical Care, Hospital of the University of Pennsylvania, Philadelphia, PA 19104;
Tumor Immunology Program, Division of Immunogenetics, German Cancer Research Center, Heidelberg, Germany;
§
Department of Microbiology, Instituto Nacional de Enfermedades Respiratorias, Mexico, Mexico City; and
¶
Microbiology and Immunology Department, Gazi University, Ankara, Turkey
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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9+/V
2+ T cells in their
blood and lungs. Here we have determined whether this T loss is
a consequence of Mtb Ag-mediated activation-induced cell death (AICD).
Using a DNA polymerase-mediated dUTP nick translation labeling assay,
5% or less of freshly isolated CD4+ 
ß or T
cells from normal healthy individuals and TB patients were apoptotic.
However, during culture Mtb Ags induced apoptosis in a large proportion
of V9+/V2+ peripheral blood T cells from
healthy subjects (30–45%) and TB patients (55–68%); this was
increased further in the presence of IL-2. By contrast, anti-CD3
did not induce any significant level of apoptosis in T cells
from healthy subjects or TB patients. Mtb Ag stimulation rapidly
induced Fas and Fas ligand (FasL) expression by T cells, and in
the presence of metalloproteinase-inhibitors >70% of T cells
were FasL+. Blockade of Fas-FasL interactions reduced the
level of Mtb-mediated T cell apoptosis by 75 to 80%.
Collectively, these findings demonstrate that Mtb-reactive T
cells are more susceptible to AICD and that the Fas-FasL pathways of
apoptosis is involved. AICD of T cells, therefore, provides an
explanation for the loss of Mtb-reactive T cells during mycobacterial
infection. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß
T cells in mycobacterial immunity has been clearly demonstrated (1, 2),
several lines of evidence suggest that T cells may also be
important.
Among T cells in normal healthy individuals, more than ß
are Mycobacterium tuberculosis (Mtb)-reactive (3, 4).
Mtb-reactive T cells express a TCR encoded by a single V
(V9) and V (V2) gene (5, 6, 7). T cells from both normal
and mycobacterium-sensitized subjects proliferate vigorously and
release cytokines in vitro in response to a variety of Mtb-derived
protein (3, 6, 8, 9, 10, 11, 12) and nonprotein (13, 14, 15, 16) Ags. Increases in
T cell numbers in mice (17, 18) and humans (19, 20) infected with Mtb
have also been reported.
Recently, we have shown that in patients with active pulmonary TB, the
numbers of V9+/V2+ Mtb-reactive T cells
in the blood and bronchoalveolar lavage were significantly reduced
compared with those in healthy PPD+ individuals and in
patients with unrelated pulmonary granulomatous diseases (21). This
correlation between the changes in the T cell repertoire and
manifestations of disease in patients with pulmonary TB is consistent
with a direct role for these cells in the immunopathogenesis of TB.
In the present study we have investigated the mechanisms that could
account for the loss or the absence of
V9+/V2+ T cells in TB patients. Possible
causes include functional inactivation (anergy), inactivation or
dysfunction of other immune cell populations necessary for T
cell activation and growth (11, 22, 23), and elimination as a
consequence of activation-induced cell death (AICD). Of these, AICD is
an attractive possibility, since programmed cell death and apoptosis of
Ag-activated T cells are known to be important mechanisms for
controlling T cell responses and maintaining homeostasis (24). The
finding that T cells from normal PPD+ individuals
(6, 25) and patients with advanced clinical forms of TB (26) can be
induced to undergo apoptosis after activation with either anti-CD3
and IL-2 (6, 25) or mycobacterial Ags (26) suggest that the loss of
T cells may be a consequence of the elimination (clonal
deletion) of Mtb-specific T cells. However, it is not known
whether this occurs in vivo, what pathways of AICD are involved, and
whether AICD can explain the loss of
V9+/V2+ T cells in patients with active
TB.
Consequently, we have investigated whether AICD is an outcome of the
exposure of T cells to Mtb both in vivo and during culture. We
have analyzed and compared T cell populations from normal
healthy individuals and TB patients for evidence of apoptotic death
directly ex vivo and after in vitro culture with different types of
Mtb-derived Ags. Our results show that compared with CD4+
ß T cells, T cells are more susceptible to Mtb Ag-mediated
AICD and that this susceptibility is increased further in the presence
of IL-2. We also show that Fas-Fas ligand (FasL) interactions are
involved in this Mtb-mediated ACID of T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The patient and control subject populations are described in
Table I
. Peripheral blood samples were
obtained from a total of 32 patients diagnosed with pulmonary TB who
were recruited from the Hospital of the University of Pennsylvania
(n = 2), the Bengbu Medical College Affiliated
Hospital (People’s Republic of China; n = 13), and
Gazi University Hospital (Ankara, Turkey; n = 17).
The diagnosis of active pulmonary TB was established in all patients
(21 men, 38 ± 16.4 yr of age, range from 21–66 yr; 11 women,
33 ± 11.1 yr of age, range from 18–52 yr) by the presence of
recent clinical symptoms of TB, positive culture of Mtb and smear test
for acid-fast bacilli (AFB) from sputum or bronco-brush samples, and
abnormal chest radiograph. All TB patients were HIV-.
Eighteen patients at the time of sampling were receiving
anti-mycobacterial therapy (ethambutol, pyrazinamide,
morphanizamide, and/or streptomyocin), the course of which varied from
<2 wk to >2 yr. For some patients it was possible to obtain more than
one blood sample at different times during drug therapy. Twelve
patients were diagnosed with inactive pulmonary TB (seven men,
44.3 ± 16.9 yr of age, range from 17–65 yr; five women, 35
± 12 yr of age, range from 2–57 yr) according to previous medical
history, abnormal chest radiographs typical of TB, and negative AFB
smear and Mtb culture. Thirty-four healthy (PPD+) subjects
were used as normal controls recruited from the University of
Pennsylvania (n = 10), Bengbu Medical College
(n = 2), and Gazi University Hospital
(n = 12), consisting of 16 men (29.8 ± 6.8 yr
of age) and 18 women (34.1 ± 7.3 yr of age). The collection,
shipping, and processing of samples from Turkey and China have been
described previously (21). In addition, PBMC samples from four healthy
subjects from a household in which one member has active pulmonary TB
(household contacts) were obtained from the Instituto Nacional de
Enfermedades Respiratorias (Mexico City, Mexico). Samples were
received and processed at University of Pennsylvania within 30 h
of collection. Informed consent was obtained from each patient and
healthy volunteer, and the protocol was approved by the human subjects
institutional review board of University of Pennsylvania.
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PBMC were isolated from heparinized venous blood from patients and control subjects by Ficoll-Hypaque density gradient centrifugation. Some PBMC samples, particularly those from Chinese patients, were cryopreserved in liquid nitrogen for up to 2 mo before evaluation. Cryopreservation did not adversely affect T cell viability or significantly change their composition (21).
mAbs and reagents
The following mouse mAbs were used. CD3 (clone, Leu 4)-PE and
anti-TCR -1(11F2)-PE were purchased from Becton Dickinson
(San Jose, CA). For lymphocyte stimulation assays the anti-CD3 Ab
OKT3 (American Tissue Type Culture Collection, Rockville, MD) was used.
The TCR pan-reactive Ab, TCR-1-FITC, and the FITC-labeled
V9-specific Ab, 7A5, were purchased from T Cell Diagnostics (Woburn,
MA). The TCRß-specific Ab, BMA031, was purchased from Behring
(Marburg, Germany) and was conjugated to either biotin or FITC. The
anti-V2 Ab, 15D, was generated in this laboratory (21) and was
conjugated to either biotin or FITC. The mouse monoclonal
anti-human Fas Abs CH-11 and Dx2 were obtained from MBL
International (Watertown, MA) and PharMingen (San Diego, CA),
respectively. Fab fragments of the mouse monoclonal anti-Fas Ab,
APO-1 (27), were used for blocking experiments. The anti-human FasL
Ab NOK-1 was purchased from PharMingen. Human TNF- neutralizing
rabbit antiserum and control nonimmune sera were purchased from Genzyme
(Cambridge, MA). The mouse anti-human CD45-FITC and CD45RO-PE Abs
(Dako Corp., Carpinteria, CA) were used as compensation control
reagents for two- and three-color flow cytometric analyses.
Fluorochrome-conjugated mouse IgG isotype control Abs were purchased
from Becton Dickinson. Phorbol ester, ionomycin, and murine IgM were
obtained from Sigma (St. Louis, MO).
Flow cytometry
Fifty-microliter aliquots of freshly isolated or cultured PBMC
(containing 2–10 x 105 cells) in PBS with 5% FCS
and 0.1% NaN3 (staining buffer) were added to individual
wells of V-bottom 96-well plates. All Ab incubations were conducted on
ice for 30 min each. Cell samples were first incubated with
biotin-conjugated Abs, washed, and incubated with
fluorochrome-conjugated streptavidin (SA-Red 670, Life Technologies,
Gaithersburg, MD) and fluorochrome-conjugated Abs. Cells were washed
and fixed with PBS containing 1% (w/v) paraformaldehyde before
analysis on a FACScan (Becton Dickinson) and using CellQuest analysis
software (Becton Dickinson). Fluorochrome-conjugated isotype-matched
mouse Abs were used as negative controls. Cell samples stained with
anti-CD3-biotin and SA-Red 670, anti-CD45-FITC, and
anti-CD45RO-PE were used as positive controls to identify and gate
on lymphocytes and as fluorochrome compensation controls.
V9+ and V2+ cells were identified and
enumerated by gating on cells reactive with a TCR pan-reactive Ab
(11F2-PE), which were then reanalyzed for cells reactive with the
V9-specific (7A5-FITC) and V2-specific (15D-biotin and SA-Red
670) Abs.
Lymphocyte culture
PBMC isolated from TB patients and normal subjects were cultured
in RPMI/10% human serum supplemented with 25 mM HEPES at 37°C
in 5% CO2. Cells were cultured at 5 x
106/ml (1 ml/well) in 24-well tissue culture plates (Life
Technologies) in the presence of 1) 1 to 100 U/ml recombinant human
IL-2 (Boehringer Mannheim, Indianapolis, IN), 2) 2.5 µg/ml
anti-CD3 Ab (OKT3), 3) 1 µg/ml of culture filtrate from H37Ra
(provided by Dr. Michael E. Ellis, Colorado State University, Fort
Collins, CO), 4) 2 µg/ml of supernatant from heat-treated H37Ra (SHT)
provided by Dr. Henry Boom (Case Western Reserve University, Cleveland,
OH), or 5) medium alone. For the majority of experiments, cells were
cultured for 48 h. In some experiments analyzing FasL expression,
cells were cultured in the presence of 5 mM EDTA to inhibit
metalloprotease-mediated cleavage of cell surface FasL (28). EDTA was
only present for the last 4 h of culture before analysis. For the
derivation of and V9+/V2+ T cell
lines, PBMC were cultured for up to 14 days by stimulating PBMC with
anti-CD3 or Mtb-SHT, adding 1 U/ml of IL-2 after 5 and 10 days of
culture.
Apoptosis assay
Apoptotic cells were identified among suspensions of freshly
isolated or cultured PBMC using a DNA polymerase-mediated dUTP nick
translation labeling (DUNTL) assay provided by Dr. J. Ashwell (National
Institutes of Health, Bethesda, MD). For evaluating TNF-induced
apoptosis, 2 to 20 ng/ml of human recombinant TNF- (PeproTech, Rocky
Hill, NJ) was added to cultures of Mtb Ag-expanded lymphocytes for
72 h before analysis. To evaluate the contribution that
endogenously produced TNF- makes to apoptotic death of T cell during
in vitro culture, neutralizing rabbit anti-human TNF- antiserum
(1/5 to 1/100 dilution) was included in some cultures. Nonimmune rabbit
serum was used as a control. As a positive control for Fas-mediated
apoptosis, cells were incubated with 200 ng of the anti-Fas IgM Ab,
CH-11, or control Ab (purified murine IgM) for 20 h before
analysis. In some experiments Fab fragments of the anti-Fas Ab,
APO-1 (0.01–10 µg/ml), were included to block Fas-mediated
apoptosis. Cells were stained with Abs for cell surface Ags, fixed with
1% paraformaldehyde/PBS, then washed in staining buffer (PBS, 2% FCS,
and 0.1% NaN3) followed by permeabilization buffer (0.1%
Triton-X 100 and 0.1% sodium citrate). Cells were resuspended in
labeling buffer (50 mM Tris (pH 7.4), 10 mM MgSO4, 0.1 mM
DTT, 1 nmol/ml dATP/CTP/GTP, and 0.7 nmol/ml dTTP) containing 40 pmol
of FITC-12-dUTP (Boehringer Mannheim, Indianapolis, IN) and either
active (2 U) or denatured DNA polymerase (Promega, Madison, WI) and
incubated at 37°C for 90 min before analysis by flow cytometry.
Stained cells were run on a FACScan and analyzed as described above.
The frequency of apoptotic (dUTP+) cells was determined by
subtracting the frequency of cells incorporating dUTP in the presence
of denatured (control) DNA polymerase from the frequency of cells
incorporating dUTP in the presence of active polymerase. The frequency
of dUTP- cells was used to determine the number of viable
cells.
Statistical analysis
For comparing the proportions of T cells freshly isolated
from PB and bronchoalveolar lavage in different patient and control
groups, Student’s t test was used. For comparison of
T cells and subsets, considered nonparametric, Mann-Whitney
U test or Wilcoxon’s signed rank sum test was used.
Values of p < 0.05 were chosen for rejection of the
null hypothesis.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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9+/V2+ T cells in
TB patients
Previously we have demonstrated a strong correlation between the
absence or loss of the major Mtb-reactive subset of T cells,
V9+/V2+ cells, and manifestations of
active pulmonary TB (21). To determine whether chemotherapy adversely
affected V9+/V2+ T cells and if drug
treatment had any beneficial effect on this population of T cells, the
number of peripheral blood V9+/V2+ T
cells was analyzed in patients during and after drug therapy (Fig. 1). Nontreated patients were those that
had not received any antimycobacterial drugs before analysis. Treated
patients received drug therapy before analysis and were divided into
two groups depending upon the duration of treatment: 2 mo or less
(average, 1.2 mo for active TB patients; n = 15)
and >2 mo (average, 8 mo for disease-free subjects
(n = 5) and 16 mo for TB patients with active
disease (n = 3)). Of the patients treated for >2
mo, the average period of treatment for three patients with active TB
was longer due to treatment failure. For five of the treated TB
patients we were able to obtain blood samples both before and 1–2 mo
after beginning drug treatment. The active disease group of patients
consisted of those whose sputum at the time of analysis was AFB smear
test and Mtb culture positive. Inactive disease patients were those
originally diagnosed with pulmonary TB who, after a period of drug
treatment and at the time of sampling, were AFB smear test and Mtb
culture negative.
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9+/V2+ T cells in TB patients with
active disease (39 ± 7% of all T cells; Fig. 1
) was, as
reported previously (21), significantly (p < 0.0001)
reduced compared with healthy individuals (84 ± 8% of all
T cells). This reduced frequency of
V9+/V2+ T cells in TB patients reflected
a decrease in absolute numbers of these cells, since as a group, the
number of peripheral blood T cells in patients with active TB
(82 ± 43 cell/µl) and nonactive TB (83 ± 40 cells/µl)
was comparable to that in normal PPD+ control subjects
(94 ± 42 cells/µl). A similar decrease in
V9+/V2+ cells was seen in the lungs of TB
patients (data not shown) (21). As we have described previously (21),
as a result of the loss of V9+/V2+ T
cells, the T cell repertoire in TB patients is dominated by
V9-/V2- T cells, which in the majority
of normal, healthy, PPD+ subjects represent only a small
proportion of the peripheral blood T cell repertoire (21). What
accounts for the increase in number of these unusual T cell
subsets in TB patients and the nature of the Ag(s) that drives their
expansion are not known. However, since the Mtb reactivity of T
cells is contained entirely within the
V9+/V2+ subset (29), it is unlikely to be
mediated by Mtb Ags.
In drug-treated TB patients decreased numbers of
V9+/V2+ cells were associated with
patients that had persistent positive cultures for Mtb (i.e., active
disease) regardless of the period of drug treatment. Reduced numbers of
V9+/V2+ T cells were seen in a group of
15 treated patients with active disease for >2 mo or less (Fig. 1) and
in three patients with positive sputum cultures for Mtb after 8, 16,
and 18 mo (active disease patient group treated for 2 mo in Fig. 1).
Persistently reduced numbers of V9+/V2+ T
cells were seen in five patients analyzed before and 30
(n = 3) or 60 (n = 2) days
post-treatment (data not shown). By comparison, the frequency of
V9+/V2+ cells in treated patients who
were sputum culture negative for Mtb (inactive disease group in Fig. 1)
was significantly higher (p < 0.001) than that in
patients with active TB, although still slightly lower than that in
normal subjects. Importantly, this recovery of
V9+/V2+ cell numbers in patients
successfully treated with antimycobacterial drugs makes it unlikely
that the loss of Mtb-reactive T cells seen in TB patients is due
to the adverse affect of antimycobacterial drugs on the survival or
growth of V9+/V2+ cells. Together, these
findings demonstrate that the loss or the absence of
V9+/V2+ T cells in TB patients correlates
with the presence of disease symptoms.
T cells exhibit increased susceptibility to spontaneous
death and AICD in vitro
To investigate the underlying basis of the loss of Mtb-reactive
T cells, we determined whether Mtb Ag stimulation resulted in
AICD and could account for the reduced number of
V9+/V2+ T cells in TB patients. Apoptotic
cells were identified and quantitated using a flow cytometry-based
assay in which DNA strand breaks, a hallmark feature of apoptosis, were
detected in cells using an in vitro DNA polymerase-mediated DUNTL
method. Freshly isolated or cultured cells were first stained with
anti-TCR Abs, fixed, permeabilized, and incubated with FITC-dUTP in
the presence of DNA polymerase. As a control, duplicate aliquots of
cells were incubated with FITC-dUTP in the presence of heat-inactivated
polymerase. The absolute numbers and proportion of viable and apoptotic
cells were determined by cell counts and flow cytometric determination
of the frequencies of CD4+ TCR-ß+,
+, or V9/V2+ T cells that were
dUTP- and dUTP+, respectively. The data
obtained and shown in Figure 2 represent
the number of viable (dUTP-) and apoptotic
(dUTP+) cells detected among 5 million PBMC before (freshly
isolated) and after 48 h of culture.
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). A similar
proportion of CD4+ ß (3.1 ± 2.2%) and
(3.0 ± 0.9%) T cells from patients with active TB was also
dUTP+ (Fig. 2). By contrast, it was possible to detect
differences in the susceptibility of CD4+ ß and
T cells from normal subjects and TB patients during short term (48 h)
in vitro culture (Fig. 2). Five different conditions were used:
complete medium alone, anti-CD3, IL-2, and two different
preparations of Mtb Ags representative of secreted (culture filtrate;
Mtb-filtrate) and cell-associated (supernatant from heat-treated
bacteria; Mtb-SHT) Ags. Both of these Mtb Ags are capable of
stimulating ß T cells, whereas the Mtb-SHT antigenic preparation
is very effective at promoting T cell growth in normal
individuals (12, 21).
In the absence of any exogenous Ag (medium alone) the level of
apoptosis (spontaneous death) in (17–24%) and V9/V2
(16–27%) T cells, from PPD+ normal subjects was slightly
higher than that in CD4+ ß (10–15%) T cells (Fig. 2). Mtb Ags induced levels of apoptosis in T cells and
V9+/V2+ cells from the same individuals
that were significantly higher (p < 0.01 for
Mtb-SHT and p < 0.02 for Mtb-filtrate) than the level of
spontaneous death. The results obtained from the analysis of T cells
from patients with nonactive TB were very similar to those described
for PPD+ normal subjects (data not shown). By contrast,
none of the stimuli used induced levels of apoptosis in
CD4+ ß T cells that were significantly higher than
those in cultures containing medium alone.
The profile of Ag-mediated apoptosis in cultured T cells from patients
with active TB was similar to that in T cells from normal subjects.
However, the overall level of T cell apoptosis was slightly higher in
PBMC from TB patients. Whereas 30 to 45% of T cells from normal
subjects were induced to undergo apoptosis by Mtb-SHT or Mtb-filtrate,
these Ags induced apoptosis in >50% (55–68%) of cells from
TB patients (Fig. 2). Although this increase in apoptotic
V9+/V2+ T cells is small, it is
significant, since there are now more dead (apoptotic) than viable
(dUTP-) cells present in these cultures, providing an
explanation for the lack of growth of these T cells when
cultured with Mtb Ags (21). Also of note was the finding that IL-2
induced levels of apoptosis in and
V9+/V2+ cells from TB patients that were
significantly higher (p < 0.01 and p <
0.02, respectively) than the level of spontaneous death, perhaps
reflecting prior activation in vivo. Expression of the
activation-associated Ags, CD25 and CD69, by a large proportion
(20–35%) of freshly isolated peripheral blood
V9+/V2+ T cells from TB patients (B. Li
and S. R. Carding, unpublished observations) is consistent with
this interpretation and suggests that IL-2-induced AICD in vitro is a
consequence of activation in vivo as a result of exposure to Mtb Ags.
As seen from the analysis of PPD+ subjects,
CD4+ ß T cells from TB patients were, compared with
T cells, markedly less susceptible to AICD during 48 h of
culture with the various stimuli. In these cultures only anti-CD3
was able to induce levels of apoptosis that were significantly higher
(p < 0.01) than those occurring in medium alone
(Fig. 2).
Collectively, the results of our analysis of apoptotic T cells suggest
that T cells, and in particular the Mtb-reactive
V9+/V2+ subset, are more susceptible to
Mtb Ag-mediated AICD than are CD4+ ß T cells. To
investigate this phenomena further, the pathways involved in AICD of
T cells were investigated.
Blockade of Fas-FasL interactions abrogates Mtb-mediated AICD of
T cells
A large amount of evidence suggests that AICD of peripheral
lymphocytes can be triggered by the interaction of the Fas molecule
with its ligand (27, 30, 31, 32). A previous study of murine T cells has
shown that T cells express higher levels of FasL mRNA than
ß T cell clones (33). However, to date the involvement of Fas and
FasL interactions in AICD of bulk polyclonal populations of human
T cells in mycobacterial disease has not been investigated.
To determine whether or how much the Fas-FasL pathway contributed to
T cell apoptosis induced by Mtb Ags, we investigated what effect
Ab-mediated blockade of Fas-FasL interactions had on Mtb Ag-mediated
apoptosis of V9+/V2+ T cells. For this
experiment two T cell lines (>98% CD3+) derived by
culturing PBMC from a PPD+ donor with the Mtb-SHT Ag
preparation for 10 to 14 days were used. The first line (Expt. 1 in
Fig. 3) comprised 45% CD4+
TCRß+ T cells and 55% TCR+ (>90%
V9+/V2+) T cells, and the second (Expt.
2, Fig. 3) consisted of 95% TCR+ (>95%
V9+/V2+) cells. These lines were
stimulated a second time with anti-CD3 or Mtb-SHT in the presence
or the absence of Fab fragments of the APO-1 Ab that blocks Fas-FasL
interactions (27). Forty-eight hours later the frequency of apoptotic
cells was determined using the DUNTL assay. The efficacy of the APO-1
Ab was demonstrated by its ability to almost completely inhibit CH-11
Ab-induced apoptosis of ß CD4+ (90%) and
(85–95%) T cell lines (Fig. 3). A final concentration of 2 µg/ml of
APO-1 Ab was shown to be optimal for inhibition of CH-11-induced
apoptosis (data not shown) and was used for inhibiting Mtb-mediated
AICD of ß and T cells.
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T
cell apoptosis and anti-CD3-induced apoptosis of CD4+
ß T cells were reduced by 70 to 85% and 65 to 75%, respectively
(Fig. 3). By contrast, the frequency of apoptotic and ß T
cells in cultures containing medium alone was not significantly reduced
in the presence of the APO-1 Ab. These results demonstrate that the
Fas-FasL pathway of AICD mediates Mtb Ag-induced apoptosis of T
cells. By contrast, the Fas-FasL pathway does not appear to be involved
in the spontaneous death of cultured or ß T cells. Finally,
the ability of Mtb-SHT to induce apoptosis in the T cell line that
comprised almost entirely (95%) T cells suggests that their
death is a result of "fratricide" (34), in which activated T
cells that express Fas (Figs. 4 and
5) and FasL (Fig. 6) induce apoptosis in one another.
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9/V2 T cells, we
decided to investigate the kinetics of induction of Fas and FasL
expression by Mtb-stimulated T cells and whether surface
expression of these molecules is regulated differently in ß vs
T cells. To our knowledge, the regulation of expression of these
molecules by bulk polyclonal populations of human T cells in
mycobacterial disease has not been investigated previously.
Fas expression by Mtb-stimulated ß and T cells
Since our analysis of T cell apoptosis demonstrated that ß
and T cells have different susceptibilities to anti-CD3- and
Mtb Ag-mediated AICD (Fig. 2), these stimuli were used to compare Fas
expression. Cell surface Fas expression by freshly isolated and
cultured peripheral blood T cells from PPD+ subjects and
patients with active pulmonary TB was detected by Ab staining and flow
cytometry. Analysis of freshly isolated cells showed that in
PPD+ healthy individuals a higher proportion of T
cells (38 ± 9%; n = 10) than of CD4+
ß T cells (12 ± 8%; n = 10) was
Fas+ (Fig. 4A). The level of Fas expression by
both ß and T cells was variable, with the level of
expression low in the majority of positive cells (Fig. 4A).
In patients with active TB, the proportions of Fas-expressing ß T
cells and T cells were similar to those in healthy subjects
(Fig. 5).
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ß CD4 and T
cells induced by anti-CD3 and Mtb-SHT were distinct (summarized in
Fig. 5). In cultures of Mtb-SHT-stimulated PBMC, Fas was up-regulated
more rapidly by T cells than by CD4+ ß T cells.
For example, at 48 h poststimulation the frequency of
Fas+ T cells was significantly (p
< 0.006) higher than that for CD4+ ß T cells (Figs. 4B and 5). In fact, the frequency of CD4+ ß
T cells that were Fas+ at this time in culture was lower
than that among freshly isolated cells (Fig. 4, A and
B). Beyond 48 h of culture the proportions of
and CD4+ ß T cells that were Fas+ were
similar, with maximal levels of positive cells seen 10 days after
activation (Fig. 5). In contrast to Mtb-SHT activation, anti-CD3
stimulation of PBMC from PPD+ subjects resulted in the
rapid (within 24 h) up-regulation of Fas by ß CD4 T cells,
which was sustained over the 14-day culture period. By contrast,
up-regulation of Fas by T cells occurred more slowly, with the
highest frequency of positive cells occurring 10 days after
stimulation.
In cultures of T cells from TB patients a similar profile of Fas
expression by ß and T cells was seen (Figs. 4B
and 5). Of note, the delayed up-regulation of Fas by T cells
from PPD+ subjects was more pronounced in TB patient
samples. Thus, differences in the temporal expression of Fas
distinguish the responses of ß and T cells to different
antigenic stimuli and Mtb-derived Ags.
FasL expression by Mtb-stimulated ß and T cells
Consistent with previous studies (28), cell surface-associated
FasL was expressed by very few (1.4% or less) freshly isolated ß
and T cells (Fig. 6). There were also no obvious differences in
the frequency of FasL+ T cells in normal or TB PBMC
samples. In contrast, it was possible to detect differences in the
kinetics and level of FasL expression by ß and T cells
during in vitro culture (Fig. 6). An increase in the frequency of
FasL+ T cells was seen within 48 h of culture,
representing a three- to fivefold increase. The stimuli used were
equivalent in their ability to induce FasL expression by T
cells, and there were no significant differences in the level of FasL
expression by T cells from PPD+ normal subjects or
TB patients. The frequency of FasL+ T cells did not
change significantly when analyzed at 13 days poststimulation, although
FasL expression did appear to be up-regulated more slowly by ß T
cells, since the highest frequency of positive cells was seen 13 days
after stimulation (data not shown).
Cell surface expression of FasL has been shown to be regulated by
metalloproteinases (28, 35), and inhibitors of these enzymes, such as
EDTA, can increase the level of expression by preventing their cleavage
from the surface. To determine whether our analysis had underestimated
the amount of FasL expressed by ß and T cells, PBMC from
PPD+ subjects were cultured with Mtb Ags in the presence of
5 mM EDTA before analysis of FasL expression. EDTA was added during the
last 4 h of the 48-h culture period. During this 4-h incubation
period, cell viability did not change significantly (data not shown). A
striking increase in FasL expression by T cells was seen in the
presence of EDTA. Approximately 70% of T cells activated in the
presence of EDTA were FasL+ compared with 10% or less in
the absence of EDTA (Fig. 6). However, there was no evidence of
stimulus (Ag)-specific modulation of FasL expression for T
cells, since the majority of T cells cultured in medium alone
were FasL+, and there was no significant increase in the
frequency of positive cells after culture with any of the stimuli.
Although the frequency of FasL-expressing ß CD4 T cells was also
increased in the presence of EDTA, it was restricted to a small
(11–23%) subset of cells. There was, however, some evidence of
Ag-specific modulation of its expression in the presence of EDTA (Fig. 6). Whereas stimulation with anti-CD3 or Mtb-SHT did not
significantly change the frequency of FasL+ ß T cells,
there was a twofold increase in FasL+ ß T cells in
response to Mtb-filtrate compared with that in cells cultured in medium
alone.
Alternative mechanisms of T cell apoptosis; TNF pathway
Since TNF is an apoptosis-associated molecule that shares amino
acid homology with FasL and has been shown to induce apoptosis of
activated lymphocytes (36, 37), we investigated whether this cytokine
might contribute to Mtb-mediated T cell apoptosis. Mtb
Ag-specific T cells lines were established by culturing PBL from
PPD+ normal subjects or TB patients in the presence of
Mtb-filtrate or Mtb-SHT antigenic preparations and were restimulated
with Ag in the presence of TNF- for 3 days before DUNTL analysis.
Mtb-stimulated CD4+ ß T cells from both
PPD+ healthy subjects and TB patients were refractory to
TNF--induced apoptosis, with 10% or less dUTP+ cells
detected. Similarly, it was not possible to detect any significant
difference in the level of or
V9+/V2+ T cell apoptosis after
stimulation with Mtb-SHT or -filtrate in the absence or the presence of
TNF- (Fig. 7). Also, the inability of
neutralizing anti-human TNF- antiserum (1/5 to 1/100) to
significantly reduce the level of spontaneous apoptosis of
CD4+ ß, , or
V9+/V2+ T cells from TB patients (data
not shown) suggests that any TNF- produced by these cells as a
result of prior activation in vivo is not responsible for the death of
these cells. We interpret these results as evidence of TNF- not
being involved in Mtb Ag-mediated AICD of T cells.
|
9+/V2+T cells
Whereas we and others have previously shown that the Mtb-SHT Ag
preparation can promote the growth of Mtb-reactive,
V9+/V2+ T cells, the data presented here
demonstrate that this Ag preparation can also promote T cell
apoptosis. To attempt to explain this apparent paradox we investigated
the possibility that the presence of additional growth factors, such as
IL-2, might influence the outcome of T cell activation by
Mtb-SHT. The rationale for this experiment was based upon the
observation by Kabelitz and colleagues that whereas anti-CD3 Abs
alone are mitogenic for V9+/V2+ T cells,
when combined with IL-2 they induce T cell death by apoptosis
(6, 25).
T cells from a PPD+ healthy subject were expanded in
vitro by stimulation with Mtb-SHT. After approximately 12 days these
cultures contained >95% V9+/V2+ cells.
The cells were then stimulated a second time with anti-CD3 or
Mtb-SHT in the absence or the presence of exogenous IL-2 (1–100U/ml)
or with IL-2 alone, and 48 h later the proportion of apoptotic
cells was determined using the DUNTL assay. At concentrations of 10
U/ml and higher, IL-2 alone was able to induce a small but significant
(p < 0.02) increse in T cell apoptosis above
that due to spontaneous death in culture (Media, in Fig. 8), reflecting, perhaps, the death of
residual activated cells from the initial in vitro stimulation. By
contrast, IL-2 had potent synergistic apoptotic activity when combined
with Mtb-SHT (Fig. 8). In the presence of IL-2 the level of apoptosis
induced by Mtb-SHT was increased by approximately twofold (26 ± 4
and 55 ± 5% dUTP+ cells in the absence and the
presence of IL-2, respectively). This synergistic activity of Mtb-SHT
and IL-2 was seen at IL-2 concentrations >1 U/ml, with the maximal
effect obtained between 10 and 100 U/ml. By contrast, other cytokines,
including TNF- (Fig. 7), IL-4, and IFN- (data not shown), alone
or in combination with Mtb-SHT, did not increase the level of T
cell apoptosis.
|
T cell
apoptosis was seen when it was combined with anti-CD3 (Fig. 8). In
the presence of 10 and 100 U/ml of IL-2, the frequency of apoptotic
T cells was significantly greater (p < 0.01)
than that after stimulation with anti-CD3 alone. Not surprisingly, the
presence of IL-2 severely reduced the ability of Mtb-SHT to promote the
growth of V9+/V2+ T cells. Whereas the
Mtb-SHT Ag preparation expanded V9+/V2+ T
cells, on the average, >50-fold after 10 to 12 days of culture (21)
(data not shown), a <5-fold increase was seen in the presence of IL-2
(data not shown). The ability of the anti-Fas Ab, APO-1, to almost
completely inhibit T cell apoptosis induced by IL-2 and Mtb-SHT
or anti-CD3 (Fig. 8) demonstrates that the synergistic effect of
IL-2 on Mtb-mediated AICD of V9+/V2+ T
cells is mediated by Fas-FasL interactions. Together, these findings
demonstrate that IL-2 influences the outcome of Mtb-SHT Ag stimulation
of T cells and, in combination with the Mtb-SHT Ag preparation,
promotes apoptotic cell death rather than growth. |
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
ß T cells, Mtb-reactive,
V9+/V2+, T cells are more
susceptible to Mtb Ag-mediated AICD, and that the Fas-FasL pathway is
involved in T cell apoptosis. These findings provide an
explanation for the loss or the absence of
V9+/V2+ T cells in patients with active
TB. The demonstration of Ag (Mtb)-mediated AICD is both consistent with
and now extends the observations of previous studies demonstrating that
T cells readily undergo apoptosis in culture with anti-CD3
and IL-2 (6, 24, 25). Interestingly, our in vitro studies show that Mtb
Ags are more effective than anti-CD3 at inducing AICD of
(V9+/V2+) T cells. This difference
may be related to differences in the outcome of signaling via the TCR
vs the CD3 complex alone. For example, since we have shown that the
presence of IL-2 potentiates anti-CD3 and Mtb-SHT-mediated
V9/V2 T cell apoptosis, it may be due to qualitative or
quantitative differences in the ability of these stimuli to induce IL-2
production by T cells or other (ß) T cells present in these
cultures.
By identifying the pathways involved in Mtb-mediated T cell
apoptosis, our findings extend those made recently by Duarte and
colleagues describing Mtb (H37Ra)-mediated T cell apoptosis (26)
in patients with advanced forms of TB. Our demonstration of the rapid
and sustained expression of FasL by activated polyclonal populations of
T cells in both healthy subjects and TB patients is in agreement
with a recent study showing that V1-expressing T cell clones
obtained from synovial fluid of patients with Lyme arthritis (38)
express high levels of FasL for a longer period after in vitro
activation than ß T cells. Up-regulation of FasL expression by
T cell clones from Lyme disease patients was also associated
with broad spectrum cytotoxicity. However, using conventional
51Cr release assays or DNA fragmentation of
[3H]TdR-labeled cells (JAM assay) to detect cell-mediated
cytotoxic activity, we were unable to demonstrate any autologous
cell-directed cytotoxicity by Mtb-stimulated FasL+
V2+ T cells (B. Li and S. R. Carding,
unpublished observations). Indeed, our finding that Mtb-mediated AICD
of T cells is abrogated by blocking Fas-FasL interactions
suggests that activation by Mtb Ags results in expression of both Fas
and FasL, making them targets for and effectors of Fas-mediated
cytotoxicity. The more rapid up-regulation and persistent expression of
Fas that occur in T cells compared with CD4+ ß
T cells after Mtb-SHT stimulation is also consistent with this
interpretation and may explain in part the increased susceptibility of
T cells to AICD.
Although the in vitro culture conditions used in this study may not
necessarily reflect those found in vivo during mycobacterial infection,
the results we have obtained suggest that the conditions under which
T cells are exposed to Mtb Ags is an important factor in
determining their response and fate. For example, patients with
clinically advanced forms of pulmonary TB will have high levels of
bacteria due to the inability to contain and prevent their spread. This
would presumably result in chronic T cell activation, high local
concentrations of IL-2, and sustained expression and production of Fas
and FasL. These are conditions that we have shown to result in AICD of
Mtb-reactive V9+/V2+ T cells. Conversely,
conditions under which bacterial growth is contained and restricted to
the lung would result in acute exposure of T cells to relatively low
amounts of Ag, resulting in transient or lower levels of T cell
activation and IL-2 production. This would produce conditions that in
the majority of individuals exposed to Mtb would favor the survival and
expansion of V9+/V2+ T cells. Our
findings that IL-2 influences the outcome of Mtb Ag stimulation of
T cells (Fig. 8) and that the magnitude, kinetics, and duration
of T cell responses to intracellular bacteria in vivo are
directly related to the dose of bacteria and the sites at which
infection is established (39) are consistent with this hypothesis.
Although Mtb Ags could directly mediate AICD of T cells, they
could also be indirectly involved. For example, by the ability of the
bacteria to modulate the activity of macrophages (22, 40) and
CD4+ ß T cells (21, 23, 41) that normally interact
with and regulate the response of T cells to Mtb. Whatever the
mechanism, the persistence of high levels of bacteria and chronic
exposure of V9+/V2+ T cells to Mtb Ags
provide an explanation for why in patients with active disease this
population of T cells remains low but recovers in patients that
respond to drug therapy. The lack of response of residual T
cells from patients with advanced clinical forms of TB to in vitro
challenge with Mtb-SHT (21) and other Ags (42) could reflect the
elimination, rather than hyporesponsiveness (anergy), of Mtb-reactive
cells in vivo as a result of chronic stimulation and AICD. Studies to
analyze the V9/V2-TCR repertoire in TB patients should provide
additional insights into T cell function in mycobacterial
disease.
The current findings may also bear on observations that in various
animal models of infectious and autoimmune disease, inflammation and
disease pathology are more rapid in onset and more severe in the
absence of T cells. For example, the inflammatory response is
accelerated in a model of orchitis in which T cells are depleted
(43). Unusual lesions and pathologies develop in tissues of
bacteria-infected (44, 45, 46) or parasite-infected (47) -deficient
or -depleted mice. Also, inflammation is more aggressive in collagen-
(48) and adjuvant-induced (49) arthritis in the absence of T
cells. Together with the finding that T cells can modulate the
activity of other immune cell populations (50, 51, 52, 53, 54, 55), it seems likely,
therefore, that they perform an immunoregulatory function, being able
to initiate as well as regulate the immune response to a variety of
pathogens. Based upon our previous analyses of influenza virus-induced
pneumonia in mice, we have proposed that T cells are part of an
inflammatory immune cell circuit made up of macrophages and ß T
cells as well as defined subsets of T cells (reviewed in Refs.
50 and 51). Through the production of specific cytokines, these
T cells are able to modulate the activity of the other cellular
components of the inflammatory immune cell circuit and to resolve the
immune response. Thus, in individuals infected with Mtb, the absence of
such an immunoregulatory (V9+/V2+)
T cell population and the inability to effectively down-modulate
inflammatory immune responses could exacerbate cell and tissue necrosis
and promote disease progression rather than protective immunity. A role
for T cells in the prevention or amelioration of immunopathology
is attractive in light of the exaggerated pathology in Mtb-infected
mice lacking T cells and in TB patients in whom Mtb-reactive
T cells are reduced in number (21).
Although our studies cannot establish whether the T cell
phenotype seen in patients with active TB is causally related to
disease, they do suggest that V9+/V2+ T
cells are an important component of the cell-mediated immune response
to Mtb. Studies are in progress to determine how Mtb-reactive
V9+/V2+ T cells can interact with and if
they can modulate the function of other immune cell populations.
Overall, the current findings are consistent with the concept that
T cells, particularly the V9+/V2+
Mtb-reactive subset, contribute to the development of protective
immunity to Mtb and that the conditions under which they react with Mtb
Ags influences the nature of their response and possibly the outcome of
infection.
|
Acknowledgments |
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|
Footnotes |
|---|
2 Current address: Department of Microbiology, Bengbu Medical College, Bengbu, Peoples Republic of China.
3 Current address: Department of Pulmonary Disease, Baskent University, Fevzi Cakmak Caddesi 10, Sokak NO:45, 06490 Bahcelievler-Ankara, Turkey.
4 Address correspondence and reprint requests to Dr. Simon R. Carding, Department of Microbiology, University of Pennsylvania, 303A Johnson Pavilion, Philadelphia, PA 19104-6076. E-mail address:
5 Abbreviations used in this paper: TB, tuberculosis; Mtb, Mycobacterium tuberculosis; PPD, purified protein derivative; AICD, activation-induced cell death; FasL, Fas ligand; AFB, acid-fast bacilli; PE, phycoerythrin; SA, streptavidin; SHT, supernatant from heat-treated H37Ra; DUNTL, dUTP nick translation labeling.
Received for publication January 6, 1998. Accepted for publication April 1, 1998.
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; dUTP-) and apoptotic
(
; dUTP+) cells as described in Materials and
Methods. The results shown represent the mean (±SD) number of
dUTP- (
