| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
*
Unité d’Oncologie Virale, Département SIDA et Rétrovirus, Institut Pasteur, Paris, and
Service des Maladies Infectieuses et Tropicales, Hôpital Militaire Bégin, Saint Mandé, France
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-producing T cells was
observed, whereas those producing IFN-
remained preserved.
Furthermore, there is a gradient of susceptibility to
activation-induced apoptosis (IL-2 < IFN- < TNF-) among
the different Th1 subsets. This gradient was detected in both CD4 and
CD8 subsets, as well as in control donors and HIV-infected patients, in
whom the susceptibility to apoptosis of IL-2 and IFN- producers was
increased compared with controls. This differential intrinsic apoptosis
susceptibility of Th1 effectors was found to be tightly regulated by
Bcl-2 expression. In HIV-infected persons, disappearance of
IL-2-producing T cells was a good indicator of disease progression and
was correlated with the progressive shrinkage of the
CD4+CD45RA+ T cell compartment and a
gradual increased susceptibility to activation-induced apoptosis of the
IL-2-producing subset. This close relationship between the
CD45RA/CD45R0 ratio, the level of type 1 cytokine production, and
susceptibility to apoptosis should be considered in HIV-infected
patients under antiviral or immune-based therapies. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, and TNF-, which are important mediators of cellular
immunity and are involved in activation of macrophages; whereas Th2
cells secrete IL-4, IL-5, IL-6, IL-10, and IL-13, which provide help to
B cells for Ig synthesis, mediate eosinophilia, and antagonize the
macrophage-activating action of Th2 cytokines. In addition, a third
subset of helper T cells has been identified, Th0 cells, which produce
both Th1 and Th2 cytokines (5). Some pathologic situations are
associated with an imbalance in Th1 and Th2 subsets, but the mechanisms
underlying this imbalance remain unclear (5). Recent reports indicate
that Th1 effectors differ from Th2 effectors in their
susceptibility to activation-induced apoptosis, with Th1 T cells
being highly sensitive to Fas-induced apoptosis (6, 7, 8).
Activation-induced apoptosis is involved in peripheral T cell deletion
and has been implicated in the loss of CD4 Th cells in AIDS (9, 10, 11, 12).
HIV infection is characterized by a persistent immune activation
responsible for the continuous recruitment of CD45R0+
effectors and the concomitant decline of the CD45RA+ naive
compartment in both CD4 and CD8 subsets (13). This is associated with
an increased rate of spontaneous and activation-induced apoptosis in
both subsets, leading to the progressive alteration in T helper
functions (14). This activation-induced deletion of Th cells is
accompanied by early impairment of cytokine synthesis. It was proposed
several years ago that a shift from Th1- to Th2-type secretion during
HIV infection might perturb the immune system and lead to progression
to AIDS (15). Since that time, many studies reported alteration in
cytokine production by T cells of HIV-infected patients but discrepant
results were found. A decrease in IL-2 production by patients’
peripheral T cells in response to Ag or mitogen stimulation or to TCR
ligation, or a decrease in constitutive IL-2 mRNA expression, has been
uniformly reported (16, 17, 18, 19, 20, 21). In contrast, IFN- production was found
to increase (18, 22, 23, 24, 25), decrease (20, 21), or remain unchanged as
well (26). Furthermore, analysis of CD4+ T cell clones
derived from AIDS patients revealed a reduced number of clones
producing IL-2 and IFN-, together with an increased number of
Th0-type T cell clones when compared with healthy controls (27, 28, 29).
The pattern of cytokines produced by an effector population can now be
analyzed using a single-cell analysis method that allows enumeration of
Th1/Th2 subsets derived from peripheral T cells stimulated in
short-term cultures and provides information on the number and the
phenotype of cells that are potentially capable of producing a given
cytokine. To evaluate whether a specific commitment to secrete certain
Th1 or Th2 cytokines is found at the T cell level in HIV-infected
patients and to investigate the basis of alterations in the cytokine
pattern, we have performed a single-cell analysis of Th1 (IL-2,
IFN-, TNF-) and Th2 (IL-4, IL-5, IL-13) CD4- and CD8-producing
cells and have determined whether perturbations in the proportions of
CD45RA/CD45R0 T cells influence the cytokine pattern in HIV-infected
patients. In addition, we have asked whether alterations in the
representation of some Th1 subsets was the consequence of a
differential susceptibility to activation-induced apoptosis. Our
results indicate that there is no commitment of T cells to secrete Th2
cytokines in HIV-infected patients, but rather, a differential
alteration in the representation of Th1 subsets. In particular, a
significant decrease in the number of IL-2-producing T cells with a
preserved production of IFN- was observed throughout HIV infection,
and this was directly related to the shrinkage of the CD45RA T cell
compartment as the infection progressed. In addition, we report that
among the different Th1 subsets, there is a gradient of susceptibility
to activation-induced apoptosis, which is regulated by Bcl-2 expression
and which contributes to the dysregulation in type 1 cytokine
production in AIDS.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Peripheral blood samples were obtained from 64 HIV-1-infected
patients at the Service for Infectious Diseases, Bégin Military
Hospital, St. Mandé, France. Clinical characteristics of these
patients are shown in Table I
. None of
the patients studied received anti-HIV protease inhibitor, but some
of them received a bi-therapy consisting of the combination of two
inhibitors of the HIV reverse transcriptase, zidovudine +
didanosine; zidovudine + lamivudine; zidovudine +
zalcitabine; and stavudine + lamivudine or didanosine. Controls
(n = 22) were HIV-seronegative healthy blood
donors (Centre National de Transfusion Sanguine, Paris, France). PBMC
were isolated from heparinized blood by centrifugation on a
Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) density gradient, washed,
and resuspended in complete medium composed of RPMI 1640 (BioWhittaker,
Verviers, Belgium) supplemented with 10% heat-inactivated FCS
(Institut Jacques Boy, Reims, France), 10 IU/ml penicillin, 10 µg/ml
streptomycin, 20 mM HEPES, and 2 mM L-glutamine.
|
The mouse mAbs specific for human surface Ags used in this study
included: anti-CD3 (IgG1k, clone SK7), anti-CD4 (IgG1k, clone
SK3), anti-CD8 (IgG1k, clone SK1) (Becton Dickinson, Pont de Claix,
France); anti-CD45RA (IgG1, clone 2H4) and anti-CD45RO (IgG2ak,
clone UCHL1) (Becton Dickinson). All mAbs were directly coupled either
to phycoerythrin (PE)3
or to FITC, except for three-color immunofluorescence analyses, in
which anti-CD8 and anti-CD3 mAb conjugated with PerCP (Becton
Dickinson) were used. Control Abs, including mouse PE-, FITC-, or
PerCP-conjugated IgG1k or IgG2ak (Becton Dickinson) were used for cell
surface labeling. Intracellular Bcl-2 protein was detected using
anti-Bcl-2 mAb (IgG1k, clone 124; 0.2 mg) coupled to FITC purchased
from Dako (Glostrup, Denmark). Mouse FITC-conjugated IgG1k (Dako) was
used as control Ab for intracellular staining. For intracellular
detection of cytokines, the following mAbs were used: IL-2 clone
MQ1-17H12, IL-4 clone MP4-25D2, IL-5 clone TRFK5, IL-10 clone JES3-9D7,
IL-13 clone JES10-5A2, TNF- clone Mab11 (purchased from PharMingen,
La Jolla, CA); and IFN- clone 25-723 (obtained from Becton
Dickinson). All mAbs were conjugated with PE and used at 1/100
dilution, except the IL-4, which was diluted at 1/1000, and the IFN-
at 1/50.
Quantification of IL-2-, IFN--, and TNF-producing T cells by
intracellular detection of cytokines
PBMC from healthy donors or from HIV-infected patients were stimulated for 16 h with PPI (50 ng/ml PMA (Sigma Chemical Co., St. Louis, MO), 100 ng/ml PHA-A (Murex Diagnostic, Paris, France), and 300 ng/ml ionomycin (Sigma Chemical Co)). Brefeldin A (Sigma Chemical Co.) was added at a concentration of 10 µg/ml during the last 12 h to inhibit cytokine secretion. Enumeration at the single-cell level of cytokine-producing peripheral T cells was performed as previously described (30). Briefly, stimulated PBMC were washed in PBS containing 1% BSA and 0.1% sodium azide (PBS-BSA-NaN3) and incubated in the same buffer with FITC-conjugated anti-CD3 or anti-CD8 mAbs and 20 µg/ml of 7-amino-actinomycin D (7-AAD) (Sigma Chemical Co.). 7-AAD staining was performed to discriminate between living and apoptotic cell (including early apoptotic cells), as previously shown (31, 32). Stained cells were further washed in PBS-BSA-NaN3 containing 20 µg/ml of actinomycin D (AD; Sigma Chemical Co.) fixed in PBS-BSA-NaN3-AD containing 1% paraformaldehyde for 15 min at +4°C. Fixed cells were then permeabilized by incubation for 15 min with 0.05% (w/v) SAP (Sigma Chemical Co.) diluted in PBS-BSA-NaN3-AD, at room temperature. Intracellular cytokine staining was then performed with PE-conjugated anti-cytokine mAbs in SAP buffer containing 20 µg/ml of AD for 30 min at 4°C. Double-stained cells were washed in PBS-BSA-NaN3-AD, fixed with 1% paraformaldehyde in PBS-BSA-NaN3-AD, and immediately applied to a FACScan flow cytometer (Becton Dickinson). For each sample, 20,000 stained cells were acquired and analyzed with LYSYS II software (Becton Dickinson). Cytokine production was evaluated on both living and apoptotic T cells.
Combined analysis in peripheral T lymphocytes of their susceptibility to activation-induced apoptosis in relation to the cytokine produced
To analyze in blood T cells the relationship between the synthesis of a given cytokine and the propensity to undergo apoptosis, PBMC were stimulated 16 h with PPI and triple stained with FITC-conjugated anti-CD3 or anti-CD8 mAbs, PE-conjugated anti-cytokine mAbs, and the nuclear dye 7-AAD, as detailed above. The spectral properties of 7-AAD allow the staining of apoptotic cells by fluorescence emission in the red channel FL-3 (650 nm < wavelength < 850 nm), and the simultaneous labeling of cell surface Ags (FL-1 and FL-2) (31, 32). For each sample, 20,000 events were immediately placed on a FACScan flow cytometer, and apoptotic cells were quantified according to their 7-AAD staining. The combination of intracellular staining with anti-cytokine mAbs and nuclear staining with 7-AAD on stimulated T cells permitted analysis of the propensity of a given type 1 subset to undergo activation-induced cell death.
Bcl-2 intracellular staining and its relation to apoptosis
Intracellular detection of Bcl-2 protein within T cell subsets was performed as described previously (33). 5 x 105 PBMCs stimulated with PPI for 16 h were washed in PBS-BSA-NaN3 and incubated with PE- and/or PerCP-conjugated mAbs specific for T cell surface Ags, and in some experiments, with the nuclear dye 7-AAD to detect apoptosis, as described above. The stained cells were further washed in PBS-BSA-NaN3, fixed in 1% paraformaldehyde (Sigma Chemical Co.) for 20 min at room temperature, washed in PBS, and then incubated for 15 min at +4°C in PBS-BSA-NaN3 containing 0.05% (w/v) SAP (Sigma Chemical Co). They were then intracellularly stained with FITC-conjugated anti-Bcl-2 mAbs and, in some experiments, with PE-conjugated anti-cytokine mAbs, in SAP buffer for 30 min at RT. Finally, the labeled cells were washed in SAP buffer, fixed with 1% paraformaldehyde in PBS, and immediately applied to a FACScan flow cytometer (Becton Dickinson). The relation between Bcl-2 expression and the percentage of apoptotic (7-AAD+) T cells was analyzed for each cytokine-positive subset with LYSYS II software (Becton Dickinson).
Stastistical analyses
Statistical analyses included the Mann-Whitney test, Wilcoxon
matched pairs test, Spearman regression analysis, and
2 test. A p value < 0.05 was
considered significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
To measure accurately the Th1 and Th2 populations and to determine
their respective proportions at different stages of HIV infection, we
used a method of single-cell analysis by flow cytometry. PBMC from
control donors or HIV-infected persons were stimulated for 16 h
with PPI, and cells were surface stained with anti-CD3 mAbs and
intracellularly stained with mAbs against the Th1 cytokines IL-2,
IFN-, and TNF- and the Th2 cytokines IL-4, IL-5, and IL-13.
Figure 1A shows representative
stainings for IL-2, TNF-, and IFN- in stimulated PBMC of a
control donor and of an HIV-infected patient belonging to group 2 (13%
< CD4 < 28%). The proportion of T cells synthesizing IL-2 was
significantly decreased in the HIV-infected donor (18.5% of CD3 T
cells were IL-2+ compared with 56% in the control donor),
whereas the percentage of T cells synthesizing IFN- was slightly
increased (30.0% of CD3 T cells were IFN-+ in the
patient compared with 20% in the control donor). The proportion of T
cells synthesizing TNF- was decreased in the patient (14.0% of CD3
T cells were TNF-+ vs 35.0% in the control donor).
|
B). Indeed, the proportion of CD3 T cells producing
IL-2 was significantly lower in HIV-infected persons compared with
control donors (median value 21.0% in patients vs 37.0% in controls,
p < 0.0003). A decrease was also found in the
proportion of CD3 T cells synthesizing TNF- (median value 27.9% in
patients vs 33.4% in controls, p < 0.01). In
contrast, the percentage of T cells producing IFN- was not
significantly modified when the whole patient population (median value
17.2% in patients vs 16.4% in controls) was taken into consideration,
although an increase in IFN--producing T cells was observed in
patients in groups 2 and 3 vs group 1 (Fig. 1B). The
decreased proportion of T cells synthesizing IL-2 and TNF- in
HIV-infected persons was an early phenomenon, detected in patients with
mild immune deficiency (group 2, 13% < CD4 < 28%). It is
noteworthy that, both in control donors and in patients, T cells that
simultaneously produced two type 1 cytokines were hardly detected (data
not shown). A similar approach was used to quantify T cells producing type 2 cytokines. The detection of IL-4, IL-5, or IL-13+ T cells was performed in the PBMC of controls (n = 8) or patients (n = 18) stimulated for 16 h with PPI. The proportion of IL-4 or IL-5+ CD3 T cells was very low in control donors (median values 1.7% for IL-4 and <1% for IL-5) and remained unchanged in patients (median values 1.6% for IL-4 and <1% for IL-5). IL-10 production was always < 1%, in control donors as well as in HIV-infected patients. T cells producing IL-13 were not detected in controls or in patients from groups 1 or 2 (median value <1%). In contrast, in some advanced patients suffering from a hyper-IgE syndrome, IL-13+ T cells were detected and their proportion could represent 26% of total CD3 T cells. Type 2-producing T cells were mainly of the CD8 phenotype (data not shown).
Taken together, these observations indicate that, under these conditions of stimulation, the previously reported perturbations in Th1/Th2 balance throughout HIV infection (34) are not detected at the peripheral T cell level at early stages of HIV infection. The only type 2 cytokine detected was IL-13, but only in some AIDS patients with hyper-IgE syndrome.
Differential alteration in CD4 and CD8 subsets from HIV-infected
persons in their synthesis of IL-2, IFN-, and TNF
For each type 1 cytokine synthesized, the respective contribution
of the CD4 and CD8 T cell subsets in their synthesis was assessed.
Because of the down-modulation of the CD4 molecule on PPI-stimulated T
cells, the percentage of CD4 T cells producing a given cytokine was
considered as the percentage of CD3+CD8- cells
producing this cytokine. Figure 2 shows
the proportion of CD4 and CD8 T cells synthesizing IL-2, IFN- or
TNF- in 16hr-PPI stimulated PBMC. In control donors, IL-2 is mainly
produced by the CD4 T cell subset whereas IFN- and TNF- are
synthesized in similar proportions by both CD4 and CD8 subsets. In
HIV-infected patients, the proportion of CD4 T cells producing IL-2
strongly decreased, particularly at advanced stages of the infection.
In contrast, the ability of the CD8 subset to synthesize IL-2 was
unchanged in HIV-infected persons (Fig. 2). The global decrease in the
proportion of IL-2-synthesizing T cells observed in patients therefore
appears to be the consequence of both a reduced number of peripheral
CD4 T cells and a reduced capacity of the remaining CD4 T cells to
synthesize IL-2 upon activation.
|
was produced in similar proportions by CD4 and CD8 T cells in
controls’ PBMC whereas the CD8 subset generally contributed in a
greater proportion to IFN- production in patients’ PBMC (Fig. 2).
As reported above, IFN- production by T cells appeared therefore to
be preserved and sometimes enhanced throughout HIV infection, due to
both an increase in the number of CD8 T cells and an increased capacity
of these cells to produce IFN-. A different situation was found for
TNF-. Both CD4 and CD8 subsets equally contributed to TNF-
synthesis in control donors. In patients, a decreased proportion of CD8
T cells producing TNF- was observed (Fig. 2). The reduction in the
proportion of T cells able to produce TNF- throughout HIV infection
therefore appears to be the consequence of both a reduced capacity of
CD8 T cells to synthesize TNF- and a reduced number of peripheral
CD4 T cells. Heterogeneity in the synthesis of type 1 cytokines among CD45RA- and CD45RA+T cell subsets
Although the CD45RA compartment exhibits a lower proliferative
activity in response to recall Ags or TCR ligation, a recent study
showed that, following PMA + ionomycin activation,
CD45RA+ T cells have a low but significant capacity to
secrete several cytokines, mainly IL-2, TNF- and GM-CSF (4, 35). The
flow cytometric analysis at the single-cell level allows identification
of a possible commitment to cytokine production of a given subset. To
define whether the CD45RA+ and CD45RA- subsets
showed a differential ability to produce type 1 cytokines, PBMC from
healthy donors were stimulated with PPI and the synthesis of IL-2,
IFN- and TNF- was measured in the CD45RA+ subset
(Fig. 3). All three cytokines were
produced by both CD45RA+ and CD45RA- subsets,
but a distinct pattern was observed for each subset. The majority of
IL-2-producing CD3 T cells was detected in the CD45RA+
compartment whereas IFN- producers were mainly detected in
the CD45RA- compartment. The TNF- producers were found
equally distributed among CD45RA+ and CD45RA-
subsets (Fig. 3A).
|
B, more than 50% of IL-2 CD4 T cell
producers and more than 60% of IL-2 CD8 T cell producers were
CD45RA+ in most of the healthy donors tested This is in
striking contrast with the situation observed for the two other
cytokines, IFN- and TNF-, which appeared to be mainly synthesized
by the CD45R0 compartment in both CD4 and CD8 subsets (Fig. 3B). Surprisingly, in spite of some alterations in
the proportions of CD4 and CD8 T cells producing those cytokines in
HIV-infected patients (Fig. 1), their distribution in the CD45RA and
CD45R0 compartments were unchanged. In particular, IL-2 was still
primarily produced by CD45RA+ cells in both CD4 and CD8
subsets (Fig. 3B).
It was reported that CD45RA molecule could be re-expressed on T cell
clones following stimulation with PMA and ionomycin (36) and that
phenotypic conversion from CD45R0 to a CD45RA could occur after PHA
long-term in vitro stimulation (3). To verify the stability of CD45RA
expression on peripheral T cells activated by PPI, the experiments
reported on Figure 3 were also performed on PBMC selectively depleted
of CD45RA or CD45R0 T cells. Both CD45RA and CD45R0 phenotypes were
found to be quite stable after 16 h of PPI stimulation (data not
shown).
The decrease in IL-2 producers throughout HIV infection is directly related to the loss of naive CD45RA+CD4+ T cells
Recent studies demonstrated that HIV disease involves the loss of
naive CD4 and CD8 T cells leading to an altered naive/memory T cell
representation (13, 30). Since we observed that IL-2 was preferentially
produced by CD45RA+ T cells, we tested whether the
decrease in IL-2 producers throughout HIV infection was related to the
loss of CD45RA+ T cells. As shown in Figs. 4A, the loss of the CD45RA
compartment detected in HIV-infected persons paralleled the decrease in
the proportion of CD3 T cells synthesizing IL-2. Analysis of the
composition in cytokine-secreting cells following PPI stimulation of
the CD45RA compartment in both CD4 and CD8 subsets is shown in Figure 4B. The most striking difference in the composition of the
CD45RA subset in patients vs controls was observed for the production
of IL-2 by CD4 T cells. Indeed, the reduction in the size of the
CD4+CD45RA+ compartment in HIV disease was
associated with the loss of IL-2 producers which represented only a
mean of 39% of the CD4+CD45RA+ subset in
patients vs 56% in controls (Fig. 4B). However, a
reduction in the relative proportions of IFN-- and TNF--producing
T cells in the CD4+ CD45RA+ compartment,
although less dramatic, was also observed in patients (3% vs 6% for
IFN- and 25% vs 34% for TNF- in patients vs controls
respectively). Surprisingly, although decreased in size, the
CD8+CD45RA+ compartment in patients was not
modified with respect to its composition in cytokine-producing subsets
(Fig. 4B). The proportion of IL-2 producers
represented 24% of the CD8+CD45RA+ subset in
patients vs 27% in controls, and that of IFN- and TNF- was 16%
vs 18% and 19% vs 21% in patients and controls respectively. It is
noteworthy that a high proportion of CD45RA+ T cells in
patients did not secrete either IL-2 or IFN- or TNF- (Fig. 4B). This nonsecreting population was particularly
important in the CD4 subset of patients where it represented 33%
of the CD4+CD45RA+ T cells compared with 3% in
controls (p < 0.0001). Although large in the
CD8+CD45RA+ subset, this population was not
significantly different in patients and controls (Fig. 4B). In the CD45R0+ populations, the
nonsecreting population was only slightly increased in HIV-infected
patients (data not shown).
|
). As expected from the
previous observations, the shrinking of the CD45RA+ T cell
compartment has a direct consequence on the production of IL-2 by CD4 T
cells, since a statistically significant correlation was found between
the percentage of CD45RA+ T cells and the percentage of
IL-2-producing CD4 T cells. The production of IFN- and TNF- were
not dependent on the alteration of the CD45RA subset (Fig. 5).
Altogether, these observations indicate that the dramatic decrease in
IL-2 production throughout HIV infection might be primarily a
consequence of the loss of naive CD45RA+ T cells, which
does not affect the production of other type 1 cytokines predominantly
synthesized by the CD45R0+ subset.
|
CD4 and CD8 T lymphocytes of HIV-infected persons show an
increased susceptibility to spontaneous and activation-induced
apoptosis (37, 38), which is correlated with the in vivo activation
state of peripheral lymphocytes and with disease progression (14).
Because the fragility of CD45RA+ T cells was found to
be greater in patients compared with controls, although less than in
the CD45RO+ subset (14), we asked whether the disappearance
of IL-2-producing T cells throughout HIV infection was related to their
susceptibility to activation-induced apoptosis. The rate of apoptosis
among the different cytokine-producing T cell subsets was quantified by
flow cytometry. PBMCs were activated with PPI for 16 h and triple
stained with surface CD3-specific mAbs, intracellular cytokine-specific
mAbs, and with the nuclear dye 7-AAD for the detection of apoptotic
cells. The median rate of apoptosis in CD3 T cells under these
conditions of activation was 40.6% in patients compared with 18.8% in
controls (p < 0.0001). Moreover, this rate of
apoptosis in CD3 T cells was found to correlate in HIV-infected donors
with disease progression, as assessed by the ex vivo CD4 percentage
(r = -0.53, p < 0.0001,
n = 58). A representative analysis of apoptosis among
cytokine-producing CD3 T cells from control donors and HIV-infected
patients is shown on Figure 6A. Interestingly, we found an
important heterogeneity in the susceptibility to activation-induced
apoptosis of Th1 CD3 T cells in control donors. A low level of
apoptosis was detected in IL-2 producers, whereas IFN- and TNF-
producers showed a higher propensity to undergo apoptosis following
activation (p < 0.05 and p <
0.001 vs IL-2, respectively; Table II).
This differential susceptibility to apoptosis of Th1 CD3 T cells was
similarly observed in HIV-infected patients. However, the rate of
apoptosis in each subset was significantly higher for IL-2 producers
(p < 0.0001 vs controls); for IFN-
producers (p < 0.0001 vs controls); and for
the TNF- producers (p < 0.0001 vs
controls) (Table II). When this same analysis was performed in the CD4
and CD8 T cell subsets, the gradient in the degree of
susceptibility to apoptosis observed among cytokine-producing CD3 T
cells (IL-2 < IFN- < TNF-) was also found at the level of
the two T cell subsets (Fig. 6B). In control donors,
IL-2 producers appeared very resistant to activation-induced apoptosis
in both CD4 and CD8 subsets. In contrast, TNF- producers were highly
susceptible to apoptosis; this was particularly evident in the CD8
subset. CD4 and CD8 cells producing IFN- were in an intermediate
position, with marked fragility of these cells in several healthy
donors (Fig. 6B). In HIV-infected patients, this
differential fragility of type 1 cytokine-producers among CD4 and CD8 T
cells followed the same order, but the rate of activation-induced
apoptosis was significantly higher in some of these producers as
compared with their counterparts in control donors. In the CD4 subset,
the proportion of apoptotic cells was increased in IL-2- and
IFN--synthesizing cells, while not significantly different from
controls in TNF--producing cells. In the CD8 subset, only the
IFN- producers exhibited a significantly higher level of apoptosis
as compared with controls (Fig. 6B).
|
|
Following activation, T lymphocytes expand and differentiate into
effectors despite the expression of death factors. Signal transduction
pathways exist, therefore, which inhibit apoptosis through the
expression of survival factors such as proteins from the Bcl-2 family
(39). To determine whether differential Bcl-2 expression may account
for the heterogeneity in the susceptibility to apoptosis between the
different cytokine producers, we compared the intracellular expression
of Bcl-2 among these different subsets following PPI activation. We
previously reported that three levels of Bcl-2 expression were detected
on freshly isolated peripheral T cells, associated with differential
susceptibility to spontaneous and Fas-mediated apoptosis (30, 33).
Figure 7A shows the analysis
of intracellular Bcl-2 expression on the three different
cytokine-producing subsets following PPI activation of PBMCs. The
representative profiles in Figure 7 were obtained with T cells from an
HIV- infected donor, but were quite similar for T cells of
control donors. Striking differences in the proportions of low (L),
normal (N), and high (H) Bcl-2-expressing T cells were detected among
the three subsets. Interestingly, IL-2 producers expressed a very high
level of Bcl-2 (89% of them were Bcl-2 H). In contrast, IFN- and
TNF- producers were equally distributed in Bcl-2 N and H subsets
(Fig. 7A). The relation between Bcl-2 expression and
the susceptibility to activation-induced apoptosis was analyzed by
quantifying, with the 7-AAD dye, the proportion of apoptotic cells
within Bcl-2 L, N, and H subsets. Similar to the situation observed for
spontaneous and Fas-mediated apoptosis of peripheral T cells (30, 33),
a correlation was found between the level of Bcl-2 expression and the
propensity of PPI-activated T cells to undergo apoptosis: 94% of Bcl-2
L cells were apoptotic after 16 h of PPI activation vs only 14%
in the Bcl-2 H cells and 50% in the Bcl-2 N cells (Fig. 7B). When analyzing the three Th1 subsets after PPI
activation, a similar correlation was found between the level of Bcl-2
expression and the rate of apoptosis, the Bcl-2 (L + N) being more
susceptible to apoptosis than the Bcl-2 H cells (Fig. 7C). Consequently, as shown in Table II, the gradient
of susceptibility to activation-induced apoptosis among the cytokine
producers (IL-2 < IFN- < TNF-) was found correlated with a
gradient in Bcl-2 expression: the resistance to apoptosis of IL-2
producers was due to the very low proportion of Bcl-2 (L + N)
cells, whereas the greater susceptibility of IFN- and TNF-
producers was related to an increased proportion of Bcl-2 (L + N)
cells (Table II). In HIV infection, a global decrease in Bcl-2
expression was detected in the three Th1 subsets, and consequently, an
increased rate of apoptosis was observed for each Th1 subset compared
with its counterpart in control donors (Table II).
|
To assess whether increased apoptosis in cytokine-producing
subsets was correlated with the observed defects in cytokine
production throughout HIV infection, the percentage of
activation-induced apoptosis in a given Th1 subset was plotted against
the percentage of this Th1 subset among CD3 T cells. As shown in Figure 8 (upper panel),
the decrease in the number of IL-2 producers was significantly
associated with increased apoptosis in this subset. A similar trend was
observed for TNF-, whereas no correlation could be observed between
the rate of apoptosis in IFN- producers and their proportion. To
define whether the increased priming for apoptosis of Th1 subsets in
patients was related to disease progression, the percentage of
activation-induced apoptosis in each subset was plotted against the ex
vivo percentage of CD4 T cells. As shown in Figure 8, lower
panel, the increased rate of apoptosis in CD3 T cells synthesizing
IL-2 or TNF- significantly correlated with the ex vivo drop of CD4 T
cells, whereas no correlation was found between the rate of apoptosis
in IFN- producers and disease progression.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Single-cell analysis of IL-2-, IFN--, and TNF--producing cells at
the CD4 and CD8 T cell levels revealed that IL-2 was the only cytokine
in which synthesis was reduced at the CD4 T cell level in HIV-infected
patients (Fig. 2). Interestingly, our study reveals that IL-2 is, for
the most part, produced by CD4+CD45RA+ T cells
(Fig. 3). The reduction in the percentage of IL-2-producing cells
throughout HIV infection appears to be a consequence of the shrinkage
of the CD4+CD45RA+ compartment and is also
associated with the impairment in cytokine synthesis of the remaining
CD4+CD45RA+ cells (Fig. 4). The progressive
reduction of CD45RA+ T cells had no consequence on IFN-
synthesis. Indeed, our approach indicated that IFN- is mainly
produced by CD45R0 CD8 T cells, a subset that is expanded throughout
HIV infection (13). As a consequence, the proportion of
IFN--producing cells in HIV-infected patients was found unchanged
and sometimes increased as compared with healthy donors (Figs. 1 and 2). Although these data are discrepant with a few reports on CD4 T cell
clones or peripheral T cells (21, 29), they are in agreement with
others showing an increased capacity of patients’ T cells to produce
IFN- or to express IFN- mRNA (18, 22, 24, 25). In fact, in
agreement with a previous report on cytokine gene expression (18), our
data show that HIV infection is associated with a clear dissociation at
the T cell level in the patterns of IL-2 and IFN- synthesis,
indicating that HIV disease is associated with alterations among Th1
subsets. Such alterations appeared to be the consequence of
modifications in the proportions of CD45RA and CD45R0 subsets. The
polarization of CD45RA T cells to IL-2 synthesis and of CD45R0 T cells
to IFN- synthesis has already been described in healthy donors (2, 3), and recent studies have confirmed that cytokine polarization was an
intrinsic characteristic of these subsets (4). In HIV infection,
impairment of IL-2 synthesis is due to a quantitative and qualitative
alteration of the CD45RA subset. The influence of the size of the
CD45RA compartment on the type of cytokine secreted during diseases was
also reported in atopic patients in whom IgE and IgA production was
found to be associated with an increase in the CD45RA+ cell
number (45).
The powerful proinflammatory cytokine TNF-, produced by
monocytes/macrophages and also by T cells, may play an important role
in activating HIV replication in patients (46, 47). Contradictory
results on TNF- production in HIV infection have been reported and
may be attributable to different methodologic approaches (18, 23, 48, 49, 50). However, an increase in TNF- production by PBMCs appears to
be associated, in advanced stages of the disease, with the occurrence
of AIDS-associated pathologies and coinfections (51, 52). Using for the
first time the approach of single-cell analysis, we show a reduction in
the percentage of TNF--producing T cells in patients compared with
controls. This reduction, independent of the shrinkage of the CD45RA
compartment, was the consequence of the decreased capacity of CD8 T
cells to synthesize TNF- combined with the reduction in the number
of CD4 T cells producing this cytokine (Figs. 1 and 2). Since the
patients did not present with active coinfections at the time of our
study, this decreased TNF- production at the T cell level is
probably the direct consequence of HIV infection. The reduced synthesis
of TNF- in HIV-infected patients could be detrimental, in view of
reports in various models of viral infection showing that TNF- can
synergize with IFN- to express a potent antiviral activity (53) and
that TNF- favors the leukocyte infiltration in virally infected
tissues (54). This hypothesis is compatible with the significant
correlation we found between the reduction in the percentage of
TNF--producing T cells and the progression of disease (Fig. 8).
Because lymphocytes from HIV-infected individuals were shown to die by
apoptosis upon stimulation in vitro (14, 37, 38, 55), we asked whether
the alterations in the representation of the Th1 subsets was the
consequence of their differential susceptibility to activation-induced
apoptosis. Exogeneous cytokines can modulate the susceptibility of
lymphocytes from HIV-infected patients to the apoptotic process (56).
Our data suggest that the intrinsic capacity of lymphocytes to produce
a given cytokine upon activation can also influence their survival. We
observed that lymphocytes committed to IFN- or TNF- production
were more sensitive to activation-induced apoptosis than lymphocytes
committed to IL-2 production. A copriming for apoptosis and IFN-
production has been previously shown to occur during the intrathymic
process of negative selection, since double-positive thymocytes
undergoing apoptosis were shown to express in situ high level of
IFN- mRNA (57). This was also suggested to occur in single-positive
thymocytes (58). In human PBMC stimulated by cross-linking of the CD4
molecule, TNF- and IFN- production were associated with the
induction of apoptosis, which could be prevented by a cytokine
synthesis inhibitor (59). Other studies proposed a role for IFN- in
the promotion of activation-induced death, which was the consequence of
an autocrine process on Th1 clones (60, 61) and a paracrine process on
Th2 clones via the up-regulation of FasL expression (61). This
copriming for apoptosis and IFN- production has not been observed in
all cases (62). Nevertheless, a recent study has shown that the
regulation of IFN- synthesis was controlled by a cysteine protease,
caspase-1, which is involved in the apoptotic pathway (63). The
copriming of lymphocytes for apoptosis and IFN- synthesis in HIV
infection could therefore represent a regulatory mechanism involved in
the selective removal of specific Th effectors. In addition, the
preferential clearance by apoptosis of TNF--secreting cells might
contribute to limit the inflammatory process.
Several mechanisms could account for the gradient of
susceptibility to activation-induced apoptosis of the Th1 subsets
(TNF- > IFN- > IL-2), which we found in control donors as
well as in HIV-infected persons, in CD4+ as well in
CD8+ subsets (Fig. 6), and which was independent of the
CD45RA+ or the CD45RO+ nature of the producing
cells (data not shown). This gradient of susceptibility to
activation-induced apoptosis tightly correlated with the expression
level of the Bcl-2 molecule (Fig. 7). This control, by Bcl-2 molecule
regulation, of the survival of cytokine-producers is in agreement with
recent data demonstrating that apoptosis induced by cytokines
preferentially involves the ceramide pathway, regulated by Bcl-2,
rather than by the Fas-induced ICE (IL-1-ßconverting enzyme) pathway,
independent of Bcl-2 (64, 65). The influence of the Fas pathway on the
control of the differential susceptibility to apoptosis of Th1 subsets
could not been determined in our study, but several reports suggested
that the control of apoptosis by Fas pathway or by molecules from the
Bcl-2 family were independent (66, 67, 68). Discordant data were recently
reported concerning the possible involvement of the Fas system in the
survival of Th1 vs Th2 cells. The report that Th1 clones are more
susceptible to apoptosis than Th2 clones was found to be related to
increased levels of FasL expression on Th1 clones (6, 61, 69). However,
other studies did not confirm these observations (70). In a recent
report, Zhang et al. (7) found that Th1 and Th2 effectors express
comparable levels of Fas and FasL, but only Th2 effectors express high
levels of FAP-1, which may act to inhibit Fas signaling. Other members
of the TNF family can also transduce the death signal (71), and the
high level of apoptosis we observed in TNF-+
lymphocytes, especially in CD8+ T cells from healthy
donors, could be the consequence of an autocrine suicide of these cells
(72, 73, 74).
IL-2 has been shown to exhibit pro- or antiapoptotic properties
according to the cellular activation status and to the cellular
microenvironment. Lenardo et al. have shown that preincubation of mouse
T cells with IL-2 increased the susceptibility of the cells to
apoptosis after TCR stimulation (75), and the requirement for IL-2 was
confirmed in human T cells in a Fas-FasL-dependent model of apoptosis
(76). In contrast, IL-2 can rescue cells from apoptosis induced by
growth factor withdrawal (38); this rescue was shown to be controlled
by molecules from the Bcl-2 family (77, 78, 79). In this study, we showed
that the intrinsic property of IL-2+ cells to overexpress
Bcl-2, as compared with IFN-- or TNF--producing cells (Fig. 7),
enabled them to be resistant to activation-induced apoptosis in normal
donors. In HIV-infected patients, an increased susceptibility to
apoptosis was observed in IL-2 producers, which was related to a
down-regulation of Bcl-2 expression. The progressive decrease in the
proportion of IL-2-synthesizing T cells was found to be correlated with
their susceptibility to activation-induced apoptosis and disease
progression. On the other hand, IFN- synthesis was found preserved
in patients despite an increased rate of apoptosis in the
IFN--producing subset. Indeed, the increased cell death of IFN-
producers is probably counterbalanced by the increased percentage of
CD8+ T cells, the main source of IFN- producers.
Altogether, these data confirm the important relationship between
apoptosis in peripheral T cells and the functional collapse of the
immune system in AIDS (12, 14, 30).
Taking into consideration the powerful efficacy of the new antiretroviral drugs, important questions are raised concerning the functional restoration of the immune system in treated HIV-infected patients. Because the functional alteration of untreated patients’ T lymphocytes was associated with their defective IL-2 production (34), the recovery of a normal proportion of IL-2 producers should be monitored during the therapy. Preliminary observations on the restoration of the immune system of advanced patients under tri-therapies, including an anti-HIV protease, indicate that recovery of the CD45RA T cell population was not observed for most of the patients, at least after 1 year (80). Consequently, and according to recent data showing that the recovery of a normal proportion of CD4+ IL-2 producers was not observed in the absence of an increase in the CD45RA+ subset (E. Ledru, H. Lecoeur, and M. L. Gougeon, unpublished observations), it might be necessary to combine IL-2 therapy with antiretroviral therapy in patients showing very low levels of CD45RA T cells before treatment. Recent reports on in vivo administration of IL-2 in antiretroviral-treated HIV-infected patients showed the efficacy of such a substitutive therapy on the recovery of CD4 T cells (81, 82, 83). Since our data strongly indicate the existence of a close relationship between the CD45RA/CD45R0 ratio, the cytokine production, and the susceptibility to apoptosis, the combined follow-up of these parameters by single-cell analysis should be included in the monitoring of the immune system of patients submitted to antiviral or immune-based therapies.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Marie-Lise Gougeon, Unité d’Oncologie Virale, Département SIDA et Rétrovirus, Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris, Cédex 15 France. E-mail address:
3 Abbreviations used in this paper: PE, phycoerythrin; AD, actinomycin D; 7-AAD, 7-amino actinomycin D; PPI, phytohemagglutinin, phorbol myristate acetate, and ionomycin; FasL, Fas ligand; SAP, saponin.
Received for publication October 3, 1997. Accepted for publication December 5, 1997.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
production. J. Immunol. 140:1401.[Abstract]
and decreased IL-2 gene expression are associated with HIV infection. J. Immunol. 154:5031.
production by T cells from HIV-infected individuals: decreased IFN- in the presence of preserved IL-4 production. J. Immunol. 157:2712.[Abstract]
. J. Acquired Immune Defic. Syndr. Hum. Retrovirol. 10:462.[Medline]
induces expression of human immunodeficiency virus in a chronically infected T-cell clone. Proc. Natl. Acad. Sci. USA 86:2365. and interleukin-1ß in human immunodeficiency virus-seropositive subjects. J. Infect. Dis. 175:1507.[Medline]
and ß inhibit virus replication and synergize with interferons. Nature 323:819.[Medline]
and accessory cells. Eur. J. Immunol. 23:1623.[Medline]
plays a critical role in induced cell death of effector T cell: a possible third mechanism of self-tolerance. J. Exp. Med. 172:1735.- and ß-chains of the IFN- receptor on human Th1 and Th2 clones. J. Immunol. 159:206.[Abstract]
-inducing factor and regulates LPS-induced IFN- production. Nature 386:619.[Medline]
ß T lymphocytes for apoptosis. Nature 353:858.[Medline]
This article has been cited by other articles:
|
|
 |
|
  E. Priceputu, I. Rodrigue, P. Chrobak, J. Poudrier, T. W. Mak, Z. Hanna, C. Hu, D. G. Kay, and P. Jolicoeur The Nef-Mediated AIDS-Like Disease of CD4C/Human Immunodeficiency Virus Transgenic Mice Is Associated with Increased Fas/FasL Expression on T Cells and T-Cell Death but Is Not Prevented in Fas-, FasL-, Tumor Necrosis Factor Receptor 1-, or Interleukin-1{beta}-Converting Enzyme-Deficient or Bcl2-Expressing Transgenic Mice J. Virol., May 15, 2005; 79(10): 6377 - 6391. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-L. Perfettini, T. Roumier, M. Castedo, N. Larochette, P. Boya, B. Raynal, V. Lazar, F. Ciccosanti, R. Nardacci, J. Penninger, et al. NF-{kappa}B and p53 Are the Dominant Apoptosis-inducing Transcription Factors Elicited by the HIV-1 Envelope J. Exp. Med., March 1, 2004; 199(5): 629 - 640. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. I. Rutitzky, G. A. Mirkin, and M. J. Stadecker Apoptosis by Neglect of CD4+ Th Cells in Granulomas: A Novel Effector Mechanism Involved in the Control of Egg-Induced Immunopathology in Murine Schistosomiasis J. Immunol., August 15, 2003; 171(4): 1859 - 1867. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. B. Alimonti, T. B. Ball, and K. R. Fowke Mechanisms of CD4+ T lymphocyte cell death in human immunodeficiency virus infection and AIDS J. Gen. Virol., July 1, 2003; 84(7): 1649 - 1661. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. AKDIS, A. TRAUTMANN, S. KLUNKER, I. DAIGLE, U. C. KUCUKSEZER, W. DEGLMANN, R. DISCH, K. BLASER, and C. A. AKDIS T helper (Th) 2 predominance in atopic diseases is due to preferential apoptosis of circulating memory/effector Th1 cells FASEB J, June 1, 2003; 17(9): 1026 - 1035. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Itescu and R. John Interactions between the recipient immune system and the left ventricular assist device surface: immunological and clinical implications Ann. Thorac. Surg., June 1, 2003; 75(90060): S58 - 65. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Jekle, O. T. Keppler, E. De Clercq, D. Schols, M. Weinstein, and M. A. Goldsmith In Vivo Evolution of Human Immunodeficiency Virus Type 1 toward Increased Pathogenicity through CXCR4-Mediated Killing of Uninfected CD4 T Cells J. Virol., May 15, 2003; 77(10): 5846 - 5854. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. D. Douglas, S. Durako, K. E. Sullivan, M. Camarca, A.-B. Moscicki, and C. M. Wilson TH1 and TH2 Cytokine mRNA and Protein Levels in Human Immunodeficiency Virus (HIV)-Seropositive and HIV-Seronegative Youths Clin. Vaccine Immunol., May 1, 2003; 10(3): 399 - 404. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Ledru, M. Fevrier, H. Lecoeur, S. Garcia, S. Boullier, and M.-L. Gougeon A nonsecreted variant of interleukin-4 is associated with apoptosis: implication for the T helper-2 polarization in HIV infection Blood, April 15, 2003; 101(8): 3102 - 3105. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Nakamoto, S. Kaneko, and K. Kobayashi Increased susceptibility to apoptosis and attenuated Bcl-2 expression in T lymphocytes and monocytes from patients with advanced chronic hepatitis C J. Leukoc. Biol., July 1, 2002; 72(1): 49 - 55. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Akari, S. Bour, S. Kao, A. Adachi, and K. Strebel The Human Immunodeficiency Virus Type 1 Accessory Protein Vpu Induces Apoptosis by Suppressing the Nuclear Factor {kappa}B-dependent Expression of Antiapoptotic Factors J. Exp. Med., November 5, 2001; 194(9): 1299 - 1312. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. C. Braun, J. M. Wang, E. Lahey, R. L. Rabin, and B. L. Kelsall Activation of the formyl peptide receptor by the HIV-derived peptide T-20 suppresses interleukin-12 p70 production by human monocytes Blood, June 1, 2001; 97(11): 3531 - 3536. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. D. Badley, A. A. Pilon, A. Landay, and D. H. Lynch Mechanisms of HIV-associated lymphocyte apoptosis Blood, November 1, 2000; 96(9): 2951 - 2964. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Somma, L. Tuosto, M. S. G. Montani, M. M. Di Somma, E. Cundari, and E. Piccolella Engagement of CD4 Before TCR Triggering Regulates Both Bax- and Fas (CD95)-Mediated J. Immunol., May 15, 2000; 164(10): 5078 - 5087. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Ledru, N. Christeff, O. Patey, P. de Truchis, J.-C. Melchior, and M.-L. Gougeon Alteration of tumor necrosis factor-alpha T-cell homeostasis following potent antiretroviral therapy: contribution to the development of human immunodeficiency virus-associated lipodystrophy syndrome Blood, May 15, 2000; 95(10): 3191 - 3198. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Fernandez-Fresnedo, M. A. Ramos, M. C. Gonzalez-Pardo, A. L. M. de Francisco, M. Lopez-Hoyos, and M. Arias B lymphopenia in uraemia is related to an accelerated in vitro apoptosis and dysregulation of Bcl-2 Nephrol. Dial. Transplant., April 1, 2000; 15(4): 502 - 510. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y.-X. Zhao, G. Lajoie, H. Zhang, B. Chiu, U. Payne, and R. D. Inman Tumor Necrosis Factor Receptor p55-Deficient Mice Respond to Acute Yersinia enterocolitica Infection with Less Apoptosis and More Effective Host Resistance Infect. Immun., March 1, 2000; 68(3): 1243 - 1251. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Cicala, J. Arthos, A. Rubbert, S. Selig, K. Wildt, O. J. Cohen, and A. S. Fauci HIV-1 envelope induces activation of caspase-3 and cleavage of focal adhesion kinase in primary human CD4+ T cells PNAS, February 1, 2000; 97(3): 1178 - 1183. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H.-J. Ankersmit, N. M. Edwards, M. Schuster, R. John, A. Kocher, E. A. Rose, M. Oz, and S. Itescu Quantitative Changes In T-Cell Populations After Left Ventricular Assist Device Implantation : Relationship to T-Cell Apoptosis and Soluble CD95 Circulation, November 9, 1999; 100 (2009): II-211 - II-215. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. S. Varadhachary, M. E. Peter, S. N. Perdow, P. H. Krammer, and P. Salgame Selective Up-Regulation of Phosphatidylinositol 3'-Kinase Activity in Th2 Cells Inhibits Caspase-8 Cleavage at the Death-Inducing Complex: A Mechanism for Th2 Resistance from Fas-Mediated Apoptosis J. Immunol., November 1, 1999; 163(9): 4772 - 4779. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Alcouffe, S. Caspar-Bauguil, V. Garcia, R. Salvayre, M. Thomsen, and H. Benoist Oxidized low density lipoproteins induce apoptosis in PHA-activated peripheral blood mononuclear cells and in the Jurkat T-cell line J. Lipid Res., July 1, 1999; 40(7): 1200 - 1210. [Abstract] [Full Text]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
28%). Numbers
in each quadrant of the dot plots correspond to the percentage of PBMCs
in each quadrant. Numbers in bold correspond to the percentage of
cytokine-positive cells among CD3+ T cells. B,
Similar analysis on PBMCs from 24 healthy subjects and 63 HIV-infected
patients, classified according to their ex vivo CD4+
percentage. Boxes and whisker plots represent range, 25th and 75th
percentiles, and median value of the percentages of cytokine-positive
cells among CD3 T cells. Arrows compare groups when p value
is < 0.05 (Mann-Whitney test).
, control donors; 
, HIV-infected patients with
CD4 > 13%; •, HIV-infected patients with CD4 
