Differential Susceptibility to Activation-Induced Apoptosis
Among Peripheral Th1 Subsets: Correlation with Bcl-2
Expression and Consequences for AIDS Pathogenesis1
Eric Ledru,* Herve ´ Lecoeur,* Sylvie Garcia,* Thierry Debord,†and Marie-Lise Gougeon2*
It has been proposed that HIV infection is associated with an imbalance in Th1 and Th2 subsets. Recent reports indicate that Th1
and Th2 effectors differ in their susceptibility to activation-induced apoptosis. To determine whether increased T cell apoptosis in
HIV-infected patients contributes to alterations in cytokine synthesis, we performed single-cell analysis of type 1 and type 2
cytokine production by CD4 and CD8 T cells, simultaneously with detection of apoptosis. We demonstrate that a differential
alteration in representation of Th1 subsets, rather than commitment of T cells to secrete Th2 cytokines, occurs throughout HIV
infection. A significant decrease in the number of IL-2- or TNF-?-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-
The Journal of Immunology, 1998, 160: 3194–3206.
expression and differentiate into memory/effector CD45RO?cells,
which are able to proliferate following subsequent exposure to
recall Ags. CD45RA?and CD45RO?T cells differ in the require-
ment for Ag-dependent activation and in the pattern of cytokine
production (2–4). Furthermore, according to the cytokine environ-
ment in which they develop and the nature of the costimulatory
signals delivered by the APCs, naive T cells can differentiate into
effector cells with polarized patterns of cytokine production. Th1
cells secrete IL-2, IFN-?, and TNF-?, which are important medi-
ators of cellular immunity and are involved in activation of mac-
rophages; 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
cells can be divided into different functional subsets on
the basis of their expression of the CD45 isoforms (1).
Upon activation, naive T cells down-regulate CD45RA
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 imbal-
ance 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 (6–8).
Activation-induced apoptosis is involved in peripheral T cell
deletion and has been implicated in the loss of CD4 Th cells in
AIDS (9–12). HIV infection is characterized by a persistent im-
mune 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 al-
teration in T helper functions (14). This activation-induced dele-
tion 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 im-
mune 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 re-
sponse to Ag or mitogen stimulation or to TCR ligation, or a de-
crease in constitutive IL-2 mRNA expression, has been uniformly
reported (16–21). In contrast, IFN-? production was found to in-
crease (18, 22–25), 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 (27–29). The
pattern of cytokines produced by an effector population can now be
*Unite ´ d’Oncologie Virale, De ´partement SIDA et Re ´trovirus, Institut Pasteur, Paris,
and†Service des Maladies Infectieuses et Tropicales, Ho ˆpital Militaire Be ´gin, Saint
Mande ´, France
Received for publication October 3, 1997. Accepted for publication December
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by grants from the Agence Nationale de Recherche sur le
SIDA (ANRS), the Fondation pour la Recherche Me ´dicale (FRM), the CNRS, the
Pasteur Institute, and the European Community (EEC) (contracts No. BMH4-CT
97-2055 and ERB-IC15-CT97-0901). E.L. was supported by the Ministe `re Franc ¸ais
de la Coope ´ration and subsequently by the FRM. S.G. was also supported by the
2Address correspondence and reprint requests to Dr. Marie-Lise Gougeon, Unite ´
d’Oncologie Virale, De ´partement SIDA et Re ´trovirus, Institut Pasteur, 28 rue du Dr.
Roux, 75724 Paris, Ce ´dex 15 France. E-mail address: firstname.lastname@example.org
Copyright © 1998 by The American Association of Immunologists0022-1767/98/$02.00
analyzed using a single-cell analysis method that allows enumer-
ation of Th1/Th2 subsets derived from peripheral T cells stimu-
lated in short-term cultures and provides information on the num-
ber and the phenotype of cells that are potentially capable of
producing a given cytokine. To evaluate whether a specific com-
mitment 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 per-
turbations in the proportions of CD45RA/CD45R0 T cells influ-
ence 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 ac-
tivation-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 con-
tributes to the dysregulation in type 1 cytokine production
Materials and Methods
Human blood samples and lymphocyte isolation
Peripheral blood samples were obtained from 64 HIV-1-infected patients at
the Service for Infectious Diseases, Be ´gin Military Hospital, St. Mande ´,
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 di-
danosine. 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 ?g/ml streptomycin, 20 mM
HEPES, and 2 mM L-glutamine.
The mouse mAbs specific for human surface Ags used in this study in-
cluded: 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 phyco-
erythrin (PE)3or to FITC, except for three-color immunofluorescence anal-
yses, in which anti-CD8 and anti-CD3 mAb conjugated with PerCP (Bec-
ton 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 cyto-
kines, 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 iono-
mycin (Sigma Chemical Co)). Brefeldin A (Sigma Chemical Co.) was
added at a concentration of 10 ?g/ml during the last 12 h to inhibit cytokine
secretion. Enumeration at the single-cell level of cytokine-producing pe-
ripheral T cells was performed as previously described (30). Briefly, stim-
ulated 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 ?g/ml of 7-amino-actino-
mycin 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-NaN3containing 20 ?g/ml of actinomycin D (AD; Sigma
Chemical Co.) fixed in PBS-BSA-NaN3-AD containing 1% paraformalde-
hyde for 15 min at ?4°C. Fixed cells were then permeabilized by incu-
bation 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 ?g/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 pro-
duction 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
To analyze in blood T cells the relationship between the synthesis of a
given cytokine and the propensity to undergo apoptosis, PBMC were stim-
ulated 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 ? 105PBMCs stimulated with PPI for 16 h
were washed in PBS-BSA-NaN3and 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% para-
formaldehyde (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
3Abbreviations 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.
Table I. Clinical characteristics of the HIV-infected patients studied
(CD4 ? 28%)a
(13% ? CD4 ? 28%)
(CD4 ? 13%)
aPatients were grouped on the basis of their ex vivo CD4 percentage, according
to the reference values of the CDC proposal (84).
bAll quantitative data are expressed as median (25th percentile-75th percentile).
cRTI, association of two reverse transcriptase inhibitors.
3195The Journal of Immunology
containing 0.05% (w/v) SAP (Sigma Chemical Co). They were then intra-
cellularly 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 cy-
tokine-positive subset with LYSYS II software (Becton Dickinson).
Statistical analyses included the Mann-Whitney test, Wilcoxon matched
pairs test, Spearman regression analysis, and ?2test. A p value ? 0.05 was
HIV infection is associated with early alterations in the
proportions of T cells producing type 1 cytokines rather than an
enrichment in T cells producing type 2 cytokines
To measure accurately the Th1 and Th2 populations and to deter-
mine their respective proportions at different stages of HIV infec-
tion, we used a method of single-cell analysis by flow cytometry.
PBMC from control donors or HIV-infected persons were stimu-
lated 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 signifi-
cantly 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).
These alterations in type 1 cytokine production were confirmed
when a panel of 62 HIV-infected patients was analyzed (Fig. 1B).
Indeed, the proportion of CD3 T cells producing IL-2 was signif-
icantly lower in HIV-infected persons compared with control do-
nors (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 syn-
thesizing 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 pro-
duced 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 (me-
dian 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 synthe-
sized 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 in-
fection. In contrast, the ability of the CD8 subset to synthesize IL-2
was unchanged in HIV-infected persons (Fig. 2). The global de-
crease 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.
IFN-? was produced in similar proportions by CD4 and CD8 T
cells in controls’ PBMC whereas the CD8 subset generally con-
tributed 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 produc-
ing TNF-? was observed (Fig. 2). The reduction in the proportion
of T cells able to produce TNF-? throughout HIV infection there-
fore appears to be the consequence of both a reduced capacity of
CD8 T cells to synthesize TNF-? and a reduced number of pe-
ripheral 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 cy-
tokines, mainly IL-2, TNF-? and GM-CSF (4, 35). The flow cy-
tometric 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
3196 DIFFERENTIAL SUSCEPTIBILITY TO APOPTOSIS AMONG Th1 SUBSETS
in the CD45RA?compartment whereas IFN-? producers were
mainly detected in the CD45RA?compartment. The TNF-? pro-
ducers were found equally distributed among CD45RA?and
CD45RA?subsets (Fig. 3A).
The important contribution of the CD45RA?subset in the pro-
duction of IL-2 was observed in both the CD4 and CD8 subsets. As
shown in Figure 3B, 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). Surpris-
ingly, 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 compart-
ments were unchanged. In particular, IL-2 was still primarily pro-
duced 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
the presence of PPI. Brefeldin A was added during the last 12 h of culture. Cells were first stained with anti-CD3 mAbs, then permeabilized and stained
with anti-cytokine mAbs. Analysis of cytokine production was performed on total (living ? apoptotic) T cells. A, FACScan dot plots from representative
control and HIV-infected patient from group 2 (13% ? CD4 ? 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
Single-cell analysis of CD3 T cells producing cytokines. PBMCs from healthy subjects or HIV-infected donors were cultured for 16 h in
3197 The Journal of Immunology
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 pref-
erentially 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 par-
alleled the decrease in the proportion of CD3 T cells synthesizing
IL-2. Analysis of the composition in cytokine-secreting cells fol-
lowing PPI stimulation of the CD45RA compartment in both CD4
and CD8 subsets is shown in Figure 4B. The most striking differ-
ence in the composition of the CD45RA subset in patients vs con-
trols was observed for the production of IL-2 by CD4 T cells.
Indeed, the reduction in the size of the CD4?CD45RA?compart-
ment 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 reduc-
tion in the relative proportions of IFN-?- and TNF-?-producing T
cells in the CD4?CD45RA?compartment, although less dra-
matic, was also observed in patients (3% vs 6% for IFN-? and 25%
vs 34% for TNF-? in patients vs controls respectively). Surpris-
ingly, although decreased in size, the CD8?CD45RA?compart-
ment 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 pa-
tients 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 ofpatientswhere it
CD4?CD45RA?T cells compared with 3% in controls (p ?
0.0001). Although large in the CD8?CD45RA?subset, this pop-
ulation 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
To determine the relationship between the loss of CD45RA?T
cells and the alterations in cytokine production throughout HIV
infection, the proportion of CD45RA?T cells was plotted against
the percentages of cytokine-producing cells in CD4 or CD8 T cells
(Fig. 5). 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 signif-
icant 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 alter-
ation of the CD45RA subset (Fig. 5). Altogether, these observa-
tions 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 pro-
duction of other type 1 cytokines predominantly synthesized by the
Differential susceptibility to activation-induced apoptosis of Th1
CD4 and CD8 T lymphocytes of HIV-infected persons show an
increased susceptibility to spontaneous and activation-induced ap-
optosis (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
stimulated as described in Figure 1. Percentages of cytokine-positive CD4 T cells were obtained on CD3?CD8?T cell subsets, because of the down-
regulation of the CD4 molecule. ‚, control donors; ?, HIV-infected patients with CD4 ? 13%; ?, HIV-infected patients with CD4 ? 13%. Horizontal
bars show median values. Statistical comparisons were performed using the Mann-Whitney test between controls and patients and the Wilcoxon matched
pairs test between T cell subsets from the same group of donors. Arrows indicate p ? 0.05.
Comparative analysis of cytokine production by CD4 T cells and CD8 T cells. PBMCs from 9 controls and 18 HIV-infected patients were
3198 DIFFERENTIAL SUSCEPTIBILITY TO APOPTOSIS AMONG Th1 SUBSETS
their susceptibility to activation-induced apoptosis. The rate of ap-
optosis 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, intracel-
lular 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). More-
over, 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 heter-
ogeneity 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 acti-
vation (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 produc-
ers (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
kine production. PPI-stimulated PBMCs were dual stained with anti-CD3
(or anti-CD8) mAbs and anti-CD45RA mAbs. Cells were then permeabil-
ized and intracellularly stained with anti-cytokine mAbs. A, Histograms
from one representative control donor. The proportion of CD3 T cells
synthesizing IL-2, TNF-?, and IFN-? was 39, 23, and 11%, respectively.
The percentages of CD45RA?T cells among each cytokine-producing
subset are indicated. B, Similar analysis performed on CD4 T cells and on
CD8 T cells from 7 controls ? and 11 HIV-infected patients (?). Hori-
zontal bars show median values. Arrows indicate statistical differences be-
tween the percentages of cytokine-producing subsets (Wilcoxon matched
pairs test, p ? 0.05). No significant difference was observed between pa-
tients and controls for each cytokine subset (Mann-Whitney test).
Contribution of the CD45RA?T cell population to cyto-
partment and the proportion of IL-2 producers in HIV infection. A, Per-
centages of CD45RA cells among total PBMCs or IL-2 T cells among CD3
T cells from 24 controls or 63 patients. Histograms represents the mean
values and SDs of the ex vivo percentage of CD45RA?cells among
PBMCs. Numbers represent the clinical groups of HIV-infected pa-
tients, and C represents control donors. B, Vertical axis compares the
percentages of CD45RA?cells among CD4 or the CD8 T cells from 7
controls or 11 patients. The proportion of IL-2-, IFN-?-, or TNF-?-
producing cells among the CD45RA T cells is indicated. T cells se-
creting none of these three cytokines are indicated as Other. Arrows
indicate significant statistical comparisons calculated by the Mann-
Whitney test. *, ?2test, p ? 0.0001.
Relationship between the decrease in the CD45RA?com-
3199The Journal of Immunology
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 sig-
nificantly 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-?-synthesiz-
ing 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).
The differential susceptibility to activation-induced apoptosis of
Th1 subsets is related to variable levels of Bcl-2 expression
Following activation, T lymphocytes expand and differentiate into
effectors despite the expression of death factors. Signal transduc-
tion pathways exist, therefore, which inhibit apoptosis through the
expression of survival factors such as proteins from the Bcl-2 fam-
ily (39). To determine whether differential Bcl-2 expression may
account for the heterogeneity in the susceptibility to apoptosis be-
tween the different cytokine producers, we compared the intracel-
lular 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 in-
tracellular Bcl-2 expression on the three different cytokine-produc-
ing 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 sub-
sets (Fig. 7A). The relation between Bcl-2 expression and the sus-
ceptibility to activation-induced apoptosis was analyzed by quan-
tifying, 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 ex-
pression 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
of CD45RA?T cells among the CD4 or the CD8 subset were plotted against the percentage of cytokine-positive cells among the same T cell subsets from
14 HIV-infected donors. Correlations were determined using the Spearman regression analysis. ?, CD4 T cells; ?, CD8 T cells; NS, not significant.
Correlation between the percentages of Th1 effectors and the proportion of CD45RA?T cells within CD4 and CD8 subsets. The percentages
3200DIFFERENTIAL SUSCEPTIBILITY TO APOPTOSIS AMONG Th1 SUBSETS
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).
intracellular staining with anti-cytokine mAbs and nuclear staining with 7-AAD. A, Representative dot plots of the staining on CD3T cells from a control
and an HIV-infected donor of group 2 are shown. The number in the left quadrant indicates the percentage of corresponding cytokine-positive cells among
living cells; the number in the right quadrant indicates the percentage of cytokine-positive cells among apopotic cells. The number in brackets indicates
the proportion of apoptotic cells among the corresponding cytokine-positive subset. B, Analysis was performed on CD4 and CD8 subsets from 9 controls
and 19 HIV-infected donors; the percentage of apoptotic cells in each cytokine-positive subset is shown. Horizontal bars show median values. Arrows
indicate statistical differences between the percentages of cytokine-producing subsets (Wilcoxon matched pairs test, p ? 0.05). The p values at the bottom
of the figure indicate the statistical comparisons between controls and HIV-infected donors (Mann-Whitney test).
Differential susceptibility to activation-induced apoptosis of Th1 subsets. PPI-stimulated PBMC were analyzed after a combination of
3201 The Journal of Immunology
The rate of apoptosis in IL-2-producing T cells is associated
with disease progression
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 pro-
ducers was significantly associated with increased apoptosis in this
subset. A similar trend was observed for TNF-?, whereas no cor-
relation 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 apo-
ptosis in each subset was plotted against the ex vivo percentage
of CD4 T cells. As shown in Figure 8, lower panel, the in-
creased 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.
HIV infection is characterized by the progressive disappearance of
the CD45RA?naive compartment and a relative increase in num-
bers of activated/memory CD45R0?cells (13). In the present
study, we first investigated how perturbations in the proportions of
these two populations would contribute to the modified pattern of
cytokine production during HIV infection. To analyze the actual
capacity of peripheral cells to produce a given cytokine, without
any in vitro selection by multiple restimulation, we enumerated
cytokine-producing cells by single-cell analysis after a short period
Table II. Susceptibility to apoptosis and Bcl-2 expression by CD3?T
cells according to cytokine productiona
(n ? 20)
% (L ? N) Bcl-2b
(n ? 6)
(n ? 56)
% (L ? N) Bcl-2b
(n ? 8)
8.7 ? 1.4
16.7 ? 3.3c
21.5 ? 3.8d
4.6 ? 1.8
16.8 ? 8.0c
24.9 ? 4.7d
21.6 ? 1.7
32.9 ? 1.8c
40.0 ? 2.1d
11.0 ? 1.9
34.6 ? 5.1c
34.7 ? 4.4c
aPBMCs, stimulated for 16 h with PMA, ionomycin, and PMA (in the presence
of brefeldin A during the last 12 h of culture), were stained with anti-CD3 mAbs (and
7-AAD for apoptosis quantification), then permeabilized and stained intracellularly
with anti-cytokine mAbs (and anti-Bcl-2 mAbs for study of Bcl-2 expression). Anal-
ysis was performed on the CD3?subset.
bMean ? SEM. L, low; N, normal.
cp ? 0.05 vs IL-2-producing cells (Wilcoxon matched pairs test).
dp ? 0.05 vs IL-2- and IFN-?-producing cells.
Intracellular detection of Bcl-2 protein within CD3 T cells according to their pattern of cytokine production. Three subsets were defined according to their
level of Bcl-2 expression: L, low Bcl-2 cells; N, normal Bcl-2 cells; H, high Bcl-2 cells. Numbers indicate the percentage of cytokine-positive cells in each
Bcl-2 subset. Data are from a representative experiment with PBMC from an HIV-infected donor from group 2. Similar results are observed with T cells
from control donors. B, Relationship between the susceptibility to apoptosis of CD3 T cells, as determined by 7-AAD incorporation, and the level Bcl-2
expression. Numbers indicate the percentage of apoptotic cells within L, N, or H Bcl-2 subsets. C, Relationship between Bcl-2 expression and the
susceptibility to apoptosis of each cytokine-producing subset. Numbers indicate the percentages of apoptotic cells within the H or (L ? N) Bcl-2 subsets.
Relationship between the differential susceptibility to activation-induced apoptosis of Th1 subsets and their level of Bcl-2 expression. A,
3202DIFFERENTIAL SUSCEPTIBILITY TO APOPTOSIS AMONG Th1 SUBSETS
of polyclonal stimulation. We report that significant alterations in
the proportions of T cells producing type 1 cytokines was observed
in patients upon HIV infection, rather than a shift from Th1 to Th2
subsets as proposed by Clerici et al. (34). Using this approach, we
found that peripheral T cells from control donors are not primed
for Th2 cytokines, since a very low proportion produce IL-4, IL-5,
or IL-13. Similar low proportions of Th2 cells were found in HIV-
infected donors. The only exceptions were observed for several
AIDS patients with hyper-IgE or Job-like syndrome, who showed
an increased proportion of peripheral IL-13 producers. When de-
tected, Th2 cytokines were mainly produced by CD8 T cells (data
not shown); our data are compatible with the reports of CD8?T
cells with a type 2 phenotype in HIV-infected persons with hyper-
IgE syndrome (40, 41) and with other reports failing to detect
increased IL-4 production in stimulated PBMC, purified CD4 T
cells, or in CD4 T cell clones from HIV-infected patients (21, 28,
42–44). Nevertheless, our study does not rule out the possibility of
an increased type 2 cytokine synthesis by non-T cells during HIV
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
revealsthatIL-2 is,for the
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?compart-
most part, producedby
ment and is also associated with the impairment in cytokine syn-
thesis of the remaining CD4?CD45RA?cells (Fig. 4). The pro-
gressive 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 ex-
panded throughout HIV infection (13). As a consequence, the pro-
portion 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 ca-
pacity 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, im-
pairment 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
percentage of activation-induced apoptosis in a given Th1 subset was plotted against the percentage of this Th1 subset among CD3 T cells. B, The
percentage of activation-induced apoptosis in each subset was plotted against the ex vivo percentage of CD4 T cells. Correlations were determined using
the Spearman regression analysis. 63 patients were tested. NS, not significant.
The decrease in the percentage of IL-2 producers and their susceptibility to apoptosis is associated with disease progression. A, The
3203The Journal of Immunology
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 impor-
tant role in activating HIV replication in patients (46, 47). Con-
tradictory results on TNF-? production in HIV infection have been
reported and may be attributable to different methodologic ap-
proaches (18, 23, 48–50). However, an increase in TNF-? pro-
duction 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 compart-
ment, was the consequence of the decreased capacity of CD8 T
cells to synthesize TNF-? combined with the reduction in the num-
ber 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 ac-
tivity (53) and that TNF-? favors the leukocyte infiltration in vi-
rally infected tissues (54). This hypothesis is compatible with the
significant correlation we found between the reduction in the per-
centage of TNF-?-producing T cells and the progression of disease
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 mod-
ulate 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 activa-
tion can also influence their survival. We observed that lympho-
cytes committed to IFN-? or TNF-? production were more sensi-
tive to activation-induced apoptosis than lymphocytes committed
to IL-2 production. A copriming for apoptosis and IFN-? produc-
tion 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 pre-
vented by a cytokine synthesis inhibitor (59). Other studies pro-
posed 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 apopto-
sis 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 prefer-
ential clearance by apoptosis of TNF-?-secreting cells might con-
tribute to limit the inflammatory process.
Several mechanisms could account for the gradient of sus-
ceptibility 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 cy-
tokine-producers is in agreement with recent data demonstrating
that apoptosis induced by cytokines preferentially involves the cer-
amide pathway, regulated by Bcl-2, rather than by the Fas-induced
ICE (IL-1-?converting 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 (66–68). Discordant data were re-
cently 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 (72–74).
IL-2 has been shown to exhibit pro- or antiapoptotic properties
according to the cellular activation status and to the cellular mi-
croenvironment. 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 (77–79). 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 pro-
gressive 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 in-
creased rate of apoptosis in the IFN-?-producing subset. Indeed,
the increased cell death of IFN-? producers is probably counter-
balanced by the increased percentage of CD8?T cells, the main
source of IFN-? producers. Altogether, these data confirm the im-
portant 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 an-
tiretroviral drugs, important questions are raised concerning the
functional restoration of the immune system in treated HIV-in-
fected patients. Because the functional alteration of untreated pa-
tients’ T lymphocytes was associated with their defective IL-2 pro-
duction (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
3204 DIFFERENTIAL SUSCEPTIBILITY TO APOPTOSIS AMONG Th1 SUBSETS
observed for most of the patients, at least after 1 year (80). Con-
sequently, 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 treat-
ment. Recent reports on in vivo administration of IL-2 in antiret-
roviral-treated HIV-infected patients showed the efficacy of such a
substitutive therapy on the recovery of CD4 T cells (81–83). Since
our data strongly indicate the existence of a close relationship be-
tween 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 mon-
itoring of the immune system of patients submitted to antiviral or
The authors thank Dr. Je ´rome Salomon for help in the collection of clinical
data, Dr. Se ´verine Boullier for advice in the intracellular cytokine staining
procedure, and Dr. Honami Naora for helpful comments on the manuscript.
We particularly acknowledge Dr. Luc Montagnier for his constant support
during this study.
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