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IMMUNOBIOLOGY
From Unité de Biologie des Rétrovirus,
Institut Pasteur, Paris, France.
The sequence of events and the mechanisms leading to the
destruction of the thymus during human immunodeficiency virus (HIV) infection are still poorly characterized. Investigated here are the
survival capacity on HIV-1 infection of the mature single-positive CD4+CD8 Depletion of CD4 T lymphocytes is a characteristic
feature of immune dysfunction during the progression of human
immunodeficiency virus (HIV) infection. Multiple mechanisms, such as a
permanent destruction1 and failure of reconstitution of
this T-cell pool,1-3 likely contribute to this depletion.
The contribution of the thymus to T-cell reconstitution during HIV
infection has been suggested by the prevalence of abundant thymic
tissue in 50% of HIV-1 infected adults4 and a larger
increase in the number of naive T cells after HAART in these
patients compared with those who have minimal thymic
tissue.5,6 The determination by polymerase chain reaction of T-cell rearrangement excision circles (TRECs) has been proposed as a way to quantify recent thymic emigrants in peripheral
blood.7 Using this approach, a low TRECs concentration was
shown to be an important predictive factor for HIV-1 disease
progression.8 The extent of thymic function is also
dependent on disease stage, as shown in macaques infected by the simian
immunodeficiency virus (SIV). For instance, a transient rebound in
thymic activity, characterized by an increase in the influx of
CD34+ precursors, was demonstrated during primary infection
and was followed, at a later stage, by thymus damage.9
Late thymic failure is reminiscent of the fact that, among patients
with advanced disease administered antiretroviral therapies, CD4 cell
counts often remain below normal despite long-term suppression of viral load.10,11
Furthermore, thymuses from pediatric patients who experienced
accelerated disease processes12,13 or from adults with
acquired immunodeficiency syndrome (AIDS)14,15 showed
severe thymocyte depletion associated with a profound disorganization
of the thymic epithelial network. Such alterations correlate with the
presence of the virus in the thymuses of humans16-18 and
animals, among them infected macaques19,20 and SCID-hu
mice.21,22
As a possible explanation for thymocyte depletion, it has been
suggested that CD34+ progenitors from HIV-infected patients
have a reduced capacity to colonize the thymus.23 However,
thymic infection also results in increased death rates of thymocytes in
human and animal models. Nevertheless, there are some discrepancies in
the literature regarding thymocyte subsets killed on infection of the
thymus and on the mechanisms involved in their death. In SCID-hu mice,
depletion of CD4+ thymocytes was attributed to the direct
killing of infected cells by a mechanism distinct from apoptosis,
whereas later stages of infection were characterized by increased
apoptosis of the CD4 This paper aims at studying the mechanisms controlling the survival
capacity of human thymocytes on HIV infection in the presence of
factors of the thymic microenvironment. We previously demonstrated that
the interaction of human thymocytes with thymic epithelial cells is
crucial for HIV replication.27 This interaction leads to
the cosecretion of 2 crucial cytokines for viral replication, tumor
necrosis factor (TNF) and interleukin-7 (IL-7), that further synergize
with IL-6, IL-1, and GM-CSF.27,28 However, this
interaction is efficient only with mature single-positive
CD4+CD8 We also showed that the deleterious effect of the virus on monocytic or
lymphocytic cells was mediated by oxidative stress, leading to a
decrease of Bcl-2 and, consequently, to apoptosis.29 However, in cells in which persistent infection can be established, compensatory mechanisms take place to maintain or increase the Bcl-2
level.29,30
In this study, we focused on the survival capacity of 2 thymocyte
subsets Reagents
Antibodies used for the selection of the thymocyte subset.
The monoclonal antibodies (mAbs) used were CD3 (X35), CD3 (UCHT1), CD8
(B9.11), CD83 (HB15A), CD10 (ALB 1), CD34 (QBEND 10), all purchased
from Beckman Coulter France S.A. Villepinte, and CD14 (M Antibodies used for immunostaining.
To characterize thymic subpopulations, we used the following conjugated
mAbs: Opticlone CD4-fluorescein isothiocyanate (FITC)/CD8-phycoerythrin (PE)/CD3-PE-CY5 (13B8.2/B9.11/UCHT-1), CD10-FITC (ALB 2), CD34-PE (QBEND-10) (Beckman Coulter France S.A), CD83-PE (HB15a), and CD14-FITC
(M Antibodies used for Western blot analysis.
Monoclonal anti-Bcl-2 (Bcl-2/100) from BD-PharMingen and
anti- Cytokines.
All the human recombinant cytokines (R&D Systems, Minneapolis, MN) were
used at the following concentrations: IL-6 and IL-1 Preparation of thymocyte subpopulations and culture
Infection of thymocytes
Measurement of apoptosis Apoptotic cells were labeled with a YO-PRO-1 iodide solution (1/200 in phosphate-buffered saline) (Interchim-Molecular Probes, Eugene, OR) or by terminal dUTP nick-end labeling (TUNEL) using a kit from Roche Molecular Biochemicals (Mannheim, Germany). The percentage of apoptotic cells was also determined on the basis of the forward scatter-side scatter profiles as previously described.37 For all techniques, analysis was carried out by flow cytometry using a XL-4C cytofluorometer (Beckman Coulter France S.A.).Western blot analysis Preparation of total extracts of thymocytes and Western blot analysis were performed as previously described.29 Densitometric analysis of the autoradiograms was carried out with a Gene Genius GG system imager (Ozyme-Syngène, Saint-Quentin Yveline, France).Statistical analysis Because of the variability in the studied parameters displayed by the different thymuses from different donors, we performed statistical analysis using the nonparametric Mann-Whitney U test. Significance level was set at P < .05.
Mature SP CD4+ thymocytes exhibited a strong capacity to survive during HIV-1 infection, in contrast to immature thymocytes We aimed first at determining ex vivo the levels of constitutive and virus-induced apoptosis of the mature single positive CD4+ CD8 CD3+ (SP
CD4+) and intermediate
CD4+CD8CD3 thymocytes, namely
the 2 subsets able to produce the virus in the thymic
microenvironment.28 Mature SP CD4+ and
immature thymocytes, the latter isolated as a pool of intermediate and
TN cells for technical convenience, were infected with a primary isolate, HIV-1B-LAIp. The cells were then maintained in
culture in the presence of IL-1, IL-6, IL-7, and TNF. In this first
experiment, the cytokines were used at the concentrations leading to
the highest level of HIV replication in each subset. Apoptosis data
presented here were obtained with the Yopro dye technique that was
perfectly related to morphologic analysis of cells based on forward
scatter-side scatter profiles. Similar results were obtained with
TUNEL analysis (data not shown). As shown in Figure
1A, the mature SP CD4+
thymocytes displayed ex vivo a relatively low constitutive rate of
apoptosis that was stable or slightly increased by infection. In
contrast, the immature subset exhibited a higher constitutive rate of
apoptosis, and these cells massively died within a few days of
infection. On the basis of 5 independent experiments, we performed
statistical analysis confirming that the difference of apoptotic levels
between control and infected cells was significantly higher in immature
than in SP CD4+ thymocytes (P < .01). Similar
resistance of SP CD4+ cells to virus-induced apoptosis was
observed using the HIV molecular clone NL4-3 (data not shown).
Furthermore, the survival capacity of mature SP CD4+ was
higher than that of immature cells in spite of higher virus production,
as revealed by p24 analysis (Figure 1B, P < .01). The
major determinant of this higher replication level in the SP
CD4+ subset is the activation level induced by TNF and IL-7
in these cells.28 There is no implication of the
expression level of the CXCR4 coreceptor used by
HIV-1B-LAIp, which is even less expressed in SP
CD4+ thymocytes than in immature thymocytes
(38,39 and data not shown).
Immature thymocytes are permissive to HIV infection at the
intermediate stage and die of apoptosis, particularly at the
CD4+CD8+CD3 CD8CD3) and intermediate
(CD4+CD8CD3). However after 11 days in culture (Figure 2, day 11), under control conditions, we
observed a decrease in the percentage of the TN and intermediate
thymocytes and the appearance of a large subset of double positive
CD4+CD8+CD3 (DP
CD3) and a few DP CD3low cells, demonstrating
a differentiation process within the culture. However, this process was
not induced to completion because the DP CD3high stage was
not attained and the SP CD4 or CD8 stage was seen only for a low
percentage of cells. Of particular interest, on HIV-1 infection, a
strong decrease in the percentage of the DP CD3 subset
occurred (P < .002), whereas an increase in the
percentage of the TN subpopulation (P < .001) and no
modification in the percentage of intermediate cells were observed
(Figure 2, day 11). These data indicate that the proportion of TN cells
among the pool of immature cells increased because the cells were not permissive to infection, whereas the percentage of intermediate cells
was unchanged because of the equilibrium between a higher rate of
differentiation from TN cells and a moderate rate of death from
infection. A more thorough analysis of the intermediate thymocytes revealed a differential survival capacity inversely related to the
level of expression of CD4 (data not shown). We observed that cells
expressing the lowest levels of CD4 and closest to the TN stage
survived infection best, in contrast to cells expressing the highest
levels of CD4 and closest to the DP stage. However, we can likely rule
out the possibility that the depletion of DP cells could have been
caused by a blockade of differentiation by the virus at the
intermediate stage because a high proportion of these cells was already
present early in the course of infection for instance, at 7 days (data
not shown)before the occurrence of virus-induced apoptosis (Figure
1). In conclusion, the death rate of immature thymocytes strongly
increased once differentiation into DP cells was achieved.
Tumor necrosis factor-
Level of interleukin-7R expression on thymocytes correlates with their differential survival capacity on infection The role of IL-7 as an antiapoptotic cytokine in infected thymocytes and the sensitivity to apoptosis of the distinct thymocyte subsets (Figure 2) suggested a difference in their responses to IL-7. We then investigated the expression of IL-7 receptor (IL-7R) in the DP CD3, immature (TN and intermediate), and SP
CD4+ thymocytes. To emphasize the detection of IL-7R, which
is internalized or masked by IL-7 produced in the thymic
microenvironment (data not shown), flow cytometry analysis was
performed on cells cultured 24 hours after cell isolation in the
absence of IL-7. As shown in Figure 5,
the population of SP CD4+ thymocytes comprises 2 subsets,
characterized as IL-7Rlow and IL-7Rhigh. The
IL-7Rlow SP CD4+ are also characterized by a
low expression of CD69 (data not shown), indicating that these cells
have not attained complete maturation.40,41 These
low-expressing cells were shown to die of apoptosis after 4 days in
culture (data not shown). In contrast, the level of IL-7R, expressed as
mean fluorescence intensity (MFI) in the IL-7Rhigh SP
CD4+ cells, was particularly high compared to those in the
DP CD3 or in the immature subpopulations
(P < .05). Given that, in our conditions, intermediate
cells were enriched with the TN cells as a pool of immature
thymocytes, we performed double labeling for both CD4 and IL-7R
to distinguish between both subsets. We observed a slightly but
significantly higher level of IL-7R on TN than on intermediates (20%
increase of MFI, mean of 4 assays; P < .03). In addition,
intermediate thymocytes included both CD4low and
CD4high cells, and the level of expression of CD4 was
inversely correlated with the level of IL-7R (data not shown).
Intermediate cells constituted the largest part of the pool of immature
thymocytes (70% as median values for 4 assays). Thus, as shown in
Figure 5, IL-7R expression was detected in a large proportion of the
immature, mostly intermediate, cells at a higher level than in DP
CD3 thymocytes (P < .05). Therefore,
differences in the level of expression of IL-7R correlated with
observed variations in the survival capacity of the studied subsets
(Figures 1, 2 and text).
Level of Bcl-2 expression correlated with the level of expression of IL-7R in the distinct thymocyte subsets We determined the expression level of Bcl-2 by flow cytometry analysis in DP CD3, immature (TN and intermediate), and
SP CD4+ thymocytes at the time of cell isolation and after
4 days of culture in the presence or absence of IL-7. As shown in
Figure 6, under the stimulation
conditions of the microenvironment (at day 0) or in the presence
of IL-7, a major part of the SP CD4+ cells are
Bcl-2high, and a smaller fraction is Bcl-2low.
As for IL-7Rlow (Figure 5), these Bcl-2low
thymocytes constitute a pool of apoptotic cells (forward scatter-side scatter profile; data not shown) regardless of whether IL-7 is present.
It is likely that the low constitutive rate of apoptosis observed
within the SP CD4+ pool (Figure 1) is related to this minor
fraction of less mature (CD69low) cells characterized by an
IL-7RlowBcl-2low phenotype. In the absence of
IL-7, Bcl-2 level decreased even in the SP CD4+ cells
previously identified as Bcl-2high (at day 0 or under IL-7
treatment), leading to 2 peaks of low Bcl-2-expressing cells. The rate
of survival decreased to 36% (forward scatter-side scatter profile).
At day 0, the SP CD4+ Bcl-2high thymocytes
exhibited a higher Bcl-2 level than immature cells or DP
CD3 thymocytes (P < .05). As previously
shown for IL-7R analysis, double labeling of immature subset for both
CD4 and Bcl-2 performed at day 0 showed that Bcl-2 was expressed at a
significantly higher level in TN than in intermediate thymocytes
(87.9% labeled TN cells vs 72.8% labeled intermediate cells, 30% MFI
decrease in intermediate cells compared with TN; mean of 4 assays;
P < .03). Furthermore, among intermediate thymocytes,
CD4high cells expressed a lower level of Bcl-2 than the
CD4low population (data not shown). As shown in Figure 6 at
day 0, immature, mostly intermediate, cells expressed a higher level of
Bcl-2 than DP CD3 cells (P < .05). We also
showed in Figure 6 that in all thymocyte subsets, Bcl-2 expression was
decreased after 4 days in culture, unless IL-7 was added to the culture
medium. This induction by IL-7 was underlined by a significant shift of
the peaks of the Bcl-2-positive cells on the x-axis in the presence of
this cytokine compared to their positions at day 0 (when IL-7 was not
in saturating conditions) and at day 4 in the absence of IL-7 (Figure
6). However, even in the presence of IL-7, the rate of survival cells
was 51% for the immature and 33% for the DP cells (forward
scatter-side scatter profiles). These data demonstrate that IL-7 is
required to induce or sustain Bcl-2 in all thymocyte subsets and that
this level of induction is directly related to the level of IL-7R
expression (Figures 5, 6).
High expression level of Bcl-2 in mature SP CD4+IL-7RhighBcl-2high is further enhanced by HIV infection, whereas the low level of Bcl-2 observed in immature thymocytes is further decreased by HIV infection We demonstrated recently that infection itself was able to induce the up-regulation of Bcl-2 in a cellular model of HIV persistence.29 We now address whether such a mechanism occurs in mature SP CD4+IL-7RhighBcl-2high thymocytes (and not in immature cells), which might further strengthen their resistance to apoptosis on HIV infection. We then compared Bcl-2 status of the control or infected SP CD4+ population or immature thymocytes after 11 days in culture. At that time of culture, among the SP CD4+ thymocytes, only the IL-7RhighBcl-2high subset was selected, as pointed out earlier. As shown in Figure 7A, Bcl-2 was analyzed by Western blot and was quantified as a ratio of Bcl-2/ -actin after densitometry
analysis of the autoradiogram. First, as a confirmation of data shown
in Figure 6, Bcl-2 is expressed at a much higher level in uninfected SP
CD4+ than in the immature thymocytes
(P < .01), especially after 11 days of culture, a time at
which immature thymocytes consisted mainly of DP CD3
cells, as previously shown in Figure 2. However, the Bcl-2
concentration in SP CD4+ cells was further increased on HIV
infection (P < .01). In contrast, in immature thymocytes,
the low concentration of Bcl-2 was further decreased by HIV infection
(P < .01). The increase in the Bcl-2 concentration
observed in infected SP CD4+ cells suggested that the
infection was able to induce an up-regulation of Bcl-2. To strengthen
this hypothesis, we performed flow cytometry analysis of both
intracellular Bcl-2 and p24 on HIV infection. This was carried out
after 11 days of infection in both SP CD4+ and immature
thymocytes as a control population dying from infection. As shown in
Figure 7B, approximately 70% of control or infected SP
CD4+ cells expressed Bcl-2. However, control cells
displayed a homogeneous population for Bcl-2 expression (MFI, 4.8),
whereas HIV-infected SP CD4+ thymocytes comprised a
subpopulation (50% of Bcl-2-positive cells) in which Bcl-2 content
was increased, as demonstrated by the shift (MFI, 8.8; indicated by the
arrow) of the corresponding peak in Figure 7B. On the contrary,
HIV-infected immature thymocytes exhibited a lower level of Bcl-2 than
uninfected control cells. Of particular interest is the observation
that p24-expression takes place in the cells overexpressing Bcl-2 under
infection (Figure 7C), suggesting that infection itself might be
responsible for a further increase in Bcl-2 level. This induction was
not significantly observed in the immature subset. The higher level of
virus production in the SP CD4+ subset was indicated by
both a higher percentage of p24 positive cells and a higher level of
p24 per cell (9.2% and MFI of 16.3 for SP CD4+ vs 4.1%
and MFI of 3.5 for immature cells) (Figure 7C). This high level of
viral replication likely accounts for the efficient induction of Bcl-2
in SP CD4+ thymocytes.
In the current report we investigated the survival capacity on HIV
infection of the SP CD4+ and intermediate
CD4+CD8 We then investigated the involvement of the factors of the thymic
microenvironment on the modulation of apoptosis on infection. We
focused on TNF and IL-7, which we showed to be crucial to sustain HIV
replication.28 We observed that though these cytokines act synergistically to trigger virus replication, they exhibit antagonistic effects on apoptosis. TNF favors apoptosis whereas IL-7 counteracts its
effect. Because the protection by IL-7 was high in the mature thymocytes and remained partial in the immature cells, we wondered whether this was due to a different level of expression of IL-7R. As
expected, IL-7R is highly expressed in the major fraction of the SP
CD4+ thymocytes at a level above that found in the immature
subpopulations. Among immature thymocytes, the TN cells constitutively
express a high level of IL-7R that is gradually down-regulated during differentiation into the intermediate and then the DP CD3 We then performed a detailed analysis of the coexpression of IL-7R and Bcl-2 in the human thymocyte subsets. Differential constitutive levels of Bcl-2 in the distinct subsets correlate with the levels of expression of IL-7R in these cells and with their survival capacity on infection. In the intermediate thymocytes, this relation is further strengthened by the fact that CD4low cells, which survive best on infection, express higher levels of IL-7R and Bcl-2 than CD4high cells. Furthermore, we strictly demonstrated that IL-7 is involved in the maintenance of a differential level of Bcl-2 in accordance with IL-7R expression in the different subsets. Therefore, Bcl-2 level appears to be of crucial importance as an underlying mechanism of IL-7-mediated protection against apoptosis in human thymocytes. In addition, it is worthwhile noting that the high constitutive level of Bcl-2 in the mature SP CD4+ thymocytes is further increased on infection in a large fraction of thymocytes. It is interesting to point out that virus replication takes place in this particular fraction. Taken together, these data suggest that the virus itself acts to sustain the production of Bcl-2, conferring high resistance to apoptosis and then persistent infection in these cells. However, the number of cells detected for p24 expression is lower than the fraction of cells in which Bcl-2 is enhanced on infection. Two possible explanations are that p24 staining leads to an underestimation of the number of HIV-1-infected cells or that up-regulation of Bcl-2 also occurs in bystander cells. In contrast, in immature thymocytes, the lower level of Bcl-2 was further decreased on infection favoring apoptosis at various degrees according to stage of differentiation; the major effect occurred within DP cells. In vivo studies are in agreement with the high resistance against
apoptosis of TN during thymus infection.9,17,19,22 In
contrast, the survival capacity of SP CD4+ thymocytes is
controversial. In SCID-hu mice, the CD4+CD8 With regard to intermediate thymocytes in vivo, it was reported in the SCID-hu mouse model that apoptosis is induced by HIV.25 In the current paper, we provide new insights by demonstrating that intermediate thymocytes display partial resistance to infection with HIV-1, which is more pronounced in the CD4low cells. Our results on the high sensitivity to apoptosis of DP cells observed in vitro are in agreement with a large number of in vivo studies demonstrating a massive reduction of this population on infection in patients17 or in animal models of AIDS.9,19,22,25,43,44 Furthermore, in SIV-infected macaques, a partial depletion of DP thymocytes correlates with a reduced expression of Bcl-2 and an increase of the apoptosis rate in these cells.9 It was unclear, however, whether this depletion was the consequence of cell death induced by a productive infection in these cells. We showed in a previous work that despite virus entry into the DP cells, HIV does not replicate because of a blockade of the activation in these cells.28 Virus entry in these cells does not modify their rate of apoptosis ex vivo (data not shown). We conclude here that infection occurs at the intermediate stage but that, after cell differentiation, cell death is particularly massive at the DP stage. We also point out here that the mechanisms underlying the resistance to virus-induced apoptosis converge toward a maintenance of a high level of Bcl-2. First, a high level of Bcl-2, correlating with a high-level IL-7R, is required before infection to resist virus-induced cell death. This is reminiscent of our previous studies showing that survival of HIV-infected Jurkat T cells was directly dependent on the basic level of expression of Bcl-2 generated by transfection in these cells.29 This is perfectly compatible with the well-known role of Bcl-2, which is to prevent the disruption of the mitochondrial membrane induced by the oxidative stress45 occurring in HIV-infected cells.29,46 We actually demonstrated that this oxidative stress can be counteracted by a high level of Bcl-229, and this might occur in thymocytes, especially in the mature SP CD4+ subset. The antagonist effect of IL-7 on TNF-induced apoptosis might also be attributed to the capacity of IL-7 to trigger the expression of the TNF receptor 2 (TNF-R2), as we previously reported for the mature SP CD4+ subset.28 TNF-R2 expression might be associated with a decreased sensitivity to TNF-induced apoptosis, as shown for peripheral T cells.47 Work is in progress in our laboratory to evaluate this possibility. In addition to a high basic level of Bcl-2 in the mature SP CD4+ cells, HIV infection further increases this level in a substantial fraction of these cells. This is reminiscent of the up-regulation of Bcl-2 at the transcriptional level that we observed in the Jurkat T-cell clones on HIV infection.29 Studies are in progress to further elucidate whether the same mechanism is involved in the up-regulation of Bcl-2 on infection in mature thymocytes. The current paper and our previous data27,28,35 lead us to
propose a model of the sequential events leading to destruction of the
thymus on HIV infection. At an early stage, mature SP CD4+
thymocytes substantially produce HIV under the activation of cytokines
cosecreted during their interaction with the thymic epithelial cells.
Infected cells are protected from apoptosis because of their high
capacity to respond to IL-7 and the subsequent high level of Bcl-2
further enhanced by infection itself. Thus, SP CD4+
thymocytes constitute a persistent HIV reservoir and not a latent one,
as reported by Brooks et al.48 Actually, there is no real discrepancy because the authors show nonnegligible HIV production in
these thymocytes isolated from HIV-infected SCID-hu mice and left
untreated for 3 days in culture.48 The infected SP
CD4+ thymocytes may give rise to an increase in the
peripheral viral load when these cells migrate as T lymphocytes into
the blood. This is consistent with the strong correlation observed
between the number of infected thymocytes in the thymic medulla and the level of peripheral viral load in SIV-infected monkeys.49
At a later stage, because of an inflammatory response to infection, TNF
produced by inflammatory cells, in combination with other cytokines,
might induce HIV production in intermediate thymocytes. Subsequently,
both CD4high intermediate and DP thymocytes will die of
apoptosis because of reduced quantity of Bcl-2 that is further
decreased by infection. Depletion of these subsets will lead to an
interruption of thymopoiesis and finally to a failure of T-cell
regeneration. This pattern is consistent with data recently reported in
FIV-infected cats showing a high level of virus in mature thymocytes
early after infection and the successive infection of immature
thymocytes associated with inflammatory response and cortical
atrophy.44 Such a sequence of events, which could
participate in T-cell loss in the periphery, might be particularly
favored in an active thymic microenvironment Now that IL-7 is envisioned as immunotherapy for AIDS, it is important to emphasize the possible role of this cytokine in virus persistence in the thymus at the first stage of infection and later in the disruption of thymopoiesis by triggering HIV replication in apoptosis-sensitive immature thymocytes. In conclusion, even in association with antiretroviral therapy, the use of IL-7 as immunotherapy for AIDS warrants prudence and the need for testing first in a relevant animal model.
We thank Dr Sonia Berrih-Aknin (Hôpital Marie Lannelongue, Le Plessis-Robinson, France) and Prof Leca (Hôpital Necker, Paris, France) for providing us with thymuses. We particularly thank Dr Gianfranco Pancino (Unité de Biologie des Rétrovirus, Institut Pasteur) for helpful discussions and Dr David Ojcius (Laboratoire de Biologie des Interactions Cellulaires, Institut Pasteur) for careful reading of the manuscript.
Submitted February 28, 2001; accepted June 8, 2001.
Supported by the Agence Nationale pour la Recherche sur le SIDA (France). E.G. was the recipient of a fellowship from Ensemble contre le SIDA (Fondation pour la Recherche Médicale, Paris, France) and a fellowship from the Agence Nationale pour la Recherche sur le SIDA.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Nicole Israël, Unité de Biologie des Rétrovirus, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris Cedex 15, France; e-mail: nisrael{at}pasteur.fr.
|
CD3+ thymocytes through
sustained induction of Bcl-2
Top
Abstract
Introduction
P9)
from BD Biosciences (Erembodegem, Belgium).
-chain was determined by using
CDw127-PE antibodies (R34.34) and IgG1-PE (679.1Mc7) as a negative
control (Beckman Coulter France S.A). For the antibodies cited,
unconjugated control IgG1 antibody (679.1Mc7; Beckman Coulter France
S.A) was used for treatment before immunostaining to avoid nonspecific
binding of fluorochrome-labeled antibodies. Bcl-2 production was
analyzed by using Bcl-2-PE antibody (Bcl-2/100) with the control
IgG1-PE (MOPC-21) from BD-PharMingen (San Diego, CA). Measurement of
intracellular p24 expression was performed with p24-FITC antibody
(KC57) with the control MsIgG1-FITC (679.1Mc7) (Beckman Coulter France
S.A). For triple immunostaining for CD4, CD8, and IL-7R or Bcl-2,
anti-CD4-PC5 (13B8.2) anti-CD8-FITC (B9.11) antibodies and their
control IgG1-PC5 and IgG1-FITC (both 679.1Mc7) were used (Beckman
Coulter France S.A). Immunostaining was analyzed using an XL-4C
cytofluorometer (Beckman Coulter France S.A).
indicates SP CD4+; 
,
immatures 
, SP CD4+/HIV; 
, immatures/HIV.
