Blood, Vol. 94 No. 5 (September 1), 1999:
pp. 1829-1833
CORRESPONDENCE
Sensitivity of CD4+ Peripheral Blood T Cells
Toward Spontaneous and CD95 (APO-1/Fas)-Induced Apoptosis in Pediatric
Human Immunodeficiency Virus Infection
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LETTER |
To the Editor:
McCloskey et al1 recently reported that spontaneous
apoptosis of T cells from human immunodeficiency virus (HIV)-infected individuals occurred only in previously activated cells independent of
CD95 receptor (R) expression or CD95 sensitivity. Using fluorescein isothiocyanate (FITC)-labeled anti-CD95 monoclonal antibody (MoAb), the
authors found that T cells dying after 72 hours of in vitro culture
were predominantly CD95-negative. However, in our own analysis of T
cells from HIV-infected children, we could identify at least 3 different cell populations in the CD4+ T-cell
subset using phycoerythrin (PE)-labeled anti-CD95 MoAb2 (and unpublished results): (1) CD95-negative cells
corresponding to resting/"naive" CD45RA+
CD45ROneg T cells; (2) CD45RA+ cells that were
recently activated and expressed low levels of both CD45RO and CD95;
(3) CD95high CD45RO+ primed/"memory" T
cells. We have previously shown that only resting/"naive" T cells
from HIV-infected children were resistant to spontaneous and
anti-CD95-induced apoptosis in vitro.2 Thus, the
CD95neg CD45RO+ cells detected by McCloskey et
al by an FITC-labeled anti-CD95 MoAb might in fact represent recently
activated, CD95-low positive CD45RO-low positive T cells if analyzed
with more sensitive staining methods using the appropriately labeled
MoAb. The conclusion that only CD95-negative T cells of HIV-infected
individuals are dying spontaneously during in vitro culture should,
therefore, be controlled by using a PE-labeled anti-CD95 MoAb.
Based on their observation of an increased percentage of CD95-negative
T cells undergoing spontaneous apoptosis in vitro, the authors
speculate that mechanisms independent of the CD95 R/ligand (L) system
may be involved in exaggerated lymphocyte apoptosis in HIV-1 infection.
Indeed, as shown in Fig 1A, spontaneous apoptosis of freshly isolated peripheral blood T cells from
HIV-infected individuals occurs already after short-term culture in
vitro (16 to 20 hours, 37°C, 5% CO2); it can be
partially blocked by addition of interleukin-2 (IL-2) to the culture
medium and is not influenced by agents that block CD95 R/L interaction,
eg, anti-APO-1 F(ab)' fragments. On the other hand, anti-CD95
MoAb-induced apoptosis is completely inhibited by preincubation of T
cells with anti-APO-1 F(ab)' and is not influenced by addition of IL-2
(Fig 1B). The profound dysregulation of the CD95 R/L-system observed in
pediatric HIV-infection3,4 is apparently not responsible
for spontaneous cell death of isolated peripheral blood T cells from
HIV-infected individuals. The CD95 system is, however, critically
involved in activation-induced cell death (AICD) following crosslinking of the T-cell receptor (TCR)/CD3-complex.3,5 Interestingly, cocultivation of freshly isolated T cells from HIV-infected individuals with both anti-CD3 and anti-CD95 MoAb did always cause a higher rate of
cell death than both of these agents alone (Fig 1C). This suggests that
the CD95 pathway is not inhibited by simultaneous stimulation of the
TCR/CD3-complex, and CD95-independent mechanisms contribute both to
spontaneous cell death and to OKT3-induced AICD (eg,
TRAIL/TRAIL-receptor mediated death6).
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| Fig 1.
Inhibition of spontaneous T-cell death but not of
anti-CD95-induced apoptosis by IL-2 and cellular activation. Isolated
peripheral blood mononuclear cells (PBMC, 2 × 105
cells/well) obtained from 10 HIV-1+ children and
adolescents were cultured in the presence or absence of either anti-CD3
MoAb (OKT3; 10 µg mL 1) and/or anti-CD95 MoAb
(anti-APO-1; 10 µg mL1) and protein A (5 ng
mL1) and were collected after 21 ± 1 hours
for determination of cell death in CD4+ and
CD8+ cells as described.2-4 The percentage of
specific anti-CD3- and anti-CD95-induced apoptosis was calculated
according to the formula: 100 × [Experimentally Induced Cell Death
(%)  Spontaneous Cell Death (%)]/[100 Spontaneous Cell
Death (%)]. In (A) and (B), data are given as arithmetic mean; error
bars indicate the standard error of the mean. (A) Recombinant human
IL-2 (30 U/mL; Biozol, Eching, Germany), but not preincubation with the
antagonistic anti-APO-1 F(ab)' fragment (5 µg
mL1),3,5 significantly reduced spontaneous
T-cell death in vitro. (B) Signaling via the CD95 receptor, but not
spontaneous cell death, was blocked by anti-APO-1 F(ab)'. (C) The
effect of T-cell activation on anti-APO-1-induced apoptosis of
freshly isolated CD4+ T cells was assessed in PBMC from 2 different patients stimulated with OKT3 and/or anti-APO-1 as described
above. Numbers in the plots indicate the percentage of apoptotic cells
that are identified by the typical change in light scatter
characteristics ("shrinking death"). In patient no. 1 OKT3
induced less apoptosis than anti-APO-1, whereas in patient no. 2 more
cell death was seen in OKT3-treated cells. However, in both experiments
the combination of these MoAbs induced more apoptosis than each of them
alone.
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Thus, there are at least 3 different mechanisms responsible for
programmed cell death of ex vivo-cultured T cells from HIV-infected individuals: (1) spontaneous cell death, which is CD95-independent but
partially inhibitable by IL-2 and represents the classical "death-by-default" induced by growth-factor
withdrawal7; (2) CD95-induced cell death, which is not
inhibitable by IL-2 nor by stimulation via the CD3/TCR-complex; and (3)
AICD via CD95-dependent and -independent pathways. It has not been
clarified yet which of these mechanisms is of major importance for
immunopathogenesis of HIV-1 infection in vivo. However, our previous
observation that an increase in sensitivity of peripheral blood T cells
toward CD95-induced apoptosis is seen already during early disease
stages4 suggests that this may be a relevant mechanism in
disturbed T-cell homeostasis, which is the central event in
immunopathogenesis of HIV-1 infection and development of acquired
immunodeficiency syndrome.8 The CD95 cell-surface receptor
is expressed at high density on a variety of different cell types,
including thymocytes and hematopoetic stem cells, and is also
functionally active at least during certain stages of cellular
activation and maturation.9,10 Immediately after starting
highly active antiretroviral treatment (HAART), which caused an
immediate increase in naive CD4+ and CD8+ T
cells in HIV-1-infected infants and children, we observed a steep
reduction in the sensitivity of peripheral blood T cells toward
anti-CD95-induced apoptosis.11 Figure
2 shows that normal CD95 sensitivity is
maintained in clinically asymptomatic patients with stable CD4 counts
during prolonged treatment with HAART, irrespective of the degree of
concomitant suppression of viral replication as reflected by plasma
viral load.
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| Fig 2.
Development of HIV-1 plasma viral load (RNA-copies per
milliliter;  ), peripheral blood CD4 counts (percent of lymphocytes;
 ), and sensitivity of CD4+ T cells toward
anti-APO-1-induced apoptosis (percent CD95-specific cell death;  )
during highly active antiretroviral therapy (HAART) in 2 children
infected perinatally with HIV-1. Patient A was a 10-year-old girl with
no previous antiretroviral therapy. After starting HAART using
lamivudine, stavudine, and nelfinavir, this patient showed an immediate
decrease in sensitivity of CD4+ cells toward
CD95-induced apoptosis, which was followed by a slow but gradual
increase in CD4 counts and a decrease in plasma viral load to levels
below the limit of detection (bDNA). Patient B was a 10-year-old boy
who switched from combined treatment with zidovudine and lamivudine to
HAART using lamivudine, stavudine, and nelfinavir. The reduction in
sensitivity of CD4+ cells toward CD95-induced apoptosis
was somewhat less pronounced, and a considerable increase in CD4 counts
was seen only after 8 months of therapy. This patient never achieved
complete repression of viral replication during HAART. However, despite
the maintenance of high plasma viral load levels, he remained
clinically well and showed a further increase in CD4 counts accompanied
by the absence of CD95-induced T-cell death in vitro.
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On the other hand, the beneficial clinical effect of IL-2
substitution12 in HIV-1-infected adults suggests that the
death-by-default mechanism induced by IL-2 deficiency might be an
important factor of disease progression in vivo. However, IL-2-treated
patients also show a partial reconstitution of the naive T-cell
compartment12,13; this argues against a decisive role
of inhibition of spontaneous cell death of peripheral blood T cells in
IL-2-induced T-cell reconstitution in HIV-infected individuals.
We previously described that both spontaneous T-cell death and CD95
sensitivity during pediatric HIV infection was confined to
primed/memory cells and did not affect resting/"naive" T
cells.2 Therefore, we recently proposed a model for the
immunopathogenesis of HIV-1 infection based on the increased activation
of the CD95 system (which is commonly observed in peripheral blood T
cells from HIV-1+ individuals), which may also contribute
to decreased T-cell regeneration in bone marrow and
thymus.14 During the initial stages of the disease, a
slight increase in loss of activated, memory-type T cells by direct,
virus-induced and
more importantlyby indirect, activation-related
mechanisms may not be sufficiently compensated for by an increase in
T-cell regeneration due to the impaired function of thymus and bone
marrow, thus leading to a constant decrease in naive T cells with
disease progression.
Thomas Böhler
Klaus-Michael Debatin
Division of Molecular Oncology
German Cancer Research
Center
Heidelberg, Germany
Children's Hospital
University of
Ulm
Ulm, Germany
Richard Linde
Children's Hospital
University of Frankfurt
Frankfurt, Germany
| |
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Response
Despite extensive study, the role of the Fas/FasL system in HIV
pathogenesis remains undetermined. While evidence exists both for1-7 and against8-12 CD95-mediated
HIV-induced cell death, the most likely explanation is that both
Fas-dependent and -independent pathways are at work, a concept
supported by recent findings from our laboratory.13
Comments by Böhler and colleagues regarding our report indicate
that they have misread the methods used and misconstrued the
conclusions drawn. Their interpretation that "only CD95-negative T
cells" were dying is incorrect; indeed, our data indicated the existence of both CD95-dependent and -independent mechanisms of apoptosis. By performing the TUNEL assay on cells labeled with anti-CD95 allophycocyanin MoAb, we observed that CD95-expressing cells
were overrepresented in the live population. This finding strongly
implicates Fas-independent mechanisms in HIV-induced cell death.
Clearly, however, a CD95-positive population was present in the
apoptotic population as well. The data presented by Böhler et al
is, in fact, in agreement with our findings. We hypothesize that those
TUNEL-positive cells which expressed CD95 may indeed be dying via a
Fas/Fas ligand induced mechanism; our point was that not all cell death
can be accounted for by this pathway.
The remark concerning the use of "FITC-labeled anti-CD95" for our
72-hour apoptosis data is also incorrect, leading them to claim that
our labeling system was not sensitive enough. The data in question were
collected with allophycocyanin-conjugated anti-CD95 MoAb, as stated. We
purposely chose allophycocyanin because (1) its emission is far from
cellular autofluorescence,14,15 (2) it has a large
extinction coefficient and a high quantum yield,16,17 and
(3) there is no spectral overlap from fluorescein (TUNEL) because the
signals generated from the two laser beams are separated temporally. In
fact, the relative "brightness" of allophycocyanin is comparable
to that of phycoerythrin.18
Regulation of apoptosis is by necessity tightly controlled, with the
expression of multiple pro- and anti-apoptotic molecules providing many
checks and balances. The interaction of a chronic viral infection with
the immune system may influence some of these regulatory pathways, thus
lending further complexity. Mechanisms of HIV-induced cell death may
differ during the disease course or following effective anti-retroviral
therapy, and may depend on specific differences in individual patients,
virus strains, treatment modalities, or other as yet unidentified
factors. In agreement with our overall conclusions, Böhler et al
state that both Fas-dependent and -independent mechanisms are
responsible for HIV-mediated apoptosis; potential alternate mechanisms
recently proposed include those mediated by TRAIL19 or by
signaling through CD4 and CXCR4.20 As stated in our
published report, our data indicate that multiple mechanisms of
apoptosis induction are active during HIV infection, a notion that is
supported by findings from other laboratories, including the data
presented by Böhler and colleagues.
Savita Pahwa
Thomas W. McCloskey
Division of Allergy and Immunology
Department of Pediatrics
North Shore University Hospital-New
York University School of Medicine
Manhasset, NY
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