CD4 T Cell Depletion Is Linked Directly to Immune Activation
in the Pathogenesis of HIV-1 and HIV-2 but Only Indirectly to
the Viral Load1
Ana E. Sousa,2* Jorge Carneiro,†Martin Meier-Schellersheim,‡Zvi Grossman,‡§and
Rui M. M. Victorino*
The causal relationships among CD4 cell depletion, HIV replication, and immune activation are not well understood. HIV-2
infection, “nature’s experiment” with inherently attenuated HIV disease, provides additional insights into this issue. We report the
finding that in HIV-2 and HIV-1 patients with a comparable degree of CD4 depletion the imbalance in the relative sizes of the naive
and memory T cell populations and the up-regulation of CD4 and CD8 cell activation markers (HLA-DR, CD38, CD69, Fas
molecules) are similar, even though the viral load in the plasma of HIV-2-infected patients is two orders of magnitude lower than
in HIV-1 patients and HIV-2 patients are known to have slower rates of CD4 T cell decline and a better clinical prognosis.
Moreover, we found a similar increase in the frequency of cycling CD4 T cells (Ki67?), which was in strong correlation with the
expression of activation markers. Finally, the level of T cell anergy, as assessed by the proliferative responses to CD3 stimulation
and to a panel of microbial Ags, proved to be comparable in HIV-1 and HIV-2 patients with a similar degree of CD4 depletion
despite large differences in viral load. Our data are consistent with a direct causal relationship between immune activation and
CD4 cell depletion in HIV disease and an only indirect relation of these parameters to the virus replication rate. Invoking the
concept of proximal immune activation and virus transmission, which links efficient transmission of virus to local cell activation
and proliferation in response to Ags and inflammation, we propose an integrative interpretation of the data and suggest that
strongly elevated immune activation induces CD4 cell depletion and not vice versa, with potential implications for the choice of
treatment strategies. The Journal of Immunology, 2002, 169: 3400–3406.
cell counts over time and eventually to AIDS. The increased turn-
over of T cells has been viewed by some as a homeostatic response
to the rapid loss of cells (1–3). An alternative assumption is that
chronic, infection-induced immune activation is the force driving
the progressive decline in CD4 cell numbers and other detrimental
effects that result in AIDS (4–11).
In HIV-2, disease progression is slower than in HIV-1, with
limited impact on the survival of the majority of infected adults
(12–14), although it apparently manifests the same clinical spec-
trum (15, 16). Both horizontal and vertical HIV-2 transmission
rates are much lower than for HIV-1 (17, 18). These epidemio-
logical findings and the reduced frequency of successful virus iso-
lation from the blood of HIV-2-infected patients (19) suggested
very low levels of viremia, which was confirmed by recently de-
uman immunodeficiency virus type 1 pathogenesis is
generally seen as a relentless destruction of CD4?T
cells by the virus leading to the observed decline in CD4
veloped methods to quantify HIV-2 RNA copies in the plasma
(20–22). Interestingly, quantitative assessment of HIV-2 DNA
documented proviral levels similar to those observed in HIV-1-
infected individuals, which was interpreted to indicate similar tar-
get cell infectivity but a decreased rate of virus production in
HIV-2 infection (23, 24).
As in HIV-1 infection, CD4 cell counts decline progressively
under HIV-2, but the decline is much slower and viremia levels are
lower at any stage of the disease (13, 21, 24). Studying HIV-2
infection offers the possibility to quantitatively reassess the signif-
icance of virological and immunological parameters in HIV patho-
genesis in an infection with an inherently attenuated virus. We
report in this work that HIV-1 and HIV-2 patients having a similar
degree of CD4 depletion displayed similar levels of T cell hyper-
activation and similar numbers of cycling cells in the peripheral
blood despite great differences in the plasma viral load. These
results and other recent reports call for reevaluation of different
hypotheses about causal relationships among virus concentration,
CD4 depletion, and activation and turnover of T lymphocytes.
Patients and Methods
Twenty-seven HIV-2-infected patients, 26 HIV-1-infected patients, and 25
healthy control individuals participated in this cross-sectional study. The
patients currently live in Portugal and attend outpatient clinics in Lisbon.
They have no known ongoing opportunistic infections or tumors. The ep-
idemiological and clinical features of these groups were described previ-
ously (25). As shown in Fig. 1, HIV-2 viremia had a maximum of 2,754
RNA copies per ml and was ?500 RNA copies/ml (detection limit) in 21
of 27 patients, as quantified using a previously described RT-PCR test (22).
The geometric mean of the viral load in HIV-1?patients was 8,476 RNA
copies/ml (range: 50–740,000) as quantified by RT-PCR (Ultrasensitive
Test; Roche Molecular Systems, Branchburg, NJ). For the purpose of this
*Clinical Immunology Unit/Institute of Molecular Medicine, Faculty of Medicine of
Lisbon, Lisbon, Portugal;†Instituto Gulbenkian de Cie ˆncia, Oeiras, Portugal;‡Lab-
oratory of Immunology, National Institute of Allergy and Infectious Diseases, Na-
tional Institutes of Health, Bethesda, MD 20892; and§Department of Physiology and
Pharmacology, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel
Received for publication April 10, 2002. Accepted for publication July 9, 2002.
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 Ministe ´rio da Cie ˆncia e Tecnologia
PRAXIS XXI and from Comissa ˜o Nacional de Luta Contra a SIDA Ministe ´rio da
Sau ´de. A.E.S. and J.C. received scholarships from PRAXIS.
2Address correspondence and reprint requests to Dr. Ana E. Sousa, Clinical Immu-
nology Unit/Institute of Molecular Medicine, Faculty of Medicine of Lisbon, Av. Prof
Egas Moniz, 1649-028 Lisbon, Portugal. E-mail address: firstname.lastname@example.org
The Journal of Immunology
Copyright © 2002 by The American Association of Immunologists, Inc.0022-1767/02/$02.00
study, the HIV-1- and HIV-2-infected patients were classified into three
groups according to the degree of CD4 depletion: ?500 CD4?T cells/?l,
200–500 CD4?T cells/?l, and ?200 CD4?T cells/?l. There were no
statistically significant differences in the mean CD4 counts between the
corresponding groups of HIV-1- and HIV-2-infected patients. The study
was approved by the Ethical Board of the Faculty of Medicine of Lisbon.
Analysis of Ki67 expression and T cell phenotype
PBMCs were isolated from fresh heparinized blood by Ficoll-Hypaque
gradient centrifugation (Life Technologies, Paisley, U.K.) immediately af-
ter venipuncture. After surface staining with anti-CD3 or anti-CD4 Tri-
Color-conjugated mAbs (Caltag Laboratories, South San Francisco, CA)
and anti-CD4 or anti-CD45RO PE-conjugated mAbs (BD Biosciences, San
Jose, CA), cells were permeabilized with a saponin buffer as previously
described (26) and stained with the mouse anti-human Ki67 (MIB-1)
FITC-labeled mAb or with isotype control (Immunotech, Marseilles,
France). At least 50,000 CD3?or 20,000 CD4?lymphocytes were ac-
quired on a FACSCalibur (BD Biosciences). The frequency of Ki67?cells
CD4?CD45RO?lymphocyte gated populations using CellQuest software
(BD Biosciences). Simultaneously, the proportions of CD4 and CD8 T
cells expressing markers of cell activation and/or differentiation were as-
sessed by flow cytometric phenotypic analysis (27) using the following
mAbs: anti-CD27, -CD28, -CD38, -CD45RA, -CD45RO, -CD62L, -CD69,
-Fas molecule (CD95), -HLA-DR (BD Biosciences).
Lymphocyte proliferation assays
PBMCs were cultured in complete medium as previously described (27) in
triplicate for 3 days with immobilized anti-CD3 mAb in the presence or
absence of soluble anti-CD28 mAb (BD PharMingen, San Diego, CA) as
well as in quadruplicate for 6 days in the presence of tetanus toxoid (Con-
naught, Swiftwater, PA), purified protein derivative (Serum Statersmistitut,
Copenhagen, Denmark), Candida albicans (Greer, Lenoir, NC), the viral
recombinant proteins gp105 and p26 from HIV-2 ROD and gp120 and p24
from HIV-1 IIIB (obtained from a baculovirus expression system by Dr. I.
Jones, provided by the Medical Research Council, U.K., AIDS Reagent
Program). Proliferation was assessed by tritiated TdR (Amersham Phar-
macia Biotech, Little Chalfont, U.K.) incorporation after a pulse of 1 ?Ci
in the last 4 h of culture and counted in a gaseous scintillation beta counter
(Packard Instrument, Meriden, CT); results are expressed as cpm.
The data are presented as arithmetic mean ? SE and were compared using
an unpaired t test or Mann-Whitney test according to the type of distribu-
tion; the Pearson’s and the Spearman’s correlation coefficients were used to
determine the correlation between two variables. Model I linear regression
was performed to establish relationships between variables. A value of p ?
0.05 was considered significant.
Naive/memory-effector distribution within the CD4 and CD8 T
HIV-1 immunodeficiency is associated with a progressive decline
of both CD4 and CD8 naive cells in the peripheral blood leading
to an imbalance in the naive/memory-effector distribution (28).
Despite the complexity of the phenotypic definition of these pop-
ulations, the simultaneous expression of CD45RA and CD62L is
currently thought to identify the majority of the naive lymphocytes
and CD45RO is considered a memory marker. We found that, at a
CD45RA?CD62L?cells within the CD4 subset does not differ
significantly between the two infections (Table I). Moreover, a
progressive reduction in the proportion of CD45RA?CD62L?
cells within the CD8 T cell population was also observed (Table I).
As has been previously described for HIV-1 disease (28), there
was a positive correlation between the declines in naive cell fre-
quencies within the CD4 and the CD8 subsets in HIV-2 (r ?
0.5779; p ? 0.002) as well as in HIV-1-infected patients (r ?
0.4601; p ? 0.018). Importantly, the decrease in the absolute num-
ber of CD45RA?CD62L?CD4 T cells in peripheral blood in the
HIV-2 cohort was also in correlation with the progressive reduc-
tion in the number of CD45RA?CD62L?CD8 T cells (r ?
0.5585; p ? 0.003).
Table I. Imbalances of the naive/memory subsets within CD4 and CD8 T cell subsetsa
CD4 T CellsCD8 T Cells
?500 CD4 cells/?l
200–500 CD4 cells/?l
?200 CD4 cells/?l
?500 CD4 cells/?l
200–500 CD4 cells/?l
?200 CD4 cells/?l
27 41.0 ? 3.5**
13 46.3 ? 5.6
8 44.7 ? 3.6††
6 24.6 ? 5.6***
222 ? 41***
371 ? 61†
150 ? 21***†††
19 ? 6***
63.2 ? 3.3
60.9 ? 5.1
59.8 ? 3.4
72.6 ? 8.3*
311 ? 47**
510 ? 56†††
199 ? 23***††
61 ? 21***
39.2 ? 3.4*** 271 ? 34
50.3 ? 4.7**†
33.5 ? 3.7*** 214 ? 49
22.7 ? 3.9*** 119 ? 27** 57.7 ? 6.6*
55.0 ? 3.0** 387 ? 45**
53.1 ? 4.4
56.2 ? 5.4*
385 ? 45†
460 ? 77***
321 ? 48*
330 ? 96
26 40.7 ? 4.2**
10 51.4 ? 4.4
7 47.1 ? 7.9†
9 24.3 ? 6.7***
25 55.5 ? 2.8
191 ? 36***
372 ? 48††
154 ? 31***†††
19 ? 9***
517 ? 50
69.5 ? 3.7**
62.3 ? 4.1
67.3 ? 7.3
79.1 ? 7.2***
56.6 ? 2.7
239 ? 35***
431 ? 25†††
205 ? 21***†††
53 ? 17***
520 ? 41
34.6 ? 2.8*** 286 ? 40
39.9 ? 3.9*** 402 ? 47
34.2 ? 5.8*** 298 ? 98
29.1 ? 5.1*** 149 ? 44** 52.7 ? 4.4
63.8 ? 2.3346 ? 38
54.5 ? 2.9*
50.6 ? 4.2
62.2 ? 6.3** 551 ? 140***
487 ? 70***
558 ? 98***
357 ? 133
216 ? 1943.5 ? 3.0
aSignificance in comparison with healthy controls: ?, p ? 0.05; ??, p ? 0.01; ???, p ? 0.001. Significance of one phase of the HIV-2 or the HIV-1 disease in
comparison with the subsequent phase: †, p ? 0.05; ††, p ? 0.01; †††, p ? 0.001.
There were no significant differences between the HIV-2 cohort and the corresponding group of the HIV-1 cohort.
ripheral blood CD4 counts (cells per microliter) in the HIV-1 and HIV-2
cohorts. All the patients were without antiretroviral therapy with the ex-
ception of the ones indicated by E (two nucleoside anti-transcriptase in-
hibitors) and ? (one protease and two nucleoside anti-transcriptase
Plasmatic viral loads (RNA copies per milliliter) and pe-
3401The Journal of Immunology
Markers of CD4 T cell activation
Having shown that, for a given level of CD4 depletion, the two
infections exhibited similar imbalances of the naive/memory-ef-
fector distribution within the CD4 and CD8 T cells, we examined
the expression of makers that are up-regulated upon T cell
HIV-2-infected patients showed significant up-regulation of the
MHC class II molecule (HLA-DR) within the CD4 T cell popu-
lation similar to the one observed in the corresponding group of
HIV-1 patients (Fig. 2a), with a similar inverse correlation to the
peripheral blood CD4 count (r ? ?0.7485 and p ? 0.0001 for
HIV-2; r ? ?0.7170 and p ? 0.0001 for HIV-1 disease). More-
over, the frequency of CD4 T cells expressing the marker of recent
cell activation CD69 was also elevated and was not significantly
different in the HIV-2 as compared with HIV-1 patients (Fig. 2a).
Activation of the CD8 subset
HIV-2 is associated with an elevated frequency of HLA-DR?cells
within the CD8 subset that increases with the progression in CD4
depletion (r ? ?0.6134; p ? 0.0009) (Fig. 2b). Furthermore, the
CD38 molecule is up-regulated in terms of both percentage of
positive cells (Fig. 2b) and mean fluorescence intensity (data not
shown). In HIV-1 infection this has been shown to be indicative of
CD4 depletion and leads to adverse prognosis independent of the
metric analysis of CD4?-gated T cells stained with CD45RA and Fas from
a 50-year-old healthy subject with 1186 CD4 T cells/?l, a 51-year-old
HIV-2-infected patient with 599 CD4 T cells/?l, and a 48-year-old HIV-
1-infected patient with 548 CD4 T cells/?l. Percentages in the upper right
quadrant of each dot plot represent the proportion of CD45RA?Fas?cells
and percentages in the lower right quadrant of each dot plot represent the
frequency of CD45RA?Fas?cells. b, Proportions of Fas-expressing cells
within the population of CD45RA?CD4 T cells in healthy subjects and in
the HIV-2- and HIV-1-infected individuals stratified in three groups ac-
cording to the CD4 counts, namely ?500 cells/?l (f), 200–500 cells/?l
(z), and ?200 cells/?l (t). c, The same flow cytometric analysis per-
formed within the CD8brightlymphocytes. d, Proportions of Fas-expressing
cells within the CD45RA?CD8 T cell subset in the same groups of sub-
jects. Bars represent mean ? SE. Significance in comparison with healthy
controls: ???, p ? 0.001; ??, p ? 0.01; ?, p ? 0.05. No significant dif-
ferences were found between the two infections.
Up-regulation of the Fas molecule (CD95). a, Flow cyto-
cell subsets. Analysis in healthy subjects as well as in HIV-2- and HIV-
1-infected individuals stratified in three groups according to the CD4
counts, namely ?500 cells/?l (f), 200–500 cells/?l (z), and ?200
cells/?l (t). a, Proportion of the CD4 T cells that express HLA-DR and the
marker of recent cell activation CD69. b, Expression of HLA-DR, CD38,
or both molecules simultaneously within the CD8 T cell subset. Bars rep-
resent mean ? SE. Significance in comparison with healthy controls: ???,
p ? 0.001; ??, p ? 0.01; ?, p ? 0.05. Significance of the HIV-2?patients
as compared with HIV-1-infected patients: §, p ? 0.05.
Frequency of activated cells within the CD4 and the CD8 T
3402T CELL ACTIVATION AND CD4 DEPLETION IN HIV PATHOGENESIS
viremia (29). Assessing the simultaneous expression of HLA-DR
and CD38 in CD8 T cells, we found a similar expansion of this
subset in both infections in the intermediate and advanced stages of
CD4 depletion, although in the early stage there was a significantly
lower expansion in the HIV-2 infection than in HIV-1 (Fig. 2b).
In contrast to HIV-1 infection, HIV-2 was not found to be as-
sociated with a significant increase in the proportion of CD8 T
cells expressing CD69 (9.2 ? 1.3 in HIV-2-infected patients vs
6.9 ? 1.2 (NS) in healthy controls and 15.8 ? 1.6 in HIV-1-
infected subjects (p ? 0.0001 and p ? 0.0023, respectively)).
Because CD69 is only transiently expressed on T cells upon acti-
vation, this difference does not have direct bearing on the extent of
overall activation in the two infections, but it does suggest differ-
ences in the pattern of CD8 activation.
Up-regulation of the Fas molecule
Fas (CD95) is thought to play a role in HIV-1-associated lympho-
cyte anergy and programmed death (30). The Fas molecule is
highly expressed in the memory-effector cell population. Measur-
ing its expression in the CD45RA?CD4?subset we found major
up-regulation in both HIV-1 and HIV-2 infections (Fig. 3, a and b),
with a strong negative correlation to CD4 counts (r ? ?0.71 and
p ? 0.0001 for HIV-2 infection; r ? ?0.65 and p ? 0.0003 for
HIV-1 infection). For both types of infection major up-regulation
of Fas was also observed within the CD45RA?CD8?T cell subset
and was found to increase with disease progression as shown in
Fig. 3, c and d (correlation with the CD4 counts: r ? ?0.72 and
p ? 0.0001 for HIV-2 infection; r ? ?0.69 and p ? 0.0001 for
HIV-1 infection). Within the CD45RA?CD4 and CD8 T cell pop-
ulations, both types of infections are associated with an increase in
the already highly expressed Fas as compared with uninfected in-
dividuals (data not shown).
Cell cycle status
Because HIV-1- and HIV-2-infected individuals with similar CD4
T cell counts have different viral loads but similar degrees of CD4
and CD8 cell activation, we asked how the two infections com-
pared in terms of T cell turnover as assessed by the expression of
the nuclear factor Ki67, which is up-regulated in all cell cycle
phases except G0(31). Increased fractions of Ki67?cells within
the CD4 subset were seen in both infections (Fig. 4), with an
inverse correlation to the blood numbers of CD4 T cells that was
not observed in the control group (r ? ?0.77 in HIV-1 and r ?
?0.72 in HIV-2 infection, with a value of p ? 0.001 in both
cases). The majority of the CD4?Ki67?T cells were found to be
CD45RO?(92 ? 0.7% and 89 ? 1.5% in HIV-1 and HIV-2 co-
Fig. 4 shows a linear regression of the frequency of Ki67?
within the CD4 subset over the logarithm of the CD4 counts,
which was statistically significant for both the HIV-1 and HIV-2
patients. In uninfected controls the percentage of Ki67?cells is
practically constant. The non-zero slopes observed in the infected
groups (?1.9 and ?1.3) were significant (p ? 0.001), indicating
that the turnover of CD4 T cells changes during HIV infection. The
slopes obtained during HIV-1 and HIV-2 infection differ by 20%
(p ? 0.05), suggesting that the impact of the two viruses may
differ in quantitative details while showing a similar trend as op-
posed to controls.
Correlation between the frequency of cycling cells and the
expression of activation markers
The frequencies of CD4 and CD8 T cells with an activation or
memory phenotype are positively correlated to the frequency of
Ki67?CD4 T cells and inversely correlated to the numbers of CD4
T cells in the blood (Table II).
Lymphoproliferative responses to mitogens or Ags
Chronic immune activation has been linked to T cell anergy in
HIV-1 infection. To investigate this issue we tested the ability of
the lymphocytes to proliferate in vitro in response to CD3 stimu-
lation in the absence and presence of CD28 costimulation and in
CD4 subset. Frequency of Ki67?cells within the CD4 subset is plotted as
a function of the logarithm of the total number of CD4 cells for 25 unin-
fected (F), 25 HIV-1-infected (E), and 24 HIV-2-infected (R) individuals.
Regression equations are as follows: controls, percentage of Ki67 ? 0.6–
0.5 log number of CD4 cells (p ? 0.05); HIV-1, percentage of Ki67 ?
5.9–1.9 log number of CD4 cells (p ? 0.001) (dashed line); HIV-2, per-
centage of Ki67 ? 4.1–1.3 log number of CD4 cells (p ? 0.001) (contin-
Relationship between cycling cells and depletion within the
Table II. Correlation (Spearman coefficients) of the proportion of CD4 T cells expressing different
activation markers with the frequency of Ki67?within the CD4 subset as well as with the total number of
peripheral blood CD4 T cells
Ki67 (%) No. of CD4 cellsKi67 (%)No. of CD4 cellsKi67 (%) No. of CD4 cells
Fas in CD45RA?
Fas in CD45RO?
aSignificant correlation at p ? 0.01.
3403The Journal of Immunology
response to several microbial Ags. HIV-2-infected patients exhib-
ited a marked decrease in these proliferative responses with pro-
gression of the disease (Fig. 5). When the proliferative responses
for infected patients from corresponding HIV-1/HIV-2 groups
were compared, they were found to be not significantly different
for any of the investigated stimulatory conditions (Fig. 5). More-
over, the respective responses to HIV-1 and HIV-2 recombinant
proteins revealed no significant reactivity, even in patients with
undiminished CD4 T cell counts.
For the same level of CD4 T cell depletion, HIV-1- and HIV-2-
infected patients exhibited similar elevations in the frequencies of
activated and cycling T cells. In contrast to previous studies (32),
we grouped HIV-1- and HIV-2-infected patients whose levels of
CD4 depletion fell in the same range and found that with this
categorization the two infections exhibit 1) a similar imbalance in
the naive/memory-effector population ratios, 2) comparable up-
regulation of CD4 and CD8 T cell activation markers (HLA-DR,
CD38, CD69, Fas molecule), 3) a similar increase in the frequency
of cycling CD4 T cells (Ki67?), which was in strong correlation
with the expression of activation markers, and 4) a similar level of
anergy, as assessed by the in vitro lymphoproliferative responses
to CD3 stimulation in the presence or absence of CD28 costimu-
lation and to a panel of microbial Ags. Considering that the two
HIV-associated immunodeficiencies are characterized by mark-
edly different plasma viral loads and are known to display different
rates of CD4 T cell decline and to have different clinical prognosis
(12–14, 24), these findings call for a reappraisal of widely held
views regarding the causal relationships among chronic immune
activation, T cell turnover, viremia, and the rate of CD4 decline.
According to one paradigm, the demand for CD4 T cell pro-
duction in response to rapid virus-mediated destruction is the di-
rect cause of increased turnover (1–3). Progressive depletion of
CD4?T cells is accordingly seen as a failure of production to keep
up with the rate of loss. This hypothesis has been challenged on
several grounds. In particular, the increase in the average T cell
turnover rate appears to be due to recurrent Ag- and inflammation-
driven expansion and subsequent contraction of a fraction of the
clonal repertoire, as was suggested by an analysis of in vivo DNA
labeling results (6, 33, 34) and by correlations between immune
activation, viral load, and CD4 counts during highly active anti-
retroviral treatment (HAART)3(35, 36). Recent observations in-
dicate that CD4 depletion during the chronic phase of HIV/SIV
infection is more directly related to the overall activation and turn-
over of T cells than to the turnover of infected CD4?cells and free
virus (37–39). These observations contradict a basic tenet of the
destruction-demand hypothesis (1–3), which links overall T cell
turnover to the turnover of infected cells and attributes depletion to
the latter. Levels of immune activation, CD4?T cell depletion,
and viremia in untreated HIV-1-infected patients have been com-
pared with those measured in HAART-failing patients who main-
tain increasing CD4 cell counts. For any level of viremia, CD4?
cell turnover rates were higher in patients with wild-type virus than
in patients with drug-resistant virus (37). Yet the turnover rates of
infected CD4?T cells in treated and untreated patients are prob-
ably not significantly different, because treatment of both with ef-
fective HAART regimens result in similar decay rates of plasma
HIV-RNA (40). Furthermore, the natural hosts of SIV, sooty
3Abbreviation used in this paper: HAART, highly active antiretroviral treatment.
in HIV-2- and HIV-1-infected patients grouped according to a progressive
degree of CD4 depletion, namely ?500 CD4 cells/?l (left), 200–500 CD4
cells/?l (middle), and ?200 CD4 cells/?l (right). PBMCs were cultured
for 3 days with immobilized anti-CD3 mAb in the absence (a) and in the
presence (b) of soluble anti-CD28 mAb, and for 6 days in the presence of
the recall Ags Candida albicans (c), purified protein derivative (d), and
tetanus toxoid (e). Proliferation was assessed by tritiated TdR incorporation
and results are expressed as cpm in the presence of a given stimulus minus
the cpm in its absence (cpm net). Each dot represents one individual. Bars
Lymphocyte proliferative responses in healthy subjects and
3404T CELL ACTIVATION AND CD4 DEPLETION IN HIV PATHOGENESIS
mangabeys and African green monkeys, show no significant in-
crease in immune activation and turnover (38, 39). Despite high-
level virus replication and rapid death of infected cells (M. B.
Feinberg, personal communication), they do not develop progres-
sive depletion of CD4 T cells.
The quantitatively similar association between CD4 depletion
and immune activation in HIV-1 and HIV-2 infections we report in
this work supports the hypothesis that immune activation drives
depletion (11). This hypothesis is further supported by the obser-
vation of reduced CD4 T cell counts and increased CD8 counts,
accompanied by elevated immune activation, in HIV-negative hu-
mans chronically infected with helminths and other parasites (41).
It is very unlikely that the similar rates of CD4?T cell turnover,
found in HIV-1 and HIV-2 patients with comparable levels of CD4
depletion, are associated with similar turnover rates of infected
cells despite the large difference in viremia levels. The lower per-
cell production of HIV-2 can account for the lower viremia levels.
But to account for similar infection rates (23, 24), HIV-2 should be
inherently much more infectious than HIV-1, and there is no ev-
idence for this (16). Although similar proviral levels were found in
HIV-1- and HIV-2-infected individuals (23, 24), a large proportion
of the provirus-containing cells in the blood of HIV-2-infected
individuals may be latently infected rather than virus-producing
cells—a cumulative measure of the infection rather than a measure
of ongoing replication.
The reasons for the generalized immune activation and the
mechanisms whereby it may affect the homeostatic regulation of T
lymphocytes are not well understood (11). Bursts of T cell prolif-
eration continuously occur, stimulated by proinflammatory factors
and Ags (6). Bursts may also occur in futile response to “homeo-
static” signals in lymphoid sites irreversibly depleted of CD4 T
cells (42). Immune activation maintains targets for viral replication
and, coupled to virus-mediated attrition, may drive the progression
of HIV disease by destabilizing or progressively changing the ho-
meostatic steady states of resting cell populations, naive and mem-
ory (4–11). Possible mechanisms include reduced production and
increased differentiation of naive T cells and net loss in the number
of resting memory T cells—especially CD4?cells—during im-
mune activation cycles. It is also conceivable that products of ac-
tivation, such as proinflammatory factors, affect the migration pat-
terns, viability, and response characteristics of the resting
lymphocytes (4, 27). The effect of immune activation on total T
cell numbers is complex. While the numbers of resting T cells
decline, the number of activated cells increases. CD8?T cells are
known to expand more extensively than CD4?T cells during im-
mune responses, and this might account for the overall increase in
CD8 T cell counts during the less-advanced stages of progression,
while CD4 counts progressively decline.
How can substantially lower viral loads in HIV-2 infection be
associated with levels of immune activation similar to those doc-
umented in HIV-1 infection? What is the basis for the slower rate
of progression of HIV-2 infection compared with HIV-1?
The “proximal activation and transmission” model (43, 44) of-
fers potential explanations. When “latently” infected memory cells
are involved in activation bursts, they spark local bursts of infec-
tion. The local nature of virus replication (“proximal activation and
transmission”) is supported by in situ analyses of HIV and TCR
molecular sequences (45–47). Infection bursts end in the death of
most activated cells, including virus-producing CD4?T cells, but
newly generated memory cells that contain provirus survive and
spark new infection bursts (11). The production of free virus dur-
ing each prolonged burst involves several rounds of infection. Be-
cause of this amplification, the amount of virus produced per burst
should be very sensitive to the efficacy of transmission (43, 44).
Because HIV-1 and HIV-2 showed a similar destructive impact
when tested in a human lymphoid tissue culture model (48), it is
likely that certain host factors limit HIV-2 replication rate in vivo,
perhaps by acting on target cells (49–51). We have reported
marked immunosuppressive effects of the HIV-2 envelope protein
(52), which may reduce the probability of virus transmission
among T cells responding to virus Ags or to other pathogens. Even
a modest reduction of this probability may have a drastic effect on
the virus produced in local infection bursts because of the ampli-
fication factor. In contrast, the contribution of each burst to sys-
temic activation, related to HIV-mediated enhancement of APC-
lymphocyte interactions, is likely to be relatively insensitive to the
amount of free virus produced in such a burst. In that case, the
systemic level of activation would reflect mainly the frequency of
bursts initiated by infected memory cells rather than the amount of
free virus produced in each burst. In contrast, the viremia level is
proportional to the product of these two parameters.
HIV-1- and HIV-2-infected individuals would thus manifest
comparable levels of immune activation and CD4 depletion when
they have accumulated comparable numbers of infected memory
cells leading to comparable frequencies of infection bursts, but
viremia levels in HIV-2 infection would remain much lower be-
cause of the inefficient replication of HIV-2 within expanding pop-
ulations of activated CD4?T cells. This interpretation can explain
our findings and is also consistent with the finding of roughly
similar amounts of proviral DNA in peripheral blood mononuclear
cells from people infected with either HIV-2 or HIV-1, despite the
large difference in RNA amounts (23, 24). Inefficient replication of
HIV-2 would likely be associated with a slower accumulation of in-
fected memory cells and therefore with slower progression of HIV-2
infection compared with HIV-1 (12, 14, 20).
Despite the overall similarity, we have noted certain quantitative
differences between HIV-1 and HIV-2 infection in the patterns of
immune activation for similar levels of CD4 depletion. Notably,
there were more CD8 T cells expressing CD69 in HIV-1-infected
patients than in HIV-2-infected patients and slight differences in
the linear regression lines of the frequency of Ki67?CD4 T cells
over the CD4 counts (see Results). These differences may be re-
lated to the large difference in the amount of viral Ag. One should
bear in mind that different activation markers are likely associated
with different immune activation events that contribute to “chronic
immune activation,” and that some of these events are bound to be
more closely associated with CD4 depletion than others.
In summary, our findings support a close linkage between im-
mune activation and CD4 cell depletion in HIV infection and only
an indirect relationship of these parameters to the virus rate of
replication. Although the destruction-replacement hypothesis has
been forcibly advocated, a strong case can be made for alternative,
immune activation-centered hypotheses. Further comparative stud-
ies of the different host/virus systems would allow a more defin-
itive delineation of cause and effect in HIV disease progression,
with implications for the choice of treatment strategies.
We gratefully acknowledge P. Gomes and M. H. Lourenc ¸o for the perfor-
mance of the viral load studies in HIV-2-infected patients and the clinical
collaboration of the following colleagues: F. Antunes, J. Azevedo, J. Car-
doso, S. Correia, M. Doroana, F. Ina ´cio, M. Lucas, L. Pinheiro, J. Poc ¸as,
J. Ribeiro, I. Santos, and L. Tavares.
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3406T CELL ACTIVATION AND CD4 DEPLETION IN HIV PATHOGENESIS