Loss of HIV-1-specific CD8+ T cell proliferation after acute HIV-1 infection and restoration by vaccine-induced HIV-1-specific CD4+ T cells.

Abstract

Virus-specific CD8(+) T cells are associated with declining viremia in acute human immunodeficiency virus (HIV)1 infection, but do not correlate with control of viremia in chronic infection, suggesting a progressive functional defect not measured by interferon gamma assays presently used. Here, we demonstrate that HIV-1-specific CD8(+) T cells proliferate rapidly upon encounter with cognate antigen in acute infection, but lose this capacity with ongoing viral replication. This functional defect can be induced in vitro by depletion of CD4(+) T cells or addition of interleukin 2-neutralizing antibodies, and can be corrected in chronic infection in vitro by addition of autologous CD4(+) T cells isolated during acute infection and in vivo by vaccine-mediated induction of HIV-1-specific CD4(+) T helper cell responses. These data demonstrate a loss of HIV-1-specific CD8(+) T cell function that not only correlates with progressive infection, but also can be restored in chronic infection by augmentation of HIV-1-specific T helper cell function. This identification of a reversible defect in cell-mediated immunity in chronic HIV-1 infection has important implications for immunotherapeutic interventions.

Full text

Page 1

The Journal of Experimental Medicine
J. Exp. Med.
Volume 200, Number 6, September 20, 2004 701–712
http://www.jem.org/cgi/doi/10.1084/jem.20041270
©
The Rockefeller University Press • 0022-1007/2004/09/701/12 $8.00
701
Loss of HIV-1–specific CD8
HIV-1 Infection and Restoration by Vaccine-induced
HIV-1–specific CD4
T Cells
?
T Cell Proliferation after Acute
?
Mathias Lichterfeld,
Marylyn M. Addo,
Eunice Pae,
Bruce D. Walker,
1
Mary N. Johnston,
Galit Alter,Alysse Wurcel,
1,4
and Marcus Altfeld
Daniel E. Kaufmann,
1
Xu G. Yu,
Daniel Cohen,
1, 3
David Stone,
1
Stanley K. Mui,
2
Gregory K. Robbins,
3
Eric S. Rosenberg,
1
1 1 1
2 1 1
1
1
Partners AIDS Research Center, Massachusetts General Hospital and Division of AIDS, Harvard Medical School,
Boston, MA 02129
Fenway Community Health Care Center, Boston, MA 02115
Lemuel Shattuck Hospital, Boston, MA 02130
Howard Hughes Medical Institute, Chevy Chase, MD 20815
2
3
4
Abstract
Virus-specific CD8
ciency virus (HIV)1 infection, but do not correlate with control of viremia in chronic infection,
suggesting a progressive functional defect not measured by interferon
Here, we demonstrate that HIV-1–specific CD8
with cognate antigen in acute infection, but lose this capacity with ongoing viral replication.
This functional defect can be induced in vitro by depletion of CD4
kin 2–neutralizing antibodies, and can be corrected in chronic infection in vitro by addition of
autologous CD4
T cells isolated during acute infection and in vivo by vaccine-mediated induction
of HIV-1–specific CD4
T helper cell responses. These data demonstrate a loss of HIV-1–specific
CD8
T cell function that not only correlates with progressive infection, but also can be restored in
chronic infection by augmentation of HIV-1–specific T helper cell function. This identifica-
tion of a reversible defect in cell-mediated immunity in chronic HIV-1 infection has important
implications for immunotherapeutic interventions.
?
T cells are associated with declining viremia in acute human immunodefi-
?
assays presently used.
?
T cells proliferate rapidly upon encounter
?
T cells or addition of interleu-
?
?
?
Key words:HIV-1 • CD8
?
T cells • CD4
?
T cells • vaccine • protective immunity
Introduction
Acute HIV-1 infection is characterized by high level plasma
viremia, which leads to an activation of the cellular im-
mune system and the rapid expansion of HIV-1–specific
CD8
T cells (1). The first appearance of these cells in the
peripheral blood is followed by a rapid and dramatic decline of
HIV-1 plasma viremia, probably reflecting the strong anti-
viral activities of these cells (2, 3). The HIV-1–specific
CD8
T cell responses detected during acute HIV-1 infec-
tion are typically low in magnitude and narrowly directed
against a paucity of viral epitopes (4–6). Thus, the apparent
antiviral activity of these cells in acute infection constitutes
a striking contrast to chronic HIV-1 infection, where high
levels of viral replication occur in the presence of strong,
?
?
polyclonal and broadly diversified HIV-1–specific CD8
cell responses, as determined by the assessment of antigen-
specific interferon
?
secretion (7–9). These data suggest a
progressive functional defect of HIV-1–specific CD8
cells in chronic infection that is not measured by assays
quantifying solely antigen-specific interferon
of T cells.
Recent data demonstrated that HIV-1–specific CD8
cells in individuals with long-term nonprogressive infection
have a strong HIV-1–specific ex vivo proliferative capacity,
whereas this effector function seems to be absent in individuals
with high level viremia (10). A similar loss of HIV-1–specific
ex vivo proliferation was also observed for HIV-1–specific
CD4
T cells, which show strong proliferative capacities in
?
T
?
T
?
production
?
T
?
Address correspondence to Marcus Altfeld, Partners AIDS Research Center,
Massachusetts General Hospital, 149 13th St., Boston, MA 02129. Phone:
(617) 724-2461; Fax: (617) 724-8586; email: maltfeld@partners.org
Abbreviations used in this paper:
FSC, forward scatter; SFC, spot-forming cell; SSC, side scatter.
CFSE, carboxyfluorescein succinimidyl ester;

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HIV-specific CD8
?
T Cell Proliferation in Acute Infection
702
acute and long-term nonprogressive infection (11–13), but
no detectable ex vivo proliferation in the presence of ongo-
ing viral replication. However, a direct functional connec-
tion between these two cell subsets has not been revealed and
functional interactions between HIV-1–specific CD4
CD8
T cells are currently insufficiently understood.
In this study, we demonstrate that HIV-1–specific CD4
and CD8 T cells in acute HIV-1 infection have strong ex
vivo proliferative capacities, which are rapidly lost in the
presence of continuing viral replication, but partially pre-
served by early institution of antiretroviral therapy. HIV-1–
specific proliferation of CD8
on the presence of IL-2–secreting antigen-specific CD4
cells, as it was restored in CD8
chronic infection in vitro by the addition of autologous
HIV-specific CD4
T cells isolated during acute infection
and more importantly, in vivo by the induction of HIV-1–
specific CD4
T helper cell responses using an HIV-1 im-
munogen. Overall, these data suggest that the proliferative
impairment of HIV-1–specific CD8
infection is not primarily due to an intrinsic functional de-
fect of these cells, but rather represents a direct conse-
quence of the progressive loss of IL-2–secreting, HIV-1–
specific CD4
T cells.
?
and
?
?
?
?
T cells critically depended
?
T
?
T cells from the phase of
?
?
?
T cells during chronic
?
Materials and Methods
Study Individuals.
recruited from the Massachusetts General Hospital, the Fenway
Community Health Care Center, or the Lemuel Shattuck Hospi-
tal were included in this study. 18 persons had primary HIV-1 in-
fection, defined by negative or incompletely positive HIV-1–spe-
cific ELISA and/or Western blot reactions in the presence of
detectable viral load or HIV-1 seroconversion within 6 mo before
study enrollment (4). Seven additional subjects with long-term
nonprogressive disease courses (CD4
and viral load of
?
1,000 copies/ml for at least 5 yr in the absence
of antiretroviral therapy) and 10 individuals with chronic progres-
sive HIV-1 infection (viral load of
cell count of
?
300 cell/
?
l) were also included. The clinical and
demographic characteristics of the study individuals are summa-
rized in Table I. In addition, cryopreserved PBMCs from 10 study
individuals who had previously participated in a clinical pilot trial
using an HIV-1 immunogen (14) were analyzed in this study. The
study was approved by the respective institutional review boards
and was conducted in accordance with human experimentation
guidelines of the Massachusetts General Hospital.
HLA Typing.
High and intermediate resolution HLA class I
typing was performed at a commercial laboratory (Dynal) by se-
quence-specific PCRs according to standard procedures. DNA
for typing was extracted using the Purgene DNA Isolation kit for
whole blood samples (Gentra Systems).
Lymphocyte Separation and Culture.
drawn in ACD tubes (Becton Dickinson). Fresh PBMCs were
separated from whole blood by Ficoll-Hypaque (Histopaque
1077; Sigma-Aldrich) density gradient centrifugation. PBMCs
were cultured in RPMI 1640 medium (Sigma-Aldrich) supple-
mented with 2 mM
l
-glutamine, 50 U/ml penicillin, 50
streptomycin, 10 mM Hepes, and 10% heat-inactivated FCS.
Recombinant human IL-2 (provided by the AIDS Research &
A total of 35 HIV-1–infected individuals
?
T cell count of
?
500/ml
?
30,000 copies/ml or CD4
?
T
Blood specimens were
?
g/ml
Reference Reagent Repository, National Institutes of Health)
was added in some assays at a concentration of 50I U/ml.
Synthetic Peptides.
410 synthetic 17–19 amino acid peptides,
overlapping by 10 amino acids and spanning the entire HIV-1
clade B 2001 consensus sequence (http://hiv-web.lanl.gov), and
peptides corresponding to described optimal HIV-1 CD8
epitopes (15), were synthesized at the Massachusetts General
Hospital Peptide Core Facility on an automated peptide synthe-
sizer using F-moc technology. 14 CMV-specific peptides that
were selected on the basis of their capacity to bind to HLA class
II DRB1
*
0401 in binding assays (16) were similarly synthesized.
ELISPOT Assay.
ELISPOT assays were performed as de-
scribed previously (4). In brief, PBMCs were plated in 96-well
polyvinylidene plates that had been precoated with 0.5
an anti–human interferon
?
mAb (Mabtech). PBMCs were added
at a concentration of 50,000–100,000 cells per well in a volume of
100
?
l RPMI 1640 medium supplemented with 10% FCS, 10
mM Hepes buffer, 2 mM
l
-glutamine, and 50 U/ml penicillin-
streptomycin. The final concentration of the peptides in every sin-
gle well was 14
?
g/ml. Plates were incubated overnight at 37
5% CO
2
, and developed on the next day as described elsewhere
(17). Wells containing PBMCs and medium with phytohemagglu-
tinin or without any peptide were used as positive or negative
controls and run in triplicate on each plate. To calculate the num-
ber of specific T cells, the number of spots in the negative control
wells was subtracted from the counted number of spots in each
well. Responses were considered positive if there were
forming cells (SFCs)/10
PBMCs and at least three times the mean
number of SFCs of the three control wells. CD8
dence of responses was determined by depletion of CD4
using the MiniMACS cell depletion system (Miltenyi Biotec).
Ex Vivo Proliferation Assay.
PBMCs were first suspended at
10
/ml in PBS and incubated at 37
carboxyfluorescein succinimidyl ester (CFSE; Molecular Probes).
After the addition of serum and washes with PBS, cells were sus-
pended at 10
/ml in medium (RPMI 1640 supplemented with
glutamine, 10% human FCS, penicillin, and streptomycin). No
exogenous cytokines were added to the medium unless otherwise
indicated. Pools of overlapping HIV-1–specific peptides represent-
ing the entire amino acid sequence of either Gag, Nef, Pol, Env, a
combined pool of Tat, Rev, Vif, Vpr, Vpu, or tetanus toxoid
(Aventis Behring) were then added at a concentration of 20 ng/ml
per peptide. On day 6, cells were harvested, washed with PBS, and
stained with mAbs (CD4 PE, CD8 APC, and CD3 PerCP; BD
Biosciences). Cells were then washed and fixed in 1% paraformal-
dehyde and subjected to flow cytometric analysis. Where indi-
cated, IL-2–neutralizing antibodies (clone MQ1-17H12; BD Bio-
sciences) or isotype control antibodies (clone A4A; Neomarkers)
were added at 10
?
g/ml. In some experiments, 10
CD4
T cells isolated and cryopreserved during acute or chronic
HIV-1 infection were thawed and added to 5
chronic HIV-1 infection. Flow cytometric data (100,000 nongated
events) were acquired on a FACSCalibur four-color flow cytome-
ter using CELLQuest software or an LSR II flow cytometer using
the FACS DiVa software (all instruments and software from BD
Biosciences). The mean background proliferation was calculated
based on the proliferating fractions in media alone. The antigen-
specific proportion of proliferating cells was calculated by subtract-
ing the proportion of proliferating cells in unstimulated samples
from the proliferating fraction in response to antigen.
Multiparameter Flow Cytometric Analysis.
five different monoclonal surface antibodies (CD25 [IL-2R
chain] PE-Cy7, CD8 APC-Cy7, CD4 APC-Cy5.5, IL-7R
?
T cell
?
g/ml of
?
C,
?
50 spot-
6
?
T cell depen-
?
T cells
6
?
C for 7 min with 0.25
?
M
6
6
autologous
?
?
10
6
PBMCs from
We used a panel of
?
?

Page 3

Lichterfeld et al.
703
chain PE, and IL-15R
sciences) in addition to staining with APC-labeled MHC class
I–peptide tetramer complexes (Beckman Coulter) and CFSE. For
intracellular cytokine stainings, cells were initially stained with
surface antibodies. After fixation and permeabilization, cells were
stained with intracellular antibodies as described previously (17).
Samples were acquired on an LSR II flow cytometric device (BD
Biosciences), using the FACS DiVa software (BD Biosciences)
according to the manufacturer’s instructions. Data analysis was
performed with the FlowJo software package (TreeStar).
Depletion and Enrichment of Selective Cellular Subsets.
of CD8
and CD4T cells as well as depletion of CD3
from whole blood samples was performed by use of the respec-
tive Rosette Sep cell separation kits (StemCell Technologies
Inc.). Depletion of CD4
from isolated PBMCs was performed
using magnetic anti-CD4
beads and the MACS cell separation
system (Miltenyi Biotec). All cell enrichment and depletion pro-
cedures were conducted by negative selection to ensure that iso-
lated cells were not labeled with antibodies.
Statistical Analysis.
Results are given as means or medians
with ranges. Statistical analysis was based on Student’s
p-value of
?
0.05 was considered significant. When two adjacent
peptides were recognized in the ELISPOT assays, we deleted the
weaker of the two responses. In the case of three adjacent pep-
tides eliciting a response, the weakest of all three peptides was de-
leted and the responses were counted as two epitopic regions.
Statistical analysis and graphical presentation was performed by
use of the GraphPad Prism software package.
?
chain Alexa 430; all from BD Bio-
Isolation
?
T cells
? ?
?
?
t
tests. A
Results
Strong Lymphoproliferative Responses of HIV-1–specific
CD8
T Cells in Primary HIV-1 Infection.
recent studies have analyzed the magnitude, breadth, and
?
A number of
protein specificity of HIV-1–specific CD8
in primary HIV-1 infection by interferon
nology or intracellular cytokine staining (4–6, 18). Here, we
extended these studies and used a flow cytometric prolifera-
tion assay based on the sequential loss of CFSE labeling in
dividing cells to determine the ex vivo proliferative capacity
of HIV-1–specific CD8
T cells in a total of 18 different
subjects with primary HIV-1 infection. In addition, 10 indi-
viduals with untreated chronic progressive HIV-1 infection
and 7 individuals with untreated long-term nonprogressive
HIV-1 infection were included as reference populations.
The demographic and clinical characteristics of the study
subjects are summarized in Table I.
Fig. 1 shows representative experimental results from in-
dividuals with long-term nonprogressive HIV-1 infection
(Fig. 1 A), chronic progressive HIV-1 infection (Fig. 1 B),
and primary HIV-1 infection (Fig. 1 C). Although similar
frequencies of dividing CD8
three study subjects after stimulation with PHA, popula-
tions of proliferating antigen-specific CD8
only seen in subjects with primary or long-term nonpro-
gressive HIV-1 infection after stimulation of cells with a
pool of overlapping peptides spanning the HIV-1 Nef pro-
tein. These proliferating CD8
HIV-1 Nef, as demonstrated by the specific staining with
MHC class I tetramers refolded with HIV-1 Nef peptides
(Fig. 1, D and E). Overall, the proportion of CD8
proliferating in response to stimulation with viral peptides
spanning the entire HIV-1 proteome reached a median
of 10.9% (range: 3.5–22%) and 23.6% (range: 11.8–59.9%)
in individuals with primary or long-term nonprogressing
HIV-1 infection, respectively. In contrast, essentially no
?
T cell responses
ELISPOT tech-
?
?
?
T cells were observed in the
?
T cells were
?
T cells were specific for
?
T cells
Table I.
Demographical and Clinical Characteristics of the Study Persons
CD4
(median, range)
?
T cell countHIV-1 RNA
(median, range)
Study
population
Age
(median, range)
Sex
(m/f ratio)EthnicityBaseline
1 yr
follow-up Baseline
1 yr
follow-up
Subjects with long-term
nonprogressing
HIV-1 infection
(
n

?
7)
47
(34–51)
5:26 Caucasians,
1 Hispanic
805 cells/
(566–1,118)
?
ln/a555 copies/ml
(
?
50–1,000)
n/a
Subjects with
primary HIV-1
infection
(
n

?
18)
46.4
(32–57)
17:1
16 Caucasians,
1 African
American,
1 Haitian
562 cells/
(307–1,141)
?l
untreated:
490 cells/?l
(342–1,050)
treated:
753 cells/?l
(434–948)
105,000 copies/ml
(216–2,500,000)
untreated:
26,300 copies/ml
(4,231–63,900)
treated:
?50 copies/ml
(all patients)
Subjects with
chronic progressive
HIV-1 infection
(n ? 10)
42.5
(37–56) 9:1
5 Caucasians,
2 African
Americans,
2 Hispanics
177 cells/?l
(20–460) n/a
42,344 copies/ml
(7,247–232,000) n/a

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HIV-specific CD8? T Cell Proliferation in Acute Infection
704
HIV-1–specific CD8? T cell ex vivo proliferation was ob-
served in study persons with chronic progressive HIV-1 in-
fection (Fig. 1 F; reference 10), whereas the proportion of
proliferating CMV-specific CD8? T cells was not reduced
in a subset of individuals with chronic HIV-1 infection
when compared with persons with acute infection (median
of 7% [range: 4–17%] vs. 9.1% [range: 7.5–23%]; P ? 0.5).
The lymphoproliferative CD8? T cell responses in pri-
mary HIV-1 infection targeted multiple viral regions, with a
median proportion of 2.5% (range: 0–16.4%), 1.6% (range:
0–15%), 0.3% (range: 0–5.3%), 0.25% (range: 0–11%), or
0.1% (range: 0–6.48%) of CD8? T cells responding to stim-
ulation with Nef, Gag, Pol, Env, or the remaining regula-
tory and accessory HIV-1 proteins (Vpr, Vpu, Vif, Rev, and
Tat), respectively. Notably, the observed pattern of lym-
phoproliferative HIV-1–specific CD8? T cells in the three
study groups was strikingly different from the corresponding
HIV-1–specific CD8? T cell responses measured using an
interferon ? ELISPOT assay. As described previously (4, 7),
the total magnitude of interferon ?–secreting, HIV-1–spe-
cific CD8? T cells in individuals with primary HIV-1 infec-
tion was significantly lower than in individuals with chronic
infection, whereas no difference in the total HIV-1–specific
CD8? T cell magnitude was found between individuals
with progressive and long-term nonprogressive chronic in-
fection (Fig. 1 G). Taken together, these data show that
HIV-1–specific CD8? T cells in primary and long-term
nonprogressive HIV-1 infection have strong ex vivo prolif-
erative capacities, whereas this effector function is absent in
chronic progressive HIV-1 infection.
Parallel Evolution of Lymphoproliferative HIV-1–specific
CD4? and CD8? T Cell Responses after Primary HIV-1 Infec-
tion.
To more closely determine the fate of HIV-1–spe-
cific CD8? T cell lymphoproliferative responses mounted
during primary HIV-1 infection, we longitudinally fol-
lowed the evolution of these responses during the ensuing
disease process, using the CFSE-based proliferation assay in
conjunction with an interferon ? ELISPOT assay. In line
with previous findings (4, 19), we observed that the total
magnitude of HIV-1–specific interferon ?–secreting CD8?
T cells increased by a median of threefold over a 1-yr pe-
riod in individuals with ongoing viral replication in the ab-
sence of antiretroviral combination therapy (Fig. 2 A). In
contrast, the proliferative capacity of these cells diminished
dramatically during the same time period in antiretroviral
therapy–naive individuals, with almost no CD8? T cells
proliferating in response to HIV-1 antigen after 1 yr of fol-
low-up. In individuals with rapid institution of antiretrovi-
ral therapy during primary HIV-1 infection (Fig. 2 B), we
observed a stable magnitude of interferon ?–secreting,
HIV-1–specific CD8? T cell responses over a 1-yr study
period (a median of 1,420 SFCs/106 PBMCs vs. a median
of 1,985 SFCs/106 PBMCs). Interestingly, the correspond-
ing proportions of CD8? T cells proliferating in response to
HIV-1 antigen declined substantially after primary HIV-1
infection, but were maintained at clearly detectable levels
and significantly exceeded the proportion of antigen-spe-
cific proliferating CD8? T cells in persons with continuing
viral replication (a median of 2.2% [range: 0.7–14.25%] vs.
a median of 0.4% [range: 0.3–1.2%], respectively; P ?
0.03), although there was no statistically significant differ-
ence between these two study cohorts at baseline during
primary infection (P ? 0.5). In line with previous reports
(12, 20), a partial conservation of lymphoproliferative re-
sponses by initiation of antiretroviral therapy during pri-
mary HIV-1 infection was also observed for HIV-1–specific
CD4? T cells, whereas the HIV-1–specific proliferation of
CD4? T cells was essentially lost 1 yr after acute HIV-1 in-
fection in individuals with continuously ongoing viral rep-
lication (Fig. 2, A and B). Overall, we observed a strong
correlation between the total proportion of proliferating
HIV-1–specific CD4? and CD8? cells (Fig. 2 C). In con-
Figure 1.
stimulation with HIV-1 peptide pools in individuals with primary,
chronic progressive, and chronic long-term nonprogressive HIV-1 infec-
tion. (A–C) Dot plots showing the flow cytometric analysis of HIV-1–
specific CD8? T cell proliferation after stimulation of PBMCs with no
stimulus, phytohemagglutinin (PHA), or a pool of overlapping peptides
spanning the entire HIV-1 Nef protein in subjects with long-term non-
progressive (A), chronic progressive (B), or primary (C) HIV-1 infection.
Values in top left corner of dot plots indicate the proportion of CFSElow
CD8? T cells. (D and E) Corresponding antigen specificity of prolifera-
tion cells. 34% of proliferating cells in the study individual in A were
binding to the HLA-A3-QVPLRPMTYK (QK10) tetramer (D), whereas
82% of the CD8? T cells proliferating in the study person in C were specific
for the HLA-B8-FLKEKGGL (FL8) tetramer (E). (F and G) Comparative
analysis of proliferation and interferon ? secretion by CD8? T cells in re-
sponse to stimulation with overlapping peptides spanning the entire HIV-1
proteome. Data from study subjects with chronic progressive HIV-1 infec-
tion (CPHI; n ? 10), chronic long-term nonprogressive HIV-1 infection
(CNPHI; n ? 7), and primary HIV-1 infection (PHI; n ? 18) are shown.
Cross-sectional assessment of CD8? T cell proliferation after

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Lichterfeld et al.
705
trast, no correlation was seen between HIV-1–specific CD8?
T cell lymphoproliferative responses and the corresponding
magnitude of HIV-1–specific, interferon ?–secreting CD8?
T cells (Fig. 2 D). Taken together, these data indicate a
parallel evolution of lymphoproliferative HIV-1–specific
CD8? and CD4? T cell responses, and suggest a potential
link between the ex vivo proliferative responses of these
two antigen-specific T cell populations.
Ex Vivo Proliferation of HIV-1–specific CD8? Cells Criti-
cally Depends on IL-2.
Previous studies indicate a decisive
role of IL-2 for maintaining the ex vivo proliferative activity
of HIV-1–specific CD4? T helper cells (21). Here, we con-
ducted a series of experiments to elucidate the relevance
of IL-2 for also sustaining ex vivo proliferative capacities of
HIV-1–specific CD8? T cells. Overall, the neutralization of
IL-2 by IL-2–specific mAbs resulted in an almost complete
abrogation of ex vivo proliferative activities of HIV-1–spe-
cific CD8? T cells from study subjects with acute HIV-1 in-
fection, whereas control antibodies did not yield similar ef-
fects (Fig. 3, A–D). This was the case both for the entire
population of CD8? T cells dividing after stimulation with
HIV-1 peptides (Fig. 3, A and C), as well as for subsets of
proliferating CD8? T cells specific for certain defined HIV-1
CD8? T cell epitopes, as determined by staining with
HIV-1 epitope-specific MHC class I tetramers (Fig. 3, B
and D). IL-2–neutralizing mABs similarly abrogated the
proliferative capacity of CMV-specific CD8? T cells (not
depicted), indicating that IL-2 dependence was not con-
fined to HIV-1–specific CD8? T cells. In addition, CD8? T
cells proliferating in response to HIV-1 peptides signifi-
cantly up-regulated the surface expression of the IL-2R?
chain. In contrast, these dividing CD8? T cells down-regu-
lated or maintained constant cell surface expression levels of
the ? receptor chains for the homeostatic cytokines IL-7
and IL-15 (Fig. 3, E and F). Taken together, these results
indicate a critical relevance of IL-2 for the ex vivo prolifera-
tive capacity of HIV-1–specific CD8? T cells.
IL-2 Production by CD4? T Cells Supports HIV-1–specific
CD8? T Cell Proliferation.
To more closely determine cell
populations supporting the ex vivo proliferative activity of
HIV-1–specific CD8? T cells in individuals with primary
HIV-1 infection, we assessed HIV-1–specific CD8? T cell
lymphoproliferative responses after the selective ex vivo re-
moval of distinct leukocyte subsets (Fig. 4, A–C). Interest-
ingly, HIV-1–specific CD8? T cell proliferation was almost
entirely blocked when lymphocyte samples had been de-
pleted of CD4? cells before the addition of HIV-1 peptides
(Fig. 4, A and B). In contrast, HIV-1–specific proliferation
was restored after coincubation of isolated CD8? T cells
with isolated CD4? T cells, indicating that CD4? T cells
are essential for the ex vivo proliferative activities of HIV-
1–specific CD8? T cells (Fig. 4, A and B). The HIV-1–spe-
cific proliferative capacity of CD8? T cells that was lost af-
ter depletion of autologous CD4? cells was also restored by
Figure 2.
specific CD8? T cell proliferative responses after
primary HIV-1 infection. (A and B) Simulta-
neous assessment of antigen-specific proliferation
and interferon ? secretion of CD8? and CD4?
T cells after stimulation with overlapping pep-
tides spanning the entire HIV-1 proteome at
baseline and after 1 yr of follow-up in study
persons with untreated (A) and treated (B) pri-
mary HIV-1 infection. (C and D) Correlation
between HIV-1–specific CD8? and CD4? T
cell lymphoproliferative responses. Proportions
of CD8? cells proliferating after exposure to
overlapping peptides spanning the HIV-1 pro-
teome were plotted against the corresponding
proportion of CD4? T cells (C) and against the
corresponding magnitude of CD8? T cell–
mediated SFCs/106 PBMCs using an interferon
? ELISPOT assay (D). Data from the cross-
sectional and longitudinal analysis were included.
Dashed lines indicate the 95% confidence inter-
val of the regression line.
Longitudinal evolution of HIV-1–

Page 6

HIV-specific CD8? T Cell Proliferation in Acute Infection
706
the addition of exogenous IL-2 (Fig. 4, D–G), indicating
that IL-2 can directly support HIV-1–specific CD8? T cells
in the absence of CD4? T helper cells. In addition, exoge-
nous IL-2 restored the ex vivo proliferative capacity of a
subset of HIV-1–specific CD8? T cells in individuals with
chronic progressive HIV-1 infection that was otherwise
lost (Fig. 4, H and I). Thus, these data show that the ex
vivo proliferative activity of HIV-1–specific CD8? T cells
critically depends on IL-2 secreted by CD4? T cells.
Antigenic Stimulation of CD4? T Cells Significantly En-
hances the Antigen-specific Proliferative Activity of HIV-1–specific
CD8? T Cells.
In our previous experiments, the ex vivo
proliferation capacity of HIV-1–specific CD8? T cells was
assessed after stimulation of PBMCs with pools of overlap-
ping peptides spanning HIV-1 proteins, which simulta-
neously elicited lymphoproliferative CD4? and CD8? T cell
responses, but did not allow for the analysis of the lympho-
proliferative activity of HIV-1–specific CD8? T cells in the
absence of concurrent CD4? T cell proliferative immune re-
sponses. We subsequently tested the ex vivo proliferative ca-
pacity of HIV-1–specific CD8? T cells that had been stimu-
lated with defined optimal HIV-1–specific CD8? T cell
epitopic peptides in the presence or absence of a simulta-
neous stimulus for HIV-1–specific CD4? T cell proliferative
responses. Fig. 5, A and B, shows results from the HLA-B8–
expressing study individual AC-31. Only a limited propor-
tion of CD8? T cells specific for the HLA-B8–restricted Nef
epitope FLKEKGGL (B8-FL8) and virtually no CD4? T
cells proliferated when PBMCs samples were stimulated
with the HLA-B8–restricted CD8? T cell epitopic peptide
B8-FL8 alone. In contrast, a dramatically stronger HIV-1–
specific CD8? T cell proliferative response was observed
after stimulation of PBMCs with the B8-FL8 peptide and
an additional HIV-1 Nef peptide (PEKEVLVWKFDSR-
LAFHH) that, when used alone, elicited a selective CD4? T
cell–mediated lymphoproliferative response, but no signifi-
cant CD8? T cell proliferation (Fig. 5, A, B, D, and E). The
enhancement of the ex vivo proliferation of HIV-1–specific
CD8? T cells by synchronized stimulation of HIV-1–spe-
cific CD4? T cells was almost entirely blocked by adding
IL-2–neutralizing antibodies (Fig. 5 C). Finally, we observed
that the ex vivo proliferation of HIV-1–specific CD8? T
cells can also be enhanced by simultaneous stimulation of
CD4? T cells specific for CMV or tetanus toxoid (Fig. 5,
D–F). Thus, these data illustrate that antigen-specific lym-
phoproliferative CD4? T cell responses significantly enhance
the ex vivo proliferative activity of HIV-1–specific CD8? T
cells in an IL-2–dependent fashion.
Autologous HIV-1–specific CD4? T Cells Isolated in Acute
Infection Can Reconstitute the Proliferative Activity of HIV-1–
specific CD8? T Cells in Chronic Infection.
demonstrate that HIV-1–specific CD8? T cell proliferation
critically depends on IL-2 produced by antigen-specific
CD4? T cells. Next, we tested whether CD4? T cells har-
vested during acute HIV-1 infection, when triggered by
HIV-1, could rescue the ex vivo proliferative activity of
HIV-1–specific CD8? T cells in chronic, untreated HIV-1
infection. Fig. 6 A shows data from study individual AC-98.
Strong CD8? T cell–mediated lymphoproliferative immune
responses were observed in acute HIV-1 infection after
PBMC stimulation with a pool of overlapping HIV-1 Nef
peptides, with a significant proportion of these proliferating
The above data
Figure 3.
CD8? T cells critically depends on IL-2. (A and B) Dot plots (A) or histo-
grams (B) showing the flow cytometric analysis of the proportion of
CD8? T cells (A) or HLA-B8-FLKEKGGL (FL8) tetramer–specific
CD8? T cells (B) proliferating in response to stimulation with a pool of
overlapping peptides spanning HIV-1 Nef in the presence or absence of
anti–IL-2 mAb. Values in top left corner of dot plots indicate the propor-
tion of CFSElow CD8? T cells. (C and D) Proportion of CD8? T cells (C)
or B8-FL8 tetramer–specific CD8? T cells (D) proliferating after exposure
to a pool of overlapping Nef peptides in the presence or absence of IL-2
antibodies. Mean and standard deviation of four experiments in four different
study persons are shown. (E) Flow cytometric analysis of the surface ex-
pression of the ? chain of the IL-2, IL-7, and IL-15 receptor in CD8? T
cells proliferating after stimulation with overlapping HIV-1 Nef peptides.
(F) Median fluorescence of antibodies directed against the ? chain of the
IL-2, IL-7, and IL-15 receptor in CD8? T cells proliferating (black bars)
or nonproliferating (white bars) after stimulation with HIV-1 Nef pep-
tides. Data reflect the mean and standard deviation of five independent
experiments in four different study subjects.
Antigen-specific ex vivo proliferation of HIV-1–specific

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Lichterfeld et al.
707
cells being specific for the HLA-A3–restricted Nef epitope
A3-QK10. These lymphoproliferative responses were al-
most completely lost during chronic HIV-1 infection, de-
spite the physical preservation of QK10-specific CD8? T
cells, as determined by staining with QK10-specific MHC
class I tetramers (Fig. 6, A and B). Yet, the addition of au-
tologous CD4? T cells isolated during acute HIV-1 infec-
tion to the PBMC sample from chronic infection rescued
the proliferation of a subset of CD8? T cells specific for the
QK10 tetramer after stimulation with a pool of overlapping
Nef peptides, whereas the addition of the same number of
autologous CD4? T cells isolated during chronic HIV-1 in-
fection did not increase the proliferative activity of HIV-1–
specific CD8? T cells in chronic infection. The added
autologous CD4? T cells from acute HIV-1 infection ex-
hibited strong HIV-1 Nef-specific lymphoproliferative ca-
pacities and IL-2 secretion (Fig. 6, A and C), whereas CD4?
T cells from the chronic disease phase were neither able to se-
crete IL-2 nor proliferate in an antigen-specific manner (not
depicted). In two additional study subjects, we observed
that HLA-A2–restricted CD8? T cells specific for the p17
Gag epitope SLYNTVATL (SL9) had almost entirely lost
their antigen-specific proliferative capacity in chronic infec-
tion, despite being detectable at high frequencies, as deter-
Figure 4.
specific CD8? T cells is supported by CD4?
T cells. (A–C) Dot plots (A) and histograms
(B) indicating the flow cytometric assess-
ment of CD8? T cells (A) or B8-FL8–specific
CD8? T cells (B) proliferating after stimula-
tion with a Nef peptide pool in the presence
or absence of indicated leukocellular sub-
sets. Cells were gated according to forward
scatter (FSC)/side scatter (SSC) characteris-
tics of the lymphocyte population in A. In
B, lymphocytes were additionally gated ac-
cording to CD8? expression and tetramer
binding. Values in top left corner of dot
plots indicate the proportion of CFSElow
CD8? T cells. A and B show one represen-
tative experiment and C indicates the mean
and standard deviation of the proportion of
CFSElow CD8? T cells in four independent
experiments (*, P ? 0.05). (D–G) Dot plots
(D) and histograms (E) showing the flow
cytometric analysis of the proliferation of
CD8? T cells (D) or B8-FL8–specific CD8?
T cells (E) in responses to stimulation with a
Nef peptide pool in whole PBMC samples,
CD4? cell–depleted PBMC samples, and in
CD4? cell–depleted PBMC samples that
were supplemented with exogenous IL-2.
Gating was performed as described for A
and B. D and E show one representative ex-
periment and F and G give the mean and
standard deviation from ten independent
experiments for bulk CD8? T cells (F) and
three different experiments for tetramer-
specific cells (G), respectively. (F and G) Left
black bars represent proliferating cells in
whole PBMC samples, middle bars show
PBMC samples depleted of CD4? cells, and
right white bars indicate PBMC samples
depleted of CD4? cells, but supplemented
with exogenous IL-2. (H and I) Rescue of
HIV-1–specific CD8? T cell proliferation
by exogenous IL-2 in chronic replicative
HIV-1 infection. (H) CD8? T cell prolifera-
tion in the presence of exogenous IL-2 after
stimulation of PBMC samples from chronic
HIV-1 infection with Nef pool peptides
(solid line) or no antigenic stimulation
(dashed line). Gating was performed accord-
ing to FSC/SSC characteristics and CD8?
Ex vivo proliferation of HIV-1–
expression. (I) Proliferation of HLA-A3-QK10 tetramer–specific CD8? T cells in the presence of IL-2 after stimulation of PBMC samples from chronic
HIV-1 infection with Nef pool peptides (solid line) or no antigenic stimulation (dashed line). Cells were gated according to FSC/SSC characteristics, as
well as CD8 expression and HLA-A3 QK10 tetramer binding. (H and I) Percentages indicate the proportion of CFSElow CD8? T cells. One representa-
tive example of four different experiments is shown.

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HIV-specific CD8? T Cell Proliferation in Acute Infection
708
mined by specific staining of CD8? T cells with SL9–MHC
class I tetramer complexes. Yet, after addition of autologous
CD4? T cells harvested during acute HIV-1 infection, the
ex vivo proliferative capacities of a subset of these cells were
rescued (Fig. 6, D and E). Again, we found that the CD4?
T cells harvested during acute infection included HIV-1–
specific CD4? T cells with strong capacities for ex vivo pro-
liferation and IL-2 secretion after encounter with HIV-1
peptides (not depicted). Moreover, the enhancement of
HIV-1–specific CD8? T cell lymphoproliferative responses
by CD4? T cells isolated during acute HIV-1 infection was
almost entirely abrogated by IL-2–neutralizing antibodies
(not depicted). Taken together, these results show that
CD4? T cells isolated during acute HIV-1 infection can res-
cue the ex vivo proliferative capacity of HIV-1–specific
CD8? T cells in chronic HIV-1 infection by an IL-2–depen-
dent mechanism.
In Vivo Reconstitution of the Proliferative Activity of HIV-1–
specific CD8? T Cells by Vaccine-induced, IL-2–secreting, HIV-
1–specific CD4? T Cells.
A number of studies have indi-
cated that the administration of inactivated gp120-depleted
HIV-1 can result in the induction of strong HIV-1–specific
CD4? T cell lymphoproliferative responses (22–25). We
showed recently in a placebo-controlled phase II clinical
trial in 10 individuals (5 receiving HIV vaccine and 5 re-
ceiving adjuvant alone), that the vaccine could elicit vigor-
ous HIV-1–specific CD4? T cell–mediated lymphoprolif-
erative immune responses in chronically infected HIV-1
persons treated with highly active antiretroviral therapy,
but did not increase the magnitude of HIV-1–specific
CD8? T cell responses when measured by an interferon ?
ELISPOT assay (14). We predicted, based on the in vitro
data described above, that the in vivo induction of HIV-1–
specific CD4? T cell proliferative responses in these indi-
viduals should result in the simultaneous development of
HIV-1–specific CD8? T cell proliferative responses, and
used cryopreserved samples from this randomized trial to
further characterize the quality of HIV-1–specific CD4? T
cell responses induced by the vaccine, as well as their im-
pact on HIV-1–specific CD8? T cell responses.
After five consecutive immunizations with gp120-
depleted, inactivated HIV, a median of 0.6% (range: 0.4–
1.9%) of CD4? T cells secreted IL-2 in response to stimula-
tion with overlapping peptides spanning HIV-1, whereas
IL-2 secretion by CD4? T cells was minimal in five recip-
ients of placebo (median proportion of 0.1% [range:
0–0.2%]; P ? 0.05). We subsequently assessed the impact
of immunization on HIV-1–specific proliferative T cell re-
sponses. In line with our previous data, HIV-1–specific
lymphoproliferative activities of CD4? T cells were negli-
gible at baseline in all 10 individuals with chronic infection.
After immunization, HIV-1–specific CD4? T cells in vac-
cinees but not in control individuals developed strong pro-
liferative capacities (Fig. 7, A and C), as described previ-
ously using a standard tritium-incorporation assay (14). In
addition, strong lymphoproliferative activities were also
observed in HIV-1–specific CD8? T cells from vaccine re-
cipients, but virtually no CD8? T cell–mediated lympho-
Figure 5.
CD8? T cells are dramatically enhanced by simultaneous
stimulation of antigen-specific CD4? T cells. (A) CD8?
and CD4? T cell proliferation after stimulation with the
HIV-1 Nef CD8? T cell epitope B8-FL8 or with the
overlapping HIV-1 Nef peptide PEKEVLVWKFDSR-
LAFHH, or both peptides together. Dot plots of one rep-
resentative flow cytometric experiment are shown. Cells
were gated according to FSC/SSC characteristics of the
lymphocyte population. (B) B8-FL8 tetramer–specific
CD8? T cells proliferating after stimulation with B8-FL8
peptide only (top) or in conjunction with the overlap-
ping Nef peptide PEKEVLVWKFDSRLAFHH (bottom).
(C) CD8? T cell proliferation after simultaneous stimula-
tion of PBMC samples with the CD8? T cell epitope B8-FL8
and the HIV-1 Nef peptide PEKEVLVWKFDSRLAFHH
in the presence of IL-2 antibodies. Cells were gated ac-
cording to FSC/SSC characteristics of the lymphocyte
population. (D and E) Proportion of CD8? T cells (D),
B8-FL8 tetramer–specific CD8? T cells (E), or CD4? T
cells (F) proliferating after stimulation with the B8-FL8
epitopic peptide in the presence or absence of concomitant
stimulation with tetanus toxoid, a CMV peptide, or the over-
lapping HIV-1 Nef peptide PEKEVLVWKFDSRLAFHH.
Mean and standard deviation from three independent experi-
ments in three different study subjects are shown.
Ex vivo proliferative activities of HIV-1–specific

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Lichterfeld et al.
709
proliferative activities were observed in control individuals
(Fig. 7, B and D). CD4? and CD8? T cell–mediated lym-
phoproliferative responses in vaccinees were confined to
those antigens contained in the immunogen, with no in-
duction of responses to envelope proteins that were re-
moved during the preparation of the antigen (26). Thus,
these data indicate that the in vivo augmentation of virus-
specific CD4? T cell responses can lead to the reconstitu-
Figure 6.
primary HIV-1 infection can restore the ex vivo prolifera-
tive activity of HIV-1–specific CD8? T cells in chronic
HIV-1 infection. (A) Flow cytometric analysis of CD8?
and CD4? T cell proliferation after stimulation with a pool
of overlapping Nef peptides in study subject AC-98 during
primary and chronic HIV-1 infection and in chronic HIV-1
infection after the addition of isolated autologous CD4? T
cells harvested during acute HIV-1 infection. Values in top
left corner of dot plots indicate the proportion of CFSElow
CD8? or CD4? T cells, respectively. (B) Corresponding his-
tograms indicating the proportion of proliferating A3-QK10–
specific CD8? T cells. (C) Intracellular cytokine staining of
CD4? T cells from acute HIV infection in study subject
AC-98 after stimulation with Nef pool peptides. Cells
were gated according to FSC/SSC characteristics. Value in
top right corner indicates proportion of IL-2? CD4? T
cells. (D and E) Proportion of CD8? T cells (D) and HIV-1
tetramer–specific CD8? T cells (E) proliferating in chronic
HIV-1 infection after stimulation with a pool of HIV-1–
specific Nef peptides in the presence (white bars) or absence
(black bars) of added autologous CD4? T cells from acute
HIV-1 infection. Data indicate the mean and standard de-
viation from three study individuals described in Results.
Autologous CD4? T cells harvested during
Figure 7.
CD8? T cell lymphoproliferative activities by vaccine-
mediated induction of IL-2–secreting, HIV-1–specific
CD4? T cells. (A and B) Dot plots reflecting the lym-
phoproliferative activity of HIV-1–specific CD4? (A)
and CD8? T (B) cells after stimulation with a pool of
overlapping Gag peptides before and after five consec-
utive administrations of an Env-depleted immunogen
or placebo. Percentages indicate the proportion of
CFSElow CD8? and CD4? T cells. (C and D) Propor-
tions of CD4? (C) and CD8? (D) T cells proliferating
in response to stimulation with HIV-1 peptides spanning
the entire HIV-1 proteome in five recipients of placebo
and the vaccine. Data from baseline and after five con-
secutive administrations of the immunogen/placebo
are shown.
In vivo reconstitution of HIV-1–specific

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HIV-specific CD8? T Cell Proliferation in Acute Infection
710
tion of HIV-1–specific CD8? T cell lymphoproliferative
immune responses in vivo.
Discussion
HIV-1–specific CD8? T cells play a critical role in the
initial control of viral replication in acute infection (27, 28).
Yet, the functional correlates for CD8? T cell–mediated
HIV-1 immune control are not well understood. Here, we
show that HIV-1–specific CD8? T cells in acute HIV-1
infection exhibit strong ex vivo proliferative capacities,
whereas this effector function is rapidly lost in the presence
of ongoing viral replication. Moreover, our data demon-
strate that lymphoproliferative CD4? T cell responses en-
hanced HIV-1–specific CD8? T cell proliferation in an IL-
2–dependent fashion, whereas no HIV-1–specific CD8? T
cell proliferation was observed in individuals with acute in-
fection after in vitro depletion of CD4? T cells. Finally, the
proliferative defect of HIV-1–specific CD8? T cell re-
sponses in chronic infection was partially corrected in vitro
by adding autologous IL-2–secreting CD4? T cells isolated
during acute infection and in vivo by the induction of HIV-
1–specific CD4? T cells using an Env-depleted immuno-
gen. Thus, these data demonstrate a progressive loss of
HIV-1–specific CD8? T cell function that is closely linked
to the loss of HIV-1–specific, IL-2–secreting CD4? T cells,
but can be rescued in vitro and more importantly in vivo by
reconstituting HIV-1–specific CD4? T cell help.
Recent data have demonstrated that HIV-1–specific
CD8? T cell responses measured by their ability of anti-
gen-specific interferon ? secretion do not differ in individ-
uals with progressive and long-term nonprogressive HIV-1
infection and are not directly associated with the level of
viral replication (7, 9). In contrast, HIV-1–specific CD8?
T cells in individuals with long-term nonprogressive infec-
tion exhibit strong antigen-dependent ex vivo prolifera-
tive capacities, whereas HIV-1–specific CD8? T cells in
subjects with progressive disease courses lose their abilities
to proliferate ex vivo in an antigen-specific manner (10).
Here, we extend these findings, demonstrating that strong
HIV-1–specific CD8? T cell–mediated lymphoprolifera-
tive immune responses are present in acute HIV-1 infec-
tion, when high level plasma viremia declines after the first
appearance of cellular immune responses against HIV-1.
HIV-1–specific CD8? T cell proliferation was rapidly lost
in individuals with ongoing viral replication, despite the
persistence of or an increase in the number of interferon
?–secreting, HIV-1–specific CD8? T cells. In contrast,
early suppression of viral replication by antiretroviral ther-
apy preserved these proliferative responses. These data
demonstrate that the ability of HIV-1–specific CD8? T
cells to proliferate in response to antigenic stimulation ex
vivo can be conserved in individuals with suppressed HIV-1
viremia, but is lost rapidly after acute infection in the pres-
ence of ongoing viral replication.
It has recently been shown that the lack of proliferative
capacity of HIV-1–specific CD4? T cells in chronic HIV-1
infection is associated with diminished IL-2 secretion by
these cells (13, 21), suggesting a potential relevance of
autocrine IL-2 secretion for maintaining HIV-1–specific
CD4? T cell lymphoproliferative responses. In addition,
antigen-dependent IL-2 secretion of HIV-1–specific CD4?
T cells is present in acute HIV-1 infection, but sequentially
lost during the ensuing disease process, which is closely
paralleled by the loss of CD4? T cell lymphoproliferative
immune responses (13, 20). Here, we show that the loss of
the proliferative capacity of HIV-1–specific CD4? T cells is
similarly paralleled by a loss of lymphoproliferative HIV-1–
specific CD8? T cell responses, suggesting a mutual func-
tional interaction of these T cell subsets. Moreover, our in
vitro data demonstrate that HIV-1–specific CD4? T cells
isolated from acute HIV-1 infection partially restored lym-
phoproliferative capacities of HIV-1–specific CD8? T cells
in chronic HIV-1 infection by an IL-2–dependent mecha-
nism, whereas no CD8? T cell proliferation was seen in the
presence of CD4? T cells from chronic HIV-1 infection,
which had lost both their antigen-specific lymphoprolifera-
tive activity and their ability to secrete IL-2. More impor-
tantly, therapeutic immunization aimed at induction of
HIV-1–specific CD4? T cells was able to repair the prolif-
eration deficiency of HIV-1–specific CD8? cells in vivo in
persons with chronic infection. In fact, the administration
of a CD4? T cell–targeted vaccine resulted in HIV-1–spe-
cific CD8? T cell responses with similar lymphoprolifera-
tive capacities, as in individuals with acute or long-term
nonprogressive HIV-1 infection. Thus, our data suggest
that the minimal proliferative capacities of HIV-1–specific
CD8? T cells in chronic HIV-1 infection are not primarily
due to a functional defect of these cells, but are rather re-
lated to insufficient support by HIV-1–specific CD4? T
helper cells. Nevertheless, even after providing IL-2–secret-
ing CD4? T helper cells, not all HIV-1–specific CD8? T
cells proliferated, suggesting that within the entire HIV-1–
specific CD8? T cell compartment, populations with dif-
ferent thresholds for antigen-specific proliferation exist.
Our data identify IL-2 secretion as the most prominent
mechanism used by CD4? T helper cells to support HIV-
1–specific CD8? T cell lymohproliferative responses, as IL-
2–neutralizing antibodies fully abrogated the proliferative
enhancement mediated by CD4? T cells. This observation
is in line with previous data showing that exogenous IL-2
can correct cell cycle perturbations, normalize the overall
intracellular protein turnover, and restore the phase-spe-
cific pattern of the expression of cell cycle–dependent pro-
teins of lymphocytes in HIV-1–infected individuals (29,
30). However, although IL-2 administration together with
highly active antiretroviral therapy has been shown to re-
sult in a significant increase in CD4? T cell counts, it was
not associated with enhancement of HIV-1–specific T cell
responses or immune-mediated control of HIV-1 infection
(31, 32). These data suggest that the HIV-1–specific im-
mune response depends less on systemic levels of IL-2, but
rather on the levels of IL-2 provided in the microenviron-
ment of the antigen-specific interaction between antigen-

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Lichterfeld et al.
711
presenting cells, virus-specific CD4? T cells, and virus-spe-
cific CD8? T cells. This is further supported by our in vivo
data demonstrating increased HIV-1–specific CD8? T cell
proliferation after the induction of IL-2–secreting, HIV-1–
specific CD4? T cell responses, strengthening the conclu-
sion that IL-2 secretion in an antigen-specific manner
appears to be more relevant for the maintenance of HIV-
1–specific proliferative CD8? T cell responses than sys-
temic levels of IL-2.
Taken together, these data demonstrate a parallel impair-
ment of both HIV-1–specific CD4? and CD8? T cell pro-
liferative responses after acute HIV-1 infection in the pres-
ence of ongoing viral replication, and suggest a critical role
of IL-2–secreting CD4? T helper cells for sustaining the
proliferative capacity of these HIV-1–specific T cell re-
sponses both in vitro and in vivo. These results provide
evidence for direct functional linkage of HIV-1–specific
CD4? and CD8? T cell responses and contribute to the
understanding of key molecular events contributing to the
immunopathogenesis of HIV-1 infection.
This study was supported by the National Institutes of Health (to
M. Altfeld, E.S. Rosenberg, and B.D. Walker), the Doris Duke
Charitable Foundation (to M. Altfeld, E.S. Rosenberg, and B.D.
Walker), the Howard Hughes Medical Institute (to B.D. Walker),
the Foundation for AIDS and Immunology Research (to X.G. Yu),
the Schweizerische Stiftung fuer medizinisch-biologische Stipen-
dien (to D.E. Kaufmann), and the Deutsche Forschungsgemeinschaft
(to M. Lichterfeld).
The authors have no conflicting financial interests.
Submitted: 25 June 2004
Accepted: 2 August 2004
References
1. Kahn, J.O., and B.D. Walker. 1998. Acute human immuno-
deficiency virus type 1 infection. N. Engl. J. Med. 339:33–39.
2. Borrow, P., H. Lewicki, B.H. Hahn, G.M. Shaw, and M.B.
Oldstone. 1994. Virus-specific CD8? cytotoxic T-lympho-
cyte activity associated with control of viremia in primary
human immunodeficiency virus type 1 infection. J. Virol. 68:
6103–6110.
3. Koup, R.A., J.T. Safrit, Y. Cao, C.A. Andrews, G. McLeod,
W. Borkowsky, C. Farthing, and D.D. Ho. 1994. Temporal
association of cellular immune responses with the initial con-
trol of viremia in primary human immunodeficiency virus
type 1 syndrome. J. Virol. 68:4650–4655.
4. Altfeld, M., E.S. Rosenberg, R. Shankarappa, J.S. Mukher-
jee, F.M. Hecht, R.L. Eldridge, M.M. Addo, S.H. Poon,
M.N. Phillips, G.K. Robbins, et al. 2001. Cellular immune
responses and viral diversity in individuals treated during
acute and early HIV-1 infection. J. Exp. Med. 193:169–180.
5. Dalod, M., M. Dupuis, J.C. Deschemin, C. Goujard, C. De-
veau, L. Meyer, N. Ngo, C. Rouzioux, J.G. Guillet, J.F.
Delfraissy, et al. 1999. Weak anti-HIV CD8(?) T-cell effec-
tor activity in HIV primary infection. J. Clin. Invest. 104:
1431–1439.
6. Cao, J., J. McNevin, S. Holte, L. Fink, L. Corey, and M.J.
McElrath. 2003. Comprehensive analysis of human immuno-
deficiency virus type 1 (HIV-1)-specific gamma interferon-
secreting CD8? T cells in primary HIV-1 infection. J. Virol.
77:6867–6878.
7. Addo, M.M., X.G. Yu, A. Rathod, D. Cohen, R.L. Eld-
ridge, D. Strick, M.N. Johnston, C. Corcoran, A.G. Wurcel,
C.A. Fitzpatrick, et al. 2003. Comprehensive epitope analysis
of human immunodeficiency virus type 1 (HIV-1)-specific
T-cell responses directed against the entire expressed HIV-1
genome demonstrate broadly directed responses, but no cor-
relation to viral load. J. Virol. 77:2081–2092.
8. Draenert, R., C.L. Verrill, Y. Tang, T.M. Allen, A.G. Wur-
cel, M. Boczanowski, A. Lechner, A.Y. Kim, T. Suscovich,
N.V. Brown, et al. 2004. Persistent recognition of autolo-
gous virus by high-avidity CD8 T cells in chronic, progres-
sive human immunodeficiency virus type 1 infection. J. Virol.
78:630–641.
9. Betts, M.R., D.R. Ambrozak, D.C. Douek, S. Bonhoeffer,
J.M. Brenchley, J.P. Casazza, R.A. Koup, and L.J. Picker.
2001. Analysis of total human immunodeficiency virus (HIV)-
specific CD4(?) and CD8(?) T-cell responses: relationship
to viral load in untreated HIV infection. J. Virol. 75:11983–
11991.
10. Migueles, S.A., A.C. Laborico, W.L. Shupert, M.S. Sab-
baghian, R. Rabin, C.W. Hallahan, D. Van Baarle, S. Kos-
tense, F. Miedema, M. McLaughlin, et al. 2002. HIV-specific
CD8? T cell proliferation is coupled to perforin expression and
is maintained in nonprogressors. Nat. Immunol. 3:1061–1068.
11. Rosenberg, E.S., M. Altfeld, S.H. Poon, M.N. Phillips, B.M.
Wilkes, R.L. Eldridge, G.K. Robbins, R.T. D’Aquila, P.J.
Goulder, and B.D. Walker. 2000. Immune control of HIV-1
after early treatment of acute infection. Nature. 407:523–526.
12. Rosenberg, E.S., J.M. Billingsley, A.M. Caliendo, S.L.
Boswell, P.E. Sax, S.A. Kalams, and B.D. Walker. 1997. Vig-
orous HIV-1-specific CD4? T cell responses associated with
control of viremia. Science. 278:1447–1450.
13. Younes, S.A., B. Yassine-Diab, A.R. Dumont, M.R. Boulas-
sel, Z. Grossman, J.P. Routy, and R.P. Sekaly. 2003. HIV-1
viremia prevents the establishment of interleukin 2–produc-
ing HIV-specific memory CD4? T cells endowed with pro-
liferative capacity. J. Exp. Med. 198:1909–1922.
14. Robbins, G.K., M.M. Addo, H. Troung, A. Rathod, K. Ha-
beeb, B. Davis, H. Heller, N. Basgoz, B.D. Walker, and E.S.
Rosenberg. 2003. Augmentation of HIV-1-specific T helper
cell responses in chronic HIV-1 infection by therapeutic im-
munization. AIDS. 17:1121–1126.
15. Brander, C., and P. Goulder. 2000. The evolving field
of HIV CRL epitope mapping: new approaches for the iden-
tification of novel epitopes. In HIV Molecular Database.
B.T.M. Korber, C. Brander, B.D. Walker, R.A. Koup, J.
Moore, B. Haynes, and G. Meyer, editors. Los Alamos Na-
tional Laboratory. Los Alamos, New Mexico. 11–120.
16. Sidney, J., S. Southwood, C. Oseroff, M.-F. Del Guercio,
and A. Sette. 1998. Measurement of MHC/peptide interac-
tions by gel filtration. Curr. Prot. Immunol. 18:18.3.2–18.3.19.
17. Altfeld, M., M.M. Addo, R.L. Eldridge, X.G. Yu, S.
Thomas, A. Khatri, D. Strick, M.N. Phillips, G.B. Cohen,
S.A. Islam, et al. 2001. Vpr is preferentially targeted by CTL
during HIV-1 infection. J. Immunol. 167:2743–2752.
18. Alter, G., G. Hatzakis, C.M. Tsoukas, K. Pelley, D. Rou-
leau, R. LeBlanc, J.G. Baril, H. Dion, E. Lefebvre, R.
Thomas, et al. 2003. Longitudinal assessment of changes in
HIV-specific effector activity in HIV-infected patients start-
ing highly active antiretroviral therapy in primary infection.
J. Immunol. 171:477–488.

Page 12

HIV-specific CD8? T Cell Proliferation in Acute Infection
712
19. Lacabaratz-Porret, C., A. Urrutia, J.M. Doisne, C. Goujard,
C. Deveau, M. Dalod, L. Meyer, C. Rouzioux, J.F. Del-
fraissy, A. Venet, and M. Sinet. 2003. Impact of antiretroviral
therapy and changes in virus load on human immunodefi-
ciency virus (HIV)-specific T cell responses in primary HIV
infection. J. Infect. Dis. 187:748–757.
20. Harari, A., S. Petitpierre, F. Vallelian, and G. Pantaleo. 2004.
Skewed representation of functionally distinct populations of
virus-specific CD4 T cells in HIV-1-infected subjects with
progressive disease: changes after antiretroviral therapy. Blood.
103:966–972.
21. Iyasere, C., J.C. Tilton, A.J. Johnson, S. Younes, B. Yassine-
Diab, R.P. Sekaly, W.W. Kwok, S.A. Migueles, A.C. Labo-
rico, W.L. Shupert, et al. 2003. Diminished proliferation of
human immunodeficiency virus-specific CD4? T cells is as-
sociated with diminished interleukin-2 (IL-2) production and
is recovered by exogenous IL-2. J. Virol. 77:10900–10909.
22. Moss, R.B., C. Brandt, W.K. Giermakowska, J.R. Savary, G.
Theofan, M. Zanetti, D.J. Carlo, and M.R. Wallace. 2003.
HIV-specific immunity during structured antiviral drug treat-
ment interruption. Vaccine. 21:1066–1071.
23. Moss, R.B., E. Webb, W.K. Giermakowska, F.C. Jensen,
J.R. Savary, M.R. Wallace, and D.J. Carlo. 2000. HIV-1-
specific CD4 helper function in persons with chronic HIV-1
infection on antiviral drug therapy as measured by ELISPOT
after treatment with an inactivated, gp120-depleted HIV-1 in
incomplete Freund’s adjuvant. J. Acquir. Immune Defic. Syndr.
24:264–269.
24. Moss, R.B., M.R. Wallace, P. Lanza, W. Giermakowska,
F.C. Jensen, G. Theofan, C. Chamberlin, S.P. Richieri, and
D.J. Carlo. 1998. In vitro p24 antigen-stimulated lymphocyte
proliferation and beta-chemokine production in human im-
munodeficiency virus type 1 (HIV-1)-seropositive subjects
after immunization with an inactivated gp120-depleted
HIV-1 immunogen (Remune). Clin. Diagn. Lab. Immunol.
5:308–312.
25. Moss, R.B., W.K. Giermakowska, M.R. Wallace, J.R. Sa-
vary, F.C. Jensen, and D.J. Carlo. 2000. Cell-mediated im-
mune responses to autologous virus in HIV-1-seropositive
individuals after treatment with an HIV-1 immunogen.
AIDS. 14:2475–2478.
26. Turner, J.L., R.J. Trauger, A.E. Daigle, and D.J. Carlo.
1994. HIV-1 immunogen induction of HIV-1-specific de-
layed-type hypersensitivity: results of a double-blind, adju-
vant-controlled, dose-ranging trial. AIDS. 8:1429–1435.
27. Schmitz, J.E., M.J. Kuroda, S. Santra, V.G. Sasseville, M.A.
Simon, M.A. Lifton, P. Racz, K. Tenner-Racz, M. Dalesan-
dro, B.J. Scallon, et al. 1999. Control of viremia in simian
immunodeficiency virus infection by CD8? lymphocytes.
Science. 283:857–860.
28. Jin, X., D.E. Bauer, S.E. Tuttleton, S. Lewin, A. Gettie, J.
Blanchard, C.E. Irwin, J.T. Safrit, J. Mittler, L. Weinberger,
et al. 1999. Dramatic rise in plasma viremia after CD8? T cell
depletion in simian immunodeficiency virus–infected macaques.
J. Exp. Med. 189:991–998.
29. Paiardini, M., D. Galati, B. Cervasi, G. Cannavo, L. Galluzzi,
M. Montroni, D. Guetard, M. Magnani, G. Piedimonte, and
G. Silvestri. 2001. Exogenous interleukin-2 administration
corrects the cell cycle perturbation of lymphocytes from
human immunodeficiency virus-infected individuals. J. Virol.
75:10843–10855.
30. Shankar, P., M. Russo, B. Harnisch, M. Patterson, P.
Skolnik, and J. Lieberman. 2000. Impaired function of circu-
lating HIV-specific CD8(?) T cells in chronic human im-
munodeficiency virus infection. Blood. 96:3094–3101.
31. The AIDS Clinical Trials Group 248 Study Team. 2004.
Daily low-dose subcutaneous interleukin-2 added to single-
or dual-nucleoside therapy in HIV infection does not protect
against CD4? T-cell decline or improve other indices of im-
mune function: results of a randomized controlled clinical
trial (ACTG 248). J. Acquir. Immune Defic. Syndr. 36:576–
587.
32. Dybul, M., B. Hidalgo, T.W. Chun, M. Belson, S.A.
Migueles, J.S. Justement, B. Herpin, C. Perry, C.W. Halla-
han, R.T. Davey, et al. 2002. Pilot study of the effects of in-
termittent interleukin-2 on human immunodeficiency virus
(HIV)-specific immune responses in patients treated during
recently acquired HIV infection. J. Infect. Dis. 185:61–68.