of June 13, 2013.
This information is current as
Gag-Specific CD8 T Cell Responses
Cells from HIV Controllers: Association with
Heterogeneity in HIV Suppression by CD8 T
Lambotte, Alain Venet and Gianfranco Pancino
Jean-François Delfraissy, Françoise Barré-Sinoussi, Olivier
Boufassa, Véronique Avettand-Fènoël, Christine Rouzioux,
Urrutia, Pierre Versmisse, Christine Lacabaratz, Faroudy
Asier Sáez-Cirión, Martine Sinet, So Youn Shin, Alejandra
2009; 182:7828-7837; ;
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The Journal of Immunology
by guest on June 13, 2013
Heterogeneity in HIV Suppression by CD8 T Cells from HIV
Controllers: Association with Gag-Specific CD8 T Cell
Asier Sa ´ez-Cirio ´n,2* Martine Sinet,3†‡So Youn Shin,3* Alejandra Urrutia,†‡Pierre Versmisse,*
Christine Lacabaratz,4†‡Faroudy Boufassa,§Ve ´ronique Avettand-Fe `noe ¨l,¶?
Christine Rouzioux,¶?Jean-Franc ¸ois Delfraissy,†‡#Franc ¸oise Barre ´-Sinoussi,*
Olivier Lambotte,†‡#Alain Venet,†‡and Gianfranco Pancino*; for the ANRS EP36 HIV
Controllers Study Group
“HIV controllers” (HICs) are rare individuals in whom HIV-1 plasma viral load remains undetectable without antiretroviral
treatment. This spontaneous viral control in HICs is usually associated to strong functional HIV-specific CD8?T cell responses.
Accordingly, we have recently shown that CD8?T cells from HICs strongly suppress ex vivo HIV-1 infection of autologous CD4?
T cells, suggesting a crucial role of this response in vivo. Knowledge of the mechanisms underlying the CD8?T cell antiviral
activity might help to develop effective T cell-based vaccines. In the present work, we further characterized the HIV-suppressive
capacity of CD8?T cells in 19 HICs. CD8?T cells from 14 of the 19 HICs showed strong HIV-suppressive capacity ex vivo. This
capacity was stable over time and was partially effective even on other primate lentiviruses. HIV-suppressive capacity of CD8?
T cells correlated strongly with the frequency of HIV-specific CD8?T cells, and in particular of Gag-specific CD8?T cells. We
also identified five HICs who had weak HIV-suppressive CD8?T cell capacities and HIV-specific CD8?T cell responses. Among
these five HICs, at least three had highly in vitro replicative viruses, suggesting that the control of viremia in these patients is not
due to replication-defective viruses. These results, on the one hand, suggest the importance of Gag responses in the antiviral
potency of CD8?T cells from HICs and, on the other hand, propose that other host mechanisms may contribute to restraining
HIV infection in HICs. The Journal of Immunology, 2009, 182: 7828–7837.
of HIV infection (4–6). The presence of Gag-specific CD8?T
cells and the breadth of their specificities have also been linked to
low HIV viremia (7–9). One of the most compelling indications of
the pressure exerted by CD8?T cell responses is the emergence of
variants that escape recognition by these cells (10–14). However,
most HIV-infected individuals have uncontrolled viremia and
eventually progress to AIDS despite strong CD8?T cell
t least partial control of HIV can be achieved by CD8?
T cells (1–3). Some HLA class I molecules, particularly
alleles B27 and B57, have been linked to better control
Rare individuals called “HIV controllers” (HICs)5spontane-
ously and durably control HIV infection in the absence of therapy,
possibly illustrating what truly effective CD8?T cell responses
can achieve (15, 16). HICs have extremely low and stable amounts
of viral DNA in their PBMC (17) and undetectable plasma viral
load (18). The protective HLA alleles B27 and B57 are overrep-
resented among these individuals (4, 6, 19–21). Despite very low
levels of Ag in blood (22), most but not all HICs have high fre-
quencies of HIV-specific CD8?T cells that preferentially target
the viral Gag protein (20, 23, 24). Studies of CD8?T cell re-
sponses in HICs have revealed important characteristics of func-
tional HIV-specific CD8?T cells in HIV infection. Contrary to
cells from viremic individuals, HIV-specific CD8?T cells from
HICs can, upon stimulation with their cognate Ag, proliferate and
generate a multifunctional response that includes perforin expres-
sion, degranulation, and chemokine/cytokine secretion (25–27).
This could be related to a peculiar activation phenotype of these
cells (21) and to constitutive telomerase activity that protects them
against senescence (28). However, how much of this is the cause
and how much the consequence of viral control and low-level im-
mune activation remains to be determined. We have recently
shown that CD8?T cells from most HICs are endowed with a
striking capacity to suppress HIV infection ex vivo (21), a property
that is likely to be relevant in vivo. To further characterize this
HIV-suppressive activity we extended our analysis to a larger
*Institut Pasteur, Unite ´ de Re ´gulation des Infections Re ´trovirales, Paris, France;
†INSERM Unite ´ 802, Le Kremlin-Bice ˆtre, France;‡Universite ´ Paris-Sud, Faculte ´
de Me ´decine Paris XI, Le Kremlin-Bice ˆtre, France;§INSERM Unite ´ 822, Ho ˆpital
Bice ˆtre, Le Kremlin-Bice ˆtre, France;¶AP-HP, CHU Necker-Enfants Malades,
Laboratoire de Virologie, Paris, France;?Universite ´ Paris-Descartes, Faculte ´ de
Me ´decine, Paris, France;#AP-HP, Ho ˆpital Bice ˆtre, Service de Me ´decine Interne
et Maladies Infectieuses, Le Kremlin-Bice ˆtre, France
Received for publication November 25, 2008. Accepted for publication April 3, 2009.
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 financially supported by the Agence Nationale de Recherches sur le
SIDA. S.Y.S. was supported by Korea Science and Engineering Foundation and the
Institut Pasteur Korea.
2Address correspondence and reprint requests to Dr. Asier Sa ´ez-Cirio ´n, Unite ´ de
Re ´gulation des Infections Re ´trovirales, Institut Pasteur, 25 rue du Dr Roux, 75725
Paris Cedex 15, France. E-mail address: email@example.com
3M.S. and S.Y.S. contributed equally to this work.
4Current address: INSERM Unite ´ 841, Faculte ´ de Me ´decine Henri Mondor, Cre ´teil
5Abbreviations used in this paper: HIC, HIV controller; moi, multiplicity of infec-
tion; PBMC, peripheral blood mononuclear cell; SFC, spot-forming cell.
Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00
The Journal of Immunology
by guest on June 13, 2013
group of 19 HICs and evaluated the relationship between this ac-
tivity and HIV-specific CD8?T cell responses.
Materials and Methods
Nineteen patients diagnosed with HIV-1 infection at least 10 years previ-
ously who had never received antiretroviral treatment and in whom ? 90%
of plasma HIV RNA assays gave values?400 copies/ml were studied (Ta-
ble I): 8 have been described elsewhere (21), and 11 were newly recruited
from the ANRS EP36 national monitoring program on HIV controllers.
The subjects were serologically HLA typed by complement-mediated lym-
phocytotoxicity testing (InGen One Lambda). All had very weak and stable
DNA load (Table I).
All of the subjects gave their written informed consent.
HIV DNA quantification
Total DNA was extracted from whole blood with QIAamp DNA minikits
(Qiagen), according to the manufacturer’s instructions. HIV-1 DNA was
then quantified by real-time PCR (LTR amplification; Agence Nationale de
Recherches sur le SIDA) (29). Four PCRs, each testing 1 ?g of total DNA,
were performed per extract in this ultrasensitive assay (threshold of 10
copies/million leukocytes) (30).
Primary cell culture
CD4?and CD8?cells were purified (?97%) from freshly isolated PBMC
by positive and negative selection, respectively, with Ab-coated magnetic
beads (Miltenyi Biotec). CD4?cells were stimulated for 3 days with phy-
tohemagglutinin (PHA) at 2 ?g/ml in the presence of IL-2 (Chiron) at 100
IU/ml. The culture medium was RPMI 1640 containing 10% FCS and
penicillin/streptomycin (100 U/ml). CD8?T cells were kept in culture
without mitogens or cytokines.
Productive infection in vitro
CD4?T cells (105) were superinfected with HIV-1 BaL (R5) in triplicate
at a multiplicity of infection (moi) of 10?3.6in 96-well plates with a spi-
noculation protocol (31). For some experiments, SIVagm.Gril, SIV-
mac.251, and HIV-2.SBL and autologous primary viruses were used for
infection. For coculture, 105CD4?T cells were mixed with 105CD8?T
cells (CD8/CD4 ratio of 1:1) at the moment of infection. After challenge
the cells were washed and cultured for 14 days. Viral replication was mon-
itored every 3–4 days in supernatants by p24 or p27 ELISA (Zeptometrix).
Infectivity assays were conducted in the presence of 100 IU/ml IL-2. We
have previously shown that the presence of this cytokine during the infec-
tivity assays did not affect the suppressive capacity of unstimulated CD8?
T cells (21).
Intracellular p24 assay
Activated CD4?lymphocytes (5 ? 104) were superinfected with HIV-1
BaL (R5) as described above. Various dilutions of virus (moi of 10?1.6to
10?2.6) were used in parallel to obtain similar levels of infection in each
individual/experiment. CD4?T cells were culture in the presence or ab-
sence of unstimulated CD8?T cells (CD8/CD4 ratio of 1:1).
Seventy-two hours after infection, cells were harvested and stained with
CD4-ECD (SFCI12T4D11) and CD8-PC5 (B9.11). Cells were then perme-
abilized (Cytofix/Cytoperm fixation and permeablization kit; BD Biosciences)
and stained with KC57-FITC (FH190-1-1) to detect intracellular HIV Ags.
Abs were from Beckman Coulter. Flow cytometry was performed with a Cy-
tomix FC500 and CXP acquisition software (Beckman Coulter).
Viral isolation from peripheral blood CD4?T cells
Between 2 and 5 ? 106CD4?T cells from each patient were activated
with PHA and IL-2 as described above. Viral production in culture super-
natants was monitored for 28 days by p24 ELISA. When required, CD4?
T cells were reactivated on day 10 with CD8-depleted PHA-preactivated
allogeneic PBMC, PHA, and IL-2. Virus-containing supernatants from
CD4 T cell cultures were titrated on mixed PHA-activated CD4?T cells
from two blood donors.
IFN-? secretion by HIV-specific CD8?T cells was quantified ex vivo with
an ELISPOT assay using appropriate stimuli (32). We used a set of 124
peptides corresponding to known optimal CTL epitopes derived from the
HIV-1 Env, Gag, Pol, and Nef proteins (National Institutes of Health HIV
Molecular Immunology Database; www.hiv.lanl.gov/content/immunology/
index.html). These peptides were synthesized by Neosystem and used at a
final concentration of 2 ?g/ml. For each subject, optimal peptides were
tested depending on the results of HLA typing with an average of 36 ? 9
peptides tested per subject. IFN-? spot-forming cells (SFCs) were counted
with a KS-ELISPOT system (Carl Zeiss Vision) and expressed as SFCs/
106PBMC after subtracting the background of control unstimulated cells.
Wells were considered positive if they contained at least 50 SFCs/106
PBMC and exhibited at least twice the background level.
Depletion of HIV-specific CD8?T cells
Depletion of CD8?T cells producing IFN-? upon stimulation with HIV
peptides was performed with an IFN-? secretion assay enrichment kit
Table I. Characteristics of HIV controllers included in the study
Median CD4 Count
aPatients are sorted in function of the suppressive capacity of their CD8?T cells (log p24 decrease, Fig. 1C). Horizontal rule separates strong and weak responders.
bHem, hemophilia; Het, heterosexual sex; IDU, injection drug use; MMS, male-male sex.
cNumber of viral load between 50 and 400 copies per total determinations with detection limit ?50 copies/ml. Viral loads of ?400 RNA copies/ml are indicated in
7829The Journal of Immunology
by guest on June 13, 2013
(Miltenyi Biotec) as recommended by the manufacturer. Briefly, purified
CD8?T cells were stimulated for 6 h with appropriate pools of specific
HIV peptides. Subsequently, the cells were labeled (5 min at 4°C) with an
IFN-? catch reagent that attached to the cell surface of all leukocytes. The
cells were then incubated for 45 min at 37°C to allow IFN-? secretion. The
secreted IFN-? was captured by the IFN-? catch reagent on the positive, se-
creting cells. These cells were subsequently labeled with a second IFN-?-
specific Ab conjugated to R-PE. The IFN-?-secreting cells were magnetically
labeled with anti-PE magnetic beads and depleted by magnetic field separa-
tion. Purity of the depleted fractions was evaluated by flow cytometry.
The following Abs were used: CD8-ECD or -PC5 (clone B9.11), CD3-PC5
(UCHT1), CD45RO-ECD (UCHL1), HLA-DR-ECD (Immu-357), and
CD38-FITC (T16), all from Beckman Coulter; and CD27-FITC (M-T271)
from BD Biosciences.
Pentamer staining and phenotyping
HIV-specific CD8?T cells were identified by using soluble PE- or
allophycocyanin-labeled peptide-HLA class 1 multimers (Proimmune;
Beckman Coulter Immunomics). The following epitopes were used: the
HLA-A*0201-restricted peptide ligands SLYNTVATL (Gag 77–85)
and ILKEPVHGV (Pol 476–484), the A*0301-restricted peptide li-
gands RLRPGGKKK (Gag 20–28) and QVPLRPMTYK (Nef 73–82),
the B*2705-restricted peptide ligand KRWIILGLNK (Gag 263–272),
and the B*5701-restricted peptide ligands KAFSPEVIPMF (Gag 162–
172), TSTLQEQIGW (Gag 240–249), and QASQDVKNW (Gag 308–
316). PBMC were incubated with pentamers (1 ?g/ml) for 30 min and
then with relevant Abs for 15 min. Cells were washed in Cell Wash (BD
Biosciences) plus 1% BSA, incubated for 10 min with FACS lysing
solution (BD Biosciences). After washing, cells were fixed in 1% para-
formaldehyde for flow cytometry with an Epics XL (Beckman Coulter)
or a FACSCanto flow cytometer (BD Biosciences) and analyzed with
RXP software (Beckman Coulter).
The proliferative capacity of HIV-specific CD8?T cells was evaluated by
flow cytometry. PBMC were stained with 0.35 ?M CFSE (Molecular
Probes) for 10 min at 37°C, and, after washing they were stimulated for 5
days with 2 ?g/ml peptide or medium alone. After labeling with pentamer,
anti-CD8, and anti-CD3 Abs, PBMC were fixed in 1% paraformaldehyde
for flow cytometry as described above.
All values throughout the text are means ? SD. Values of p were calcu-
lated with the rank sum test. Correlations were identified by simple linear
regression analysis and Spearman’s rank correlation test. SigmaStat 3.5
software was used (Systat Software).
replicative HIV-1 BaL. Circles represent the average (n ? 3 independent infections) peak p24 values detected in culture supernatants for each individual.
Horizontal lines indicate median values. B, PHA-activated CD4?T cells from HICs A6 and A13 were superinfected with HIV-1 Bal and left alone (central
panels) or cocultured with autologous unstimulated CD8?T cells (right panels). Three days later the level of infection was determined by quantifying
intracellular p24 on CD8?cells. C, The HIV-suppressive capacity of CD8?T cells, as determined by the log fold decrease in the level of secreted p24
(CD4 vs CD4/CD8 cell cultures), was compared in the 19 HICs (F), 13 chronically HIV-infected subjects with viremia ?7000 copies/ml (Œ), and 8
HAART-treated patients with virologic control (plasma HIV RNA ?50 copies/ml) for ?23 mo (f). Horizontal lines indicate median values. D, p24
production in culture supernatants (mean ? SD, n ? 3) at the peak of viral replication after superinfection of CD4?T cells from HIV controllers with
equivalent infectious doses (moi of 10?3.6) of HIV-1 Bal or filtered supernatants containing autologous HIC viruses. CD4?T cells were cultured alone
(filled bars) or in the presence of non prestimulated CD8?T cells (open bars).
A, PHA-activated CD4?T cells were infected, in the absence (F) or presence (E) of autologous unstimulated CD8?T cells (1:1 ratio), with
7830HETEROGENEITY OF CD8 T CELL HIV SUPPRESSION IN HIV CONTROLLERS
by guest on June 13, 2013
Unstimulated CD8?T cells from most HICs have strong
In a previous study we found that undetectable viremia in 9 out of
10 HICs was associated with a remarkably strong capacity of their
circulating CD8?T cells to control in vitro HIV-1 infection of
autologous CD4?T cells (21). To extend this observation, we used
the same viral suppression assay to assess the ex vivo anti-HIV
capacity of CD8?T cells from 19 HICs, 11 of whom were newly
recruited for this study and 8 were retested (Table I). Viral repli-
cation was readily detected in the supernatants of purified CD4?T
cells from all 19 HICs after PHA activation and challenge with
HIV-1 BaL (Fig. 1A). A marked reduction in HIV-1 infection (un-
detectable in eight HICs) was generally observed when autologous
unstimulated CD8?T cells from HICs were added to the culture
(Fig. 1A). The associated CD8?T cell-mediated decrease in the
level of HIV proteins was due to the absence of infected CD4?T
cells in the coculture (Fig. 1B). As a whole, the HIV-suppressive
capacity of CD8?T cells from HICs (2.79 ? 1.31 log p24 de-
crease, CD8/CD4 vs CD4) was much stronger than that of cells
both from viremic individuals (0.82 ? 0.53 log p24 decrease,
CD8/CD4 vs CD4), confirming our previous results (21), as well
as from HAART-treated individuals with undetectable viral load
(0.62 ? 0.63 log p24 decrease, CD8/CD4 vs CD4) (Fig. 1C). In
particular, CD8?T cells from 14 of the 19 HICs suppressed HIV
far more strongly (log p24 decrease ?2) than did cells from both
viremic and treated individuals (Fig. 1C). These subjects are re-
ferred to below as strong responder HICs. Longitudinal analysis
(?12 mo) of CD8?T cell antiviral activity in five strong responder
HICs included in our previous study suggested that this HIV-suppres-
sive capacity is a stable characteristic (Table II). In contrast, here we
identified five “weak responder” HICs (Table I) whose CD8?T cells
could not efficiently control HIV infection of autologous CD4?T
cells (log p24 decrease ?2) (Fig. 1, B and C): the HIV-suppressive
capacity of these subjects’ CD8?T cells was not stronger than that of
viremic or HAART-treated patients (Fig. 1C). We have reported that
susceptibility of CD4?T cells from HICs to in vitro HIV infection
no significant differences were found either between weak responder
and strong responder HICs (p ? 0.331) (Fig. 1A).
To determine whether the weak HIV-suppressive activity observed
in certain HICs was due to our use of a laboratory-adapted HIV strain,
we analyzed the capacity of nonstimulated CD8?T cells from weak
responders A13 and A19 and from strong responder A21 to suppress
superinfection of their own CD4?T cells by autologous viruses pre-
viously obtained in primary culture of these individuals’ cells (see
below). CD8?T cells from strong responder A21 equally controlled
CD4?T cell superinfection by HIV-BaL and by autologous virus
(Fig. 1D). In contrast, the weak CD8-mediated HIV suppression in
subject A13 was not improved when his autologous virus was used to
challenge his CD4?T cells (0.01 vs 0.16 log p24 decrease with HIV-
BaL and the autologous virus, respectively) (Fig. 1D). CD8?T cells
from weak responder A19 showed a stronger capacity to inhibit in-
fection by autologous viruses (0.33 vs 1.76 log p24 decrease for HIV
BaL and autologous virus infection, respectively) (Fig. 1D), although
the level of suppression did not reach that observed in strong respond-
ers. Interestingly, while most HICs were infected by subtype B vi-
ruses, subject A19 was infected by HIV-1 subtype A2 (Table I).
Therefore, although the use of nonautologous viruses might lead to an
underestimation of the HIV-suppressive activity of CD8?T cells, it
was unlikely to explain the differences observed between weak and
CD8-mediated HIV-suppressive capacity in HICs correlates
with the frequency of IFN-?-producing cells
We examined whether the difference between strong and weak re-
sponder HICs was associated with a difference in the magnitude of
HIV-specific CD8?T cell responses. To quantify the HIV-specific
CD8?T cell response, we used the standard determination of the
frequency of IFN-?-secreting CD8?T cells upon stimulation with
HICs (log p24 decrease ?2) (WR). An average of 36 ? 9 peptides were tested in each subject, depending on the results of HLA typing. Each symbol
corresponds to the sum of SFCs/106PBMC obtained with individual peptides described as being restricted by HLA Ags. Horizontal lines are median values
for each group. B, Correlation between the HIV-suppressive capacity of CD8?T cells from HICs (log p24 decrease as shown in Fig. 1C) and their frequency
of IFN-?-producing CD8?T cells upon HIV peptide stimulation. Each symbol represents one HIC. Vertical dashed line separates weak responder and
strong responder HICs. C, Percentage of HIV-specific cells (based on HIV multimer and CD8 expression) from strong and weak responder HICs that
expressed ex vivo HLA-DR and CD38, coexpressed CD27 and CD45RA, or proliferated (and lost CFSE labeling) after 5 days of peptide stimulation. Each
symbol represents one specificity for one HIC. Horizontal lines are mean values for each group.
A, Frequencies of HIV-specific IFN-?-secreting CD8?T cells in strong responder HICs (log p24 decrease ?2) (SR) and in weak responder
Table II. Log p24 decrease (CD4 vs CD4/CD8 of 1:1) during
follow-up (?12 mo) of HICs
Median Value First Sample Last Sample
aNumber of blood samples analyzed.
7831The Journal of Immunology
by guest on June 13, 2013
appropriate HLA-defined optimal HIV-1 Env, Gag, Pol, and Nef pep-
tides in an ELISPOT assay. The numbers of IFN-?-secreting cells
were heterogeneous (Fig. 2A), in agreement with recent reports (20,
24). The highest frequencies of HIV-specific CD8?T cells were ob-
served in strong responders (8517 ? 4038 vs 1058 ? 903 SFCs/106
PBMC in weak responder HICs, p ? 0.0014) (Fig. 2A). The fre-
quency of HIV-specific CD8?T cells in HICs was not significantly
different, as a whole (6843 ? 4866 SFCs/106PBMC), from that ob-
for 18 patients with ?3 years of infection and plasma viral load
response in weak responder HICs was similar to that in HAART-
treated patients (865 ? 1071 SFCs/106PBMC for 11 patients with
?2 years of treatment and plasma viral load ?50 RNA copies/ml,
p ? 0.50; and Ref. 32).
Interestingly, we found a strong correlation between the fre-
quency of IFN-?-producing CD8?T cells upon peptide stimula-
tion and the HIV-suppressive capacity of unstimulated CD8?T
cells (Spearman 0.835, p ? 0.00001) (Fig. 2B). This supports the
possibility that the ex vivo anti-HIV activity of CD8?T cells from
HICs is driven by HIV-specific cells, in keeping with an MHC
class I-mediated mechanism (21). This correlation further distin-
guished strong and weak responder HICs (Fig. 2B).
We explored whether differences could also be observed be-
tween strong and weak responder HICs at the phenotypical level of
their HIV-specific CD8?T cells. Due to the low frequency of
these cells in weak responders, we could perform these analyses
only in three of them. HIV-specific CD8?T cells from strong
responder HICs possessed a discordant activation phenotype with
high expression of the activation marker HLA-DR associated with
a low CD38 expression (Fig. 2C), in keeping with our previous
study (21). In contrast, the expression of both activation markers
was low in the cells from weak responders (Fig. 2C), a phenotype
that is found in HAART subjects (21). HIV-specific CD8?T cells
from both strong and weak responders had high proliferative po-
tential (Fig. 2C), which is a hallmark of a high-quality HIV-spe-
cific CD8?T cell response in HICs (26). Interestingly, we found
in weak responders an increase of a subpopulation of HIV-specific
CD8?T cells characterized by the coexpression of CD27 and
CD45RA (Fig. 2C). We have recently reported that this subpopu-
lation is characteristically abundant in HIV patients treated during
acute primary HIV infection and may represent a stable quiescent
long-term memory pool (33).
CD8-mediated HIV-suppressive capacity in HICs correlates
strongly with the magnitude of Gag-specific CD8?T cell
The response to Gag contributed most (average, 51.8%) to the total
HIV-specific CD8?T cell response (Fig. 3A). In strong responder
HICs the contribution of the Gag response was 56.8% on average
compared with 37.9% in weak responder HICs (p ? 0.14). Re-
sponses to Nef peptides also contributed significantly to the overall
CD8?T cell response in HICs (average, 31.8% in strong respond-
ers and 31.2% in weak responders) (Fig. 3A). The contributions of
Env and Pol responses were much smaller (7.9% and 8.7%, re-
spectively, in the whole HIC population) (Fig. 3A). The contribu-
tions of the responses to the different HIV proteins were not dif-
ferent in HICs than in viremic patients (not shown), although a
tendency was observed to a greater contribution of Gag responses
in strong responder HICs than in viremics (average, 38%; p ?
0.08). The magnitude of the Gag response was higher in HICs
(3682 ? 2969 SFCs/106PBMC) than in viremics (1703 ? 2061
SFCs/106PBMC, p ? 0.05). In contrast, Gag responses contrib-
uted less and Nef responses more to the total HIV-specific CD8?
T cell response in HAART-treated patients (14.3% and 60.6% of
Gag and Nef responses) than in HICs (p ? 0.014 and p ? 0.025,
We then examined the influence of the specificity of HIC CD8?
T cells on the efficiency of HIV suppression. The correlation be-
tween the HIV-suppressive capacity of nonstimulated CD8?T
cells and the frequency of IFN-?-producing CD8?T cells upon
peptide stimulation was strongest for Gag peptides (Spearman
that was due to CD8?T cells secreting IFN-? upon stimulation with Gag,
Nef, Env, and Pol peptides. Each symbol represents one HIC. Circles rep-
resent strong responders; squares represent weak responders. Horizontal
dashed lines are mean values for each group. B and C, Correlation between
the HIV-suppressive capacity of CD8?T cells from HICs and their fre-
quency of IFN-?-producing CD8?T cells upon stimulation with Gag and
Nef peptides, respectively. Each symbol represents one HIC. Vertical
dashed line separates weak responder and strong responder HICs.
A, Percentage of the HIV-specific CD8?T cell response
7832HETEROGENEITY OF CD8 T CELL HIV SUPPRESSION IN HIV CONTROLLERS
by guest on June 13, 2013
0.907, p ? 0.00001) (Fig. 3B). This correlation was unlikely to be
due to a bias for HLA-B57-restricted Gag responses since in the 13
individuals carrying this HLA allele, HIV-specific CD8?T cell
responses targeting HLA-B57 restricted Gag epitopes represented,
on average, 26 ? 12% of their total response. Other responses
were either restricted by HLA-B57 but not directed at Gag (19 ?
18%) or restricted by other alleles and directed at Gag (28 ? 28%
of the response) or at other proteins (27 ? 20%). As mentioned,
Nef was also a main target of the HIV-specific CD8?response;
however, only a weak correlation was found with the magnitude of
Nef responses (Spearman 0.473, p ? 0.040) (Fig. 3C). Further-
more, this correlation with Nef responses was completely lost
when weak responder HICs were excluded from the analyses
(Spearman 0.070, p ? 0.797). Most interestingly, in the group of
strong responder HICs, the CD8?T cell anti-HIV capacity still
correlated more tightly with the magnitude of Gag responses (Spear-
man 0.812, p ? 0.00001) than with the total frequency of IFN-?-
producing CD8?T cells (Spearman 0.634, p ? 0.007). Overall these
results suggest that the numbers of CD8?T cells responding to Gag
epitopes influence the capacity of CD8?T cells from HICs to sup-
press HIV infection of autologous CD4?T cells.
To evaluate more directly the impact of Gag responses in the
HIV-suppressive activity of CD8?T cells from strong responder
HICs, we first tried to compare the HIV-suppressive capacity of
FACSAria-sorted pentamer-positive cell fractions. Unfortunately,
and despite a fairly good viability, functionality of these cells was
compromised. Hence, we compared the relative weight of Gag and
Nef responses by assessing the HIV-suppressive capacity of CD8?
T cell fractions depleted of either one response or the other. These
experiments were performed with cells from three HICs (A3, A6,
and A11) with similar numbers of HIV-specific cells (11,270,
12,473, and 12,612 SFC/106PBMC, respectively) and a contribu-
tion of the Gag response to the total HIV-CD8?T cell response
close to 50% (49.8%, 63.0%, and 55.3%, respectively). CD8?T
cells isolated from HICs were stimulated with 1) a pool of all the
optimal HIV-1 peptides that were recognized in individual
ELISPOT assays (not shown); (2) a pool of Gag peptides only; (3) a
pool of Nef peptides only. As shown in Fig. 4A, the suppression of
HIV infection observed when autologous unstimulated CD8?T
cells from strong responder HICs were added to CD4?T cell
cultures was lost when the CD8?T cells that produced IFN-? upon
stimulation with the complete pool of recognized HIV peptides
were removed. In the case of A3, both the cell fractions depleted
of Gag-specific or Nef-specific CD8?T cells retained strong HIV-
suppressive capacity (Fig. 4B). For A11, depletion of Gag-specific
cells caused the nearly complete loss of HIV-suppressive capacity,
whereas depletion of Nef-specific cells had no effect (Fig. 4B). For
A6, the depletion of Gag-specific cells also caused a strong loss of
HIV-suppressive capacity (Fig. 4B). Removal of Nef-specific cells
occasioned a more modest loss of HIV-suppressive capacity. In
summary, although the respective contributions of Gag and Nef
responses were difficult to quantify precisely, Gag-specific CD8?
T cells seemed to strongly contribute to the HIV-suppressive ca-
pacity of CD8?T cells in all three strong responder HICs evalu-
ated, in agreement with the correlations described above. In con-
trast, the contribution of Nef responses was more variable.
CD8-mediated HIV-suppressive capacity in strong responder
HICs is broad
We have already reported a broad capacity of CD8?T cells from
strong responder HICs to effectively control superinfection by dif-
ferent HIV-1 subtypes (21). Interestingly, CD8?T cells from
strong responder HICs also partially suppressed infection of CD4?
T cells by other human lentiviruses such as HIV-2, SIVmac, and
SIVagm (Fig. 4C). At least some of the HIV-1 epitopes recognized
by HIV-specific CD8?T cells from strong responders HICs were
conserved in the other lentiviruses used in our experiments (not
shown). In accordance with a MHC-mediated mechanism, the ca-
pacity to suppress SIV infection was totally lost when CD8?T
cells were separated from autologous CD4?T cells by semiper-
meable membranes (not shown), as was shown in the case of
A6 with HIV-1 Bal. CD4?T cells were cultured alone or in the presence (CD4/CD8 of 1:1) of non-prestimulated CD8?T cells or CD8?T cells depleted
of HIV-specific CD8?T cells. These results are representative of experiments with 5 HICs. B, HIV-suppressive capacity of CD8?T cells (mean ? SD,
n ? 3), as determined by the log fold decrease in the level of secreted p24 (CD4 vs CD4/CD8, 1:1 cell cultures), after depletion of HIV-specific cells (black
bars), Gag-specific (open bars), or Nef specific (gray bars) cell fractions. Pie charts at the bottom represent the relative contribution of Gag and Nef
responses to the total HIV-specific CD8?T cell response. C, CD4?T cells from HIV controllers were infected with replicative HIV-1.BaL (black bars),
SIVagm.Gril (open bars), SIVmac.251 (gray bars), or HIV-2.SBL (patterned bars) and cultured alone or with autologous unstimulated CD8?T cells. Viral
replication was monitored by p24 or p27 ELISA on culture supernatants. Bars indicate the level of suppression at the peak of viral replication when CD8?
T cells were present in the culture (mean ? SD, n ? 3). n.d., Experiment not done.
A, p24 production in culture supernatants (mean ? SD, n ? 3) at the peak of viral replication after superinfection of CD4?T cells from
7833The Journal of Immunology
by guest on June 13, 2013
Weak responder HICs carry infectious replicative viruses
A recent report by Hatano et al. suggested that low level viral
replication is ongoing in most HICs (22). We thus examined
whether differences between strong responder and weak responder
HICs might exist at a virological level. Ultrasensitive viral load
tests were not available for this study. However, given the long
documented virological follow-up of the patients in the study, we
had access to multiple RNA viral load determinations for all HICs
(Table I). The length of the follow-up and the number of viral load
determinations were similar for strong responder and weak re-
sponder HICs (p ? 0.309 and p ? 0.515, respectively; Table I).
Interestingly, historical plasma viral load results showed that small
blips of viral RNA were more frequently detected during fol-
low-up among strong responder HICs than among HICs with weak
CD8?T cell responses, who appeared to control HIV infection
more tightly (p ? 0.016; Fig. 5A and Table I).
HIV-1 DNA level in blood cells, which is a stable parameter that
gives an estimation of the HIV-1 reservoir size (34), was available
for most HICs (Table I). Despite the differences in the frequency
of viral RNA blips mentioned above, proviral DNA levels were
very low in all the HICs, regardless of the strength of their CD8?
T cell responses (Fig. 5B). We then investigated whether autolo-
gous viral replication might be activated upon stimulation of
CD4?T cells from HICs. Surprisingly, replication-competent vi-
ruses were more readily detected in the supernatants of activated
CD4?T cells from weak responders than from strong responders
(Fig. 5C). Moreover, autologous virus production upon CD4?T
cell stimulation correlated negatively with the HIV-suppressive
capacity of CD8?T cells (Spearman ?0.635, p ? 0.01). We ob-
tained enough autologous viruses from weak responder HICs A13,
A19, and A22 to test their infectivity. These viruses were able to
spread and infect heterologous CD4?T cells as efficiently as other
laboratory-adapted strains and primary isolates (Fig. 5D). Their
titers (6.1, 5.6, and 5.5 50% tissue culture-infective dose/ml for
vA13, vA19, and vA22, respectively) were also similar (5.4
TCID50/ml for both BaL and NL4.3, and 6.1 TCID50/ml for
v30007). Therefore, at least some HICs with weak CD8?T cell
responses carry viruses highly replicative in vitro. This is in agree-
ment with recent reports showing that defective or attenuated vi-
ruses do not generally account for the control of viral replication in
Here we show that the HIV-suppressive capacity of CD8?T cells
from HIV controllers is stable over time and is associated with the
magnitude of HIV-specific CD8?T cell responses, in particular to
those directed against Gag. We also identify a group of HICs who
carry infectious viruses and are able to durably control HIV infec-
tion despite a weak HIV-suppressive capacity of their CD8?T
Most of the HIC subjects in our study (14 of 19) had CD8?T
cells with marked and stable HIV-suppressive capacities (strong
responder HICs, p24 log decrease ?2) that we have never ob-
served in viremic (21) or HAART-treated individuals. The protec-
tive HLA alleles B27 and/or B57 were present in all strong re-
sponder HICs. However, CD8?T cells from a subgroup of HICs
cells at inclusion, for strong responder HICs (SR, F) and weak responder HICs (WR, ?). Each symbol represents one HIC. C, Correlation between peak
p24 production detected in the supernatant of 105CD4?T cells from weak responder (?) and strong responders HICs (F) upon PHA stimulation (mean
of three values) and HIV-suppressive capacity of CD8?T cells from 16 HICs. Each symbol represents one HIC. The dashed line represents the background
level. D, Kinetics of viral replication (3, 7, and 10 days postinfection) after infection (1.2 ng of p24/106cells) of CD4?T cells from a single healthy blood
donor. Viruses from subjects A13 and A19 were obtained at 10 and 14 days, respectively, of culture of PHA-activated CD4?T cells. Viruses from
A22 were obtained after 8 days of culture of PHA-activated CD4?T cells and 5 additional days of culture in the presence of heterologous
PHA-activated CD4?T cells. Open bars represent laboratory-adapted viruses, gray bars primary isolates, and black bars HIC-derived viruses. The
mean and SD are shown (n ? 3).
A, Frequency of viral load determinations with values ?50 HIV RNA copies/ml of plasma during follow-up and (B) total HIV-DNA in blood
7834HETEROGENEITY OF CD8 T CELL HIV SUPPRESSION IN HIV CONTROLLERS
by guest on June 13, 2013
had only weak HIV-suppressive capacity. In agreement with recent
reports (20, 24), the HICs we studied had heterogeneous levels of
HIV-specific CD8?T cells, as estimated by the frequency of IFN-
?-producing CD8?T cells. The magnitude of the HIV-specific
CD8?T cell response correlated strongly with the capacity of
CD8?T cells from HICs to control HIV infection of autologous
CD4?T cells in vitro. Accordingly, the lowest frequencies of IFN-
?-producing CD8?T cells were found in weak responder HICs.
Some underestimation of the CD8?T cell response in HICs may
come for the use of peptides derived from consensus sequences for
ELISPOT determinations, or of a laboratory-adapted HIV strain
for HIV-suppression analyses. However, CD8?T cells from two
weak responder HICs had limited suppressive capacity even when
autologous viruses were used, which further supported a truly
weak CD8?T cell response in these individuals. We cannot ex-
clude that control of viremia in weak responders may be due to
robust HIV-specific CD8?T cell responses in lymphoid tissues,
and actually Ferre and collegues have recently shown that HICs
have polyfunctional HIV-specific T cell responses in rectal mucosa
that were frequently stronger than in blood (38). However, the
presence in this study of a few HICs with very weak responses
both in the blood and in the rectal mucosa is interesting. Although
a weak high quality CD8?T cell response might be sufficient to
control viremia in vivo, it seems unlikely to be the case in weak
responder HICs. The absence of viral blips during the follow-up of
weak responders and our finding that at least some of these HICs
carry viruses that are highly infectious in vitro and readily detect-
able upon in vitro activation endorse the idea of a very tight and
active host-restraint of HIV-1 infection. Our phenotypical analyses
of the HIV-specific CD8?T cells in weak responders showed an
increased proportion of a CD27?CD45RA?subset of cells, pre-
viously observed in patients treated during primary HIV infection,
and that might represent a quiescent and stable memory pool able
to proliferate and acquire effector capacities upon Ag stimulation
(33). Although these cells may provide an effective response in the
eventuality of viral replication, their increased proportion in weak
responder HICs together with the low expression of HLA-DR sug-
gest a long period without antigenic stimulation of the CD8?T cell
Therefore, an alternative mechanism is probably responsible for
controlling HIV-1 in these HICs. The lower antiviral activity of
CD8?T cells in weak responder HICs did not seem to be com-
pensated for by other cell populations within PBMC (e.g., NK cells
or ?? T cells), as illustrated by HIV-suppressive experiments
where nonstimulated PBMC (depleted of CD4?cells), used in-
stead of CD8?T cells, were also unable to control HIV superin-
fection of autologous CD4?T cells (not shown). Interestingly,
persistent lack of low-level detectable viremia in one HIC has been
recently associated to low levels of HIV Abs and remarkably low
levels of T cell activation (22). Further virologic studies (such as
viral sequencing or determination of tissular viral replication) and
the analysis of innate responses and regulatory T cells (39) might
help to identify new mechanisms of control in HICs.
Unlike the cells from weak responder HICs, CD8?T cells from
strong responder HICs had a broad capacity to suppress superin-
fection of their own CD4?T cells by a wide range of HIV-1 strains
(21) and, at least partially, by other lentiviruses. This could be
related to the presence of HIV-specific CD8?T cells targeting
epitopes located within highly conserved regions of the virus. Re-
sponses against Gag and Nef epitopes together accounted for the
bulk of total CD8?T cell responses in strong responder HICs, and
no phenotypic differences were observed between Gag-specific
and Nef-specific CD8?T cell responses in these individuals (21).
Interestingly, we observed a strong correlation between the HIV-
suppressive capacity of CD8?T cells in strong responder HICs
and the number of Gag-specific CD8?T cell responses. Moreover,
the analysis of the relative HIV-suppressive capacity of the Gag
response in three strong responder HICs showed that, for all three
HICs, Gag-specific CD8?T cells possess the strongest antiviral
capacities. Thus, Gag responses appear to be strongly involved in
the antiviral potency of CD8?T cells. This is in agreement with a
report showing evidence of CD8?T cell selective pressure on gag
in HICs (40). Increasing evidence suggests that Gag-specific
CD8?and CD4?T cell responses are associated with better con-
trol of HIV viremia (7–9, 20, 41). Gag epitopes are presented on
the surface of infected CD4?T cells early after viral entry, before
DNA integration and viral protein synthesis (42), and this might
allow Gag-specific CD8?T cells to recognize and eliminate in-
fected cells before the infection is properly established and before
Nef-mediated down-regulation of MHC class I molecules occurs
(43). Other factors such as functional avidity (41, 44) or lytic gran-
ule loading (45) might contribute to an enhanced HIV-suppressive
capacity of Gag-specific CD8?T cells.
No correlation was found between HIV-suppressive capacity of
CD8?T cells in strong responder HICs with Nef-specific CD8?T
cell responses. However, this observation does not preclude a con-
tribution of responses targeting Nef (or other viral proteins) to the
HIV-suppressive capacity of CD8?T cells. Actually, our experi-
ments of selective depletion of HIV-specific cell fractions showed
variable capacities (from strong to none) of Nef-specific CD8?T
cells from HICs to suppress HIV infection, perhaps depending on
the frequency of the Nef responses that were targeting epitopes
restricted by HLA-B57. Along these lines, escaping mutations are
also found in Nef epitopes in HICs, although less frequently than
in Gag epitopes (46).
Escaping mutations in epitopes located within structurally im-
portant regions of the virus could limit the capacity of the virus to
mutate to escape immune pressure, as variations in these regions
have a fitness cost (13, 47). Although we did not directly address
this issue, the difficulties to detect HIV-1 replication in the culture
supernatants of activated CD4?T cells from strong responder
HICs might reflect the impact of the pressure exerted by CD8?T
cell responses on viral fitness. Nevertheless, we cannot exclude
that, given the extraordinary antiviral potency of CD8?T cells
from strong responder HICs, the few remaining CD8?T cells in
the ?97% pure CD4?T cell fractions used in these experiments
were enough to efficiently suppress autologous virus replication.
Several important questions await answers; that is, mainly
whether the potent CD8?T cell response observed in most HICs
precedes or follows initial viremic control, and how such a potent
CD8?T cell response is maintained. The association presented
here between blips in plasma viral RNA and stronger CD8?T cell
responses in HICs must be considered with care because of the
limited number of weak responder HICs, but it is tempting to spec-
ulate that CD8?T cell control of HIV might involve a feedback
mechanism whereby occasional blips (or ongoing low-level viral
replication) are needed to boost the antiviral response. The in-
creased telomerase activity in these cells would further ensure their
persistence (28). Two scenarios can be envisaged: 1) if viremia is
controlled by a common mechanism in weak and strong responder
HICs, the presence of the protective HLA B27 and B57 alleles may
help to sustain control over time, in the eventuality of viral escape,
through the establishment of a robust CD8?T cell response; 2)
different mechanisms are responsible for initial control of HIV
infection in weak and strong responder HICs. Detailed longitudinal
studies of HICs will be necessary to answer these questions.
7835The Journal of Immunology
by guest on June 13, 2013
We thank all the members of the French National Agency for Research on
AIDS and Viral Hepatitis (ANRS) EP36 HIV Controllers study group for
helpful discussions. We thank Chiraz Hamimi for technical help. We also
thank Dr. Laurence Meyer, Dr. Daniel Se ´re ´ni, Dr. Caroline Lascoux, Dr.
Olivier Taulera, Jeannine Delgado, Dr. Franc ¸ois Bricaire, Dr. Miche `le Ben-
tata, Dr. Pascale Kousignian, Miche `le Pauchard, Dr. Alain Krivitzky, Pa-
tricia Honore ´, Marie-The ´re `se Rannou, Dr. Jean-Paul Viard, Dr. David Zuc-
man, Nade `ge Velazquez, and all the other physicians and nurses who cared
for the patients. We especially thank the subjects who participated in this
study for their cooperation.
The authors have no financial conflicts of interest.
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