JOURNAL OF VIROLOGY, Nov. 2010, p. 12082–12086
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 84, No. 22
Reduction of Immune Activation with Chloroquine Therapy
during Chronic HIV Infection?
Shannon M. Murray,1Carrie M. Down,1† David R. Boulware,2William M. Stauffer,2
Winston P. Cavert,2Timothy W. Schacker,2Jason M. Brenchley,1§
and Daniel C. Douek1*
National Institutes of Health, National Institute of Allergy and Infectious Diseases, Vaccine Research Center,
Human Immunology Section, Bethesda, Maryland,1and Division of Infectious Diseases and
International Medicine, Department of Medicine, University of Minnesota, Minneapolis, Minnesota2
Received 14 July 2010/Accepted 1 September 2010
Increased levels of activated T cells are a hallmark of the chronic stage of human immunodeficiency virus
(HIV) infection and are highly correlated with HIV disease progression. We evaluated chloroquine (CQ) as a
potential therapy to reduce immune activation during HIV infection. We found that the frequency of CD38?
HLA-DR?CD8 T cells, as well as Ki-67 expression in CD8 and CD4 T cells, was significantly reduced during
CQ treatment. Our data indicate that treatment with CQ reduces systemic T-cell immune activation and, thus,
that its use may be beneficial for certain groups of HIV-infected individuals.
Chronic HIV infection is characterized by multifaceted sys-
temic immune activation, including increased frequencies of
activated T cells (9, 17) and increased turnover of T cells (5, 12,
18) that correlate directly with disease progression (8, 9). T-cell
immune activation is also associated with lower gains in CD4
T-cell count in HIV-infected individuals even while they are on
antiretroviral therapy (ART) that appears to suppress viral
replication (10). Thus, therapies that reduce immune activa-
tion may be of benefit, particularly for such individuals. Three
clinical studies have been conducted using hydroxychloroquine
monotherapy for patients with HIV infection (6, 21, 22), and
the studies showed that hydroxychloroquine-treated patients
had decreased viral loads as well as decreased serum interleu-
kin-6 (IL-6) levels, heightened levels of which are correlated
with disease progression (13). However, these studies did not
examine other parameters of immune activation. Chloroquine
(CQ) is known to suppress immune activation by a number of
mechanisms, including inhibition of intracellular toll-like re-
ceptor (TLR) signaling and inflammatory cytokine secretion
(11, 19). In vitro, CQ has been shown to reduce HIV infection-
induced T-cell immune activation (14). Here, we report results
using samples from a clinical study of HIV-infected individuals
treated with CQ monotherapy, where we examine multiple
parameters of immune activation during the course of CQ
Study design and sample collection. A double-blind, ran-
domized placebo-controlled trial testing the effects of chloro-
quine in chronically HIV-infected persons was conducted with
informed consent and approval by the University of Minnesota
Institutional Review Board. Thirteen chronically HIV-infected
individuals were randomized, with six receiving 250 mg of
chloroquine daily, three receiving 500 mg of chloroquine
daily, and four receiving placebo for 2 months. Inclusion
criteria included CD4 T-cell counts of ?250 cells/ml. All
were ART naïve except for four patients who had been off
ART for at least 16 months. Peripheral blood was taken at
study initiation, month 1 posttreatment, and month 2 post-
treatment. Viral RNA loads and CD4 T-cell counts were
taken for each patient at each visit.
Cell surface staining for immune activation and intracellu-
lar staining for Ki-67. Peripheral blood mononuclear cells
(PBMCs) were isolated with Ficoll-Paque centrifugation and
viably cryopreserved. Cells were thawed and washed in RPMI
medium supplemented with 10% fetal calf serum (FCS). Aqua
(Invitrogen), a viability dye, was first added to 106cells, and
then the following fluorochrome- or quantum dot (QDOT)-con-
jugated antibodies against cell surface markers (obtained from
Becton Dickinson, Pharmingen, or Coulter or provided by Mario
Roederer, NIH) were added: CD3-Cy7 phycoerythrin (PE),
PE (TRPE), HLA-DR-Cy7 allophycocyanin (APC), CD19-Pa-
cific Blue (PacBlue), and CD38-APC. Cell surface staining
followed by intracellular staining for Ki-67 was performed as
previously described (16). Cells were then resuspended in 1%
paraformaldehyde (PFA) in phosphate-buffered saline (PBS)
before flow cytometric analysis or cell sorting. Cells were an-
alyzed by 10-parameter flow cytometry with an LSRII instru-
ment (BD), and CD27?CD45RO?memory CD4 T cells were
sorted for quantitative PCR for viral DNA using a FACS Aria
cell sorter (BD). Intracellular cytokine staining of PBMCs for
tumor necrosis factor (TNF), gamma interferon (IFN-?), and
IL-2-producing HIV-specific memory T cells was performed as
* Corresponding author. Mailing address: Human Immunology
Section, NIH Vaccine Research Center, 40 Convent Dr., Room
4500, Bethesda, MD 20892-3005. Phone: (301) 594-8484. Fax: (301)
480-2565. E-mail: firstname.lastname@example.org.
† Present address: Virginia Commonwealth University, School of
Medicine, 1101 East Marshall Street, Sanger Hall, Room 1-007, Rich-
mond, VA 23298.
§ Present address: National Institutes of Health, National Institute
of Allergy and Infectious Diseases, Laboratory of Molecular Microbi-
ology, Bethesda, MD 20892.
?Published ahead of print on 15 September 2010.
previously described (4). Plasma lipopolysaccharide (LPS) lev-
els were quantified in triplicate using the Limulus amebocyte
assay (Lonza) as previously described (3). Plasma was diluted
1:10 in endotoxin-free water and heated to 80°C for 15 min
before use in the assay.
The Wilcoxon matched-pairs test was used to compare dif-
ferent time points within subjects. The analysis included sam-
ples from eight CQ- and three placebo-treated individuals (Ta-
ble 1) instead of the initially enrolled subjects due to two
subjects for whom only a baseline time point sample was ob-
tained. Due to the small sample size of the placebo group, the
Wilcoxon test was not performed for month 2 of the placebo
arm. Graph Pad Prism version 5.0 was used to perform anal-
Decreased HLA-DR?CD38?CD8 memory T cells in CQ-
treated subjects. As the percentages of HLA-DR?CD38?
CD8 memory T cells strongly correlate with HIV disease pro-
gression, we chose to evaluate this parameter first during the
course of CQ treatment. We found that there was a significant
decrease in the percentages of HLA-DR?CD38?CD8 mem-
ory T cells in the CQ-treated subjects at both month 1 and
month 2 after CQ administration relative to baseline (Fig. 1).
HLA-DR?CD38?cells comprised between 1.9% and 14.3%
of total CD8 memory T cells, defined as CD27highCD45RO?,
CD27loCD45RO?, or CD27loCD45RO?. These ranges were
comparable to those previously reported during HIV infection
for HLA-DR?CD38?CD8 T cells (8). Most of the previous
studies of HLA-DR?CD38?CD8 T cells during HIV infec-
tion have evaluated T-cell activation in the total T-cell popu-
lation, which included both memory and naïve cells. Here, we
were able to discriminate activation specifically within the
memory T-cell population by multiparameter flow cytometry.
At month 1 after CQ administration, the median percentage of
HLA-DR?CD38?CD8 memory T cells decreased from 8.0%
to 4.35% of total memory CD8 T cells (Fig. 1A) (median
change, 3.65%; P ? 0.047). At month 2 after CQ administra-
tion, the decrease relative to baseline was sustained in the
CQ-treated group, with a median of 5.5% of memory CD8 T
cells (Fig. 1A) (median change, 2.5%; P ? 0.016). Data for the
placebo recipients are shown in Fig. 1D. The median change
from baseline to month 1 for the placebo controls was 2.05%
(P ? 0.75), and that from baseline to month 2 was 1.85% (Fig.
1D). There were no significant changes in the frequencies of
HLA-DR?CD38?CD8 memory T cells in the placebo-treated
individuals (Fig. 1D).
We next examined the frequency of HLA-DR?CD38?CD4
memory T cells. Previous reports indicate that while the per-
centage of these cells is increased with HIV infection, this does
not serve as a prognostic marker for disease progression, as
does the percentage of CD38?HLA-DR?CD8 memory T
cells (1). We found that CQ treatment did not affect the per-
centage of HLA-DR CD38-coexpressing CD4 memory T cells
(Fig. 1B) (P ? 0.813). Similar data were found for placebo
controls (Fig. 1E). Furthermore, we found that there were no
significant changes in plasma viral RNA for the CQ- (Fig. 1C)
or placebo-treated (Fig. 1F) subjects or in cell-associated viral
DNA (data not shown) over the 2-month period.
Decreased Ki-67 expression in CD4 and CD8 memory T
cells at month 1 after CQ treatment. We then evaluated the
fraction of T cells in the cell cycle by measuring the percentage
TABLE 1. Study subjects and sample collectiona
No. of mo
(log10HIV RNA copies/ml)
Day 0Mo 1 Mo 2Day 0 Mo 1Mo 2Day 0b
CQ3xxxNaı ¨ve4.89 5.09 5.01 462514
CQ5xxx22.14.624.52 4.31 311 224
CQ10x*x Naı ¨ve4.77 NA 4.74271313
CQ13xxxNaı ¨ve 4.174.573.9 486512
Naı ¨ve4.074.03NA401 NA
aSymbols and abbreviation: x, sample was collected; *, sample collection was missed for a reason unrelated to subject health; ?, death; NA, sample not available.
bThe baseline CD4 cell count was the CD4 cell nadir for each subject at study initiation.
VOL. 84, 2010NOTES12083
of Ki-67?T cells. At baseline, the median percentage of pro-
liferating CD8 T cells of total CD8 memory T cells was 2.4%
for the CQ-treated group, and after 1 month of CQ treatment,
there was a significant decrease in the percentage of Ki-67?
memory CD8 T cells, to a median of 1.3% (Fig. 2A) (median
change, 1.1%; P ? 0.034). After the second month of CQ
treatment, the Ki-67?memory CD8 T-cell population relative
to baseline was not significantly altered (Fig. 2A) 9median
change, 0.1%; P ? 0.397). The data for the placebo recipients
are shown in Fig. 2C. The median decrease from baseline to
month 1 was 0.14% (P ? 0.75), and there was a median in-
crease of 1.17% at month 2.
The CD4 memory Ki-67?T-cell population exhibited a
trend similar to that observed for the CD8 memory Ki-67?
T-cell population. After 1 month of treatment, a significant
decrease in Ki-67 expression was observed, from a median of
3.2% to 1.5% of total memory CD4 T cells (Fig. 2B) (P ?
0.047). There was no significant difference from baseline to
month 2 (Fig. 2B) (median, 2.0%; P ? 0.375). Data for the
placebo controls are shown in Fig. 2D. There was a 0.53%
decrease in the median at month 1 (P ? 0.75) and then a
1.89% increase at month 2 (Fig. 2D). We also examined mem-
ory B cells and found a decrease in Ki-67 expression at month
2 post-CQ treatment with a trend toward significance (P ?
0.06) (data not shown).
Decreased plasma LPS levels at month 1 after CQ treat-
ment. We next sought to measure plasma LPS levels during the
course of CQ treatment. Plasma LPS levels are elevated during
chronic HIV infection and are correlated with immune activa-
tion (3). We therefore measured LPS in the plasma and found
a significant decrease in LPS levels from a median of 60.2 to
55.2 pg/ml (Fig. 3A) (median change, 5 pg/ml; P ? 0.037) after
1 month of CQ treatment. After a second month of CQ ad-
ministration, however, the levels were not significantly differ-
ent from baseline, with a median of 57.9 pg/ml at month 2 (Fig.
3A) (median change, 2.3 pg/ml; P ? 0.822). There was a me-
dian increase of 10.31 pg/ml in the placebo recipients from
baseline to month 1 (Fig. 3B) (P ? 0.25). At month 2, there
was a median increase in LPS levels in placebo controls of 0.88
pg/ml (Fig. 3B).
Preservation of HIV-specific T-cell responses during CQ
treatment. We measured both CD4 and CD8 T-cell responses
to Gag, Pol, Env, and Nef HIV antigens, by using overlapping
peptide pools for HIV clade B viruses. In all cases, we found no
significant changes in any TNF-, IFN-?-, or IL-2-producing
HIV- or cytomegalovirus (CMV)-specific CD8 or CD4 T cells
during the complete course of CQ treatment or in the placebo
control group (data not shown).
In summary, we found that administration of CQ during
chronic HIV infection resulted in decreased immune activation
as measured by a parameter closely correlated with disease
progression, the percentage of CD38?HLA-DR?CD8 mem-
ory T cells. In addition, Ki-67 expression was reduced in both
CD4 and CD8 memory T-cell populations during the first
phase of CQ treatment. While our study was limited by the
small number of placebo controls, we have extended previous
studies of CQ therapy to show decreases in T-cell immune
activation with CQ treatment.
CD38?HLA-DR?CD8 T cells are increased in frequency
during chronic viral infections (2) and on stimulation with
FIG. 1. CD8 T-cell activation is decreased in HIV-infected individuals during chloroquine treatment. Percentages of CD38?HLA-DR?CD8
and CD4 memory T cells for chloroquine- (A and B) or placebo-treated (D and E) subjects are shown. Plasma viral RNA loads are also shown
for chloroquine- (C) and placebo-treated (F) subjects. Each symbol, as indicated in Table 1, represents an individual study subject at day 0, month
1, or month 2 post-chloroquine or placebo treatment. n.d., not determined; CQ, chloroquine.
12084NOTES J. VIROL.
TLR3 and TLR9 ligands (7). CQ is an inhibitor of signaling by
the endosomal TLRs, TLR3, TLR7, TLR8, and TLR9 (11).
Thus, it is plausible that suppression of intracellular TLR sig-
naling resulted in the decrease in CD38?HLA-DR?CD8 T
cells with CQ treatment in these HIV-infected subjects. This
could occur by suppression of intracellular TLR signaling in
monocytes and dendritic cells (DCs) which, in turn, results in
the reduction in CD38?HLA-DR?CD8 T cells, as previously
shown to occur with DC–T-cell cocultures with CQ in vitro
(14). It is unclear why the decrease in CD38 and HLA-DR
expression was sustained during the complete course of CQ
treatment, whereas Ki-67 and LPS levels were decreased only
after the first month of treatment. However, the latter two pa-
rameters followed similar patterns of suppression and, there-
fore, may be more directly causally related.
We have identified parameters of immune activation that
are altered by CQ alone, in the absence of ART. It is possible
that differences in CQ dosage and regimen and the limited
statistical power of our study account for why we did not
observe decreases in viral load as reported in other studies.
Since our study, another report showed that a daily dosage of
250 mg CQ, as was given to the majority of the participants
in our study, was not sufficient to decrease viral load (20).
The effects of CQ in combination therapy studies have been
reported to be qualitatively different than the antiretroviral
effects and not simply additive (15). Taken together, our
results suggest that CQ intervention suppresses key aspects
FIG. 3. Plasma LPS levels in HIV-infected individuals are de-
creased during the first but not the second month of chloroquine
treatment. Plasma LPS levels were determined as described in the text.
P values were derived using the Wilcoxon matched-pairs test.
FIG. 2. Ki-67 expression by CD8 and CD4 memory T cells is decreased in HIV-infected individuals during the first but not the second month
of chloroquine treatment. The percentages of CD8 or CD4 memory T cells expressing Ki-67 for chloroquine- (A and B) or placebo-treated (C and
D) subjects are shown. Each symbol is representative of each subject, as described in Table 1. n.d., not determined; CQ, chloroquine.
VOL. 84, 2010 NOTES12085
of HIV disease pathogenesis that are correlated with disease
This work was supported in part by the Intramural Program of the
National Institute of Allergy and Infectious Diseases, National Insti-
tutes of Health (5U01-AI1027661-19); the Minnesota Medical Foun-
dation; and the International Center for Antiviral Research and Epi-
demiology (I CARE).
We thank Martha Nason for assistance with statistical analyses and
Christine Fietzer, Kathy Fox, and Max Schmeling for assistance in
clinical care and sample preparation.
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