Contrasting Effects of Low-Dose IL-2 on Vaccine-Boosted
Simian Immunodeficiency Virus (SIV)-Specific CD4?and
CD8?T Cells in Macaques Chronically Infected
Janos Nacsa,* Yvette Edghill-Smith,* Wen-Po Tsai,* David Venzon,†Elzbieta Tryniszewska,*§
Anna Hryniewicz,* Marcin Moniuszko,*¶Audrey Kinter,‡Kendall A. Smith,?and
IL-2, the first cytokine discovered with T cell growth factor activity, is now known to have pleiotropic effects on T cells. For
example, it can promote growth, survival, and differentiation of Ag-selected cells, or facilitate Ag-induced cell death of T cells when
Ag persists, and in vivo, it is thought to contribute to the regulation of the size of adaptive T cell response. IL-2 is deficient in HIV-1
infection and has been used in the management of HIV-1-infected individuals undergoing antiretroviral therapy. In this study, we
investigated how continuous low-dose IL-2 affected the CD4?and CD8?T cell response induced by two inoculations of a ca-
narypox recombinant SIV-based vaccine candidate in healthy macaques chronically infected with SIVmac251. These macaques had
normal levels of CD4?T cells at the beginning of antiretroviral therapy treatment. Vaccination in the presence of IL-2 significantly
augmented Gag-specific CD8?T cell responses, but actually reduced Gag-specific CD4?T cell responses. Although IL-2 at low doses
did not change the overall concentration of circulating CD4?or CD8?T cells, it expanded the frequency of CD4?CD25?T cells.
Depletion of the CD4?CD25?T cells in vitro, however, did not result in a reconstitution of Gag-specific CD4?responses or
augmentation of SIV-specific CD8?T cell responses. Thus, we conclude that the decrease in virus-specific CD4?T cell response may
be due to IL-2-promoted redistribution of cells from the circulation, or due to Ag-induced cell death, rather than suppression by a T
regulatory population. The Journal of Immunology, 2005, 174: 1913–1921.
the drugs are administered continuously. However, despite long-
term suppression of viral replication, ART cannot cure the persis-
tent viral infection, so that if the drugs are discontinued, viral rep-
lication resumes in most patients (1, 2). Because intracellular
pathogens are combated by cell-mediated immunity (3–5), and
both preclinical and clinical studies have demonstrated that CD8?
T cells are important to control viral infections (6–9), we have
explored methods to attempt to boost antiviral immune reactivity,
particularly the virus-specific CD4?and CD8?T cell responses.
The IL-2 molecule is the principal cytokine with T cell growth-
promoting activity (10) responsible for the proliferative clonal ex-
pansion of Ag-selected cells (11), as well as for promoting the
ith the introduction of antiretroviral therapy (ART)2
the replication of HIV and SIV can be suppressed to
undetectable levels for prolonged intervals, provided
differentiation and survival of effector T cells (12, 13). In addition,
IL-2 has been implicated in both “positive” and “negative” selec-
tion of self-reactive cells in the thymus (14, 15), as well as the
development of a subset of CD4?CD25?cells, which are thought
to suppress reactivity with self peptides, termed T regulatory cells
(T-regs) (16, 17).
IL-2 as immune therapy of HIV-1 infection has been investi-
gated in several phase I and II clinical trials and is now under
investigation in large phase III clinical trials in chronic HIV in-
fection designed to test whether high doses (9–15 million IU/
day ? 600 ?g to 1 mg) of IL-2 given intermittently (5 days every
8 wk) together with ART can delay progression to clinical immu-
nodeficiency by comparison with ART alone (18–30). However,
this IL-2 dosing regimen is poorly tolerated, due to signs and
symptoms of the systemic inflammatory response syndrome (31).
Detailed basic studies exploring the effects of IL-2 on T cell
proliferation in vitro have demonstrated that resting peripheral T
cells do not express the high-affinity trimeric IL-2R until activated
by specific Ags (32, 33). Moreover, the concentrations of IL-2
necessary to saturate the trimeric high-affinity IL-2R are very low,
?100 pM, and most of the toxicity of high-dose IL-2 therapy is
attributable to the effects of IL-2 binding to the IL-2R ?- and
?c-chain heterodimers expressed by NK cells, which have a 100-
fold lower affinity for IL-2 (34).
Therefore, we have chosen a different therapeutic approach with
IL-2 therapy. Our clinical studies have determined that IL-2 at low
doses, ?2 million IU/day (?133 ?g/day), can be given safely,
without systemic side effects, for intervals as long as 6 mo to 1
year (35, 36). Moreover, the peak plasma IL-2 concentrations
attained after a s.c. low-dose injection of IL-2, although low, ?25
*Animal Models and Retroviral Vaccines Section, and†Biostatistics and Data Man-
agement Section, National Cancer Institute, and‡Laboratory of Immunoregulation,
National Institute of Allergy and Infectious Disease, Bethesda, MD 20892;§Third
Department of Pediatrics, and¶Department of Allergology and Internal Diseases,
Medical University of Bialystok, Bialystok, Poland; and?Division of Immunology,
Department of Medicine, Weill Medical College of Cornell University, New York,
Received for publication August 16, 2004. Accepted for publication November
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.
1Address correspondence and reprint requests to Dr. Genoveffa Franchini, Animal
Models and Retroviral Vaccines Section, National Cancer Institute, 41/D804, Be-
thesda, MD 20892. E-mail address: email@example.com
2Abbreviations used in this paper: ART, antiretroviral therapy; T-reg, T regulatory
cell; gpe, gag-pol-env; ICS, intracellular cytokine staining.
The Journal of Immunology
Copyright © 2005 by The American Association of Immunologists, Inc.0022-1767/05/$02.00
pM, are still high enough to saturate most high-affinity IL-2Rs
expressed by Ag-activated T cells, and are still too low to bind to
dimeric lower-affinity IL-2Rs expressed by 90% of NK cells. IL-2
given as an immune stimulant together with ART in a randomized
controlled trial resulted in an accelerated return of naive CD4?T
cells compared with ART alone, and had an excellent safety profile
during the 6-mo administration (37).
In the past few years, we have developed a model for HIV
immune-based therapies (38) in macaques either chronically or
acutely infected with SIVmac251 (39, 40) and tested whether fur-
ther enhancing virus-specific immune response by vaccination fa-
vors control of viral replication in the absence of ART. Because
IL-2Rs are only expressed on T cells recently activated by Ags, we
tested this cytokine simultaneously with a T cell vaccine in ART-
treated macaques, which we expected to have low viral Ag levels.
Vaccination imparted a transient virological benefit and IL-2 given
with the vaccine increased the virus-specific CD8?T cell response
(40). However, in that study, we found that the virus-stimulated
proliferative responses, as well as the frequency of CD4?T cells
capable of producing IL-2, were actually decreased by IL-2 ad-
Because in the above-mentioned study, most animals were im-
mune suppressed, we wished to investigate whether IL-2 would
have the same effect in immunocompetent SIVmac251-infected
macaques and whether we could validate, using a larger animal
number, that virus-specific CD8?T cell responses would be en-
hanced, together with a reduction in helper responses. As well, we
wanted to determine whether the decreased CD4?T cell-prolifer-
ative responses corresponded with a decrease in the frequency of
virus-specific CD4?T cells, or whether it was attributable to in-
trinsic proliferative defects. Lastly, we investigated whether the
CD4?CD25?T cell population, which is augmented by high-dose
intermittent IL-2 treatment (16), could have suppressive T-reg ac-
The results detailed herein confirmed our previous study in that
IL-2 significantly increased the number of virus-specific functional
CD8?T cell responses, as monitored by tetramer staining and
flow-cytometric quantification of SIV-activated cells capable of
producing TNF-? and IFN-?. However, IL-2 therapy also signif-
icantly decreased the magnitude of virus-specific CD4?T cell re-
sponses as monitored by either Ag-driven lymphocyte prolifera-
tion assays or intracellular staining for cytokine production. As
well, there was a significant increase in circulating CD4?CD25?
T cells in IL-2-treated animals. However, in vitro depletion studies
did not demonstrate a greater suppressive activity mediated by this
cell population in IL-2-treated macaques.
Materials and Methods
Animals and treatments
All animals were colony-bred rhesus macaques (Macaca mulatta). The
animals were housed and handled in accordance with the standards of the
American Association for the Accreditation of Laboratory Animal Care.
All animals were seronegative for simian T cell lymphotropic virus type 1
and herpesvirus B before the study, and their Mamu-A*01 MHC allele
status was determined by PCR with specific primers (45).
Two naive macaques were used for the IL-2 pharmacokinetics study
(see Fig. 1). The remaining 11 macaques were previously inoculated with
the same stock of pathogenic SIVmac251 (561) (46), and their prior treat-
ment is summarized in Table I and described in Refs. 47 and 48. At the
time of this study, all animals were aviremic, so that they were analogous
to long-term nonprogressors. Macaques 3065, 3078, 3148, 3067, and 3075
were previously treated with ART, which had been suspended for ?1 year.
All macaques were initiated on ART that consisted of i.v. administration of
didanosine (10 mg/kg/day), oral administration of stavudine twice a day
(1.2 mg/kg/dose), and s.c. administration of R-9-(2-phosphonome-
thoxypropyl)adenine (20 mg/kg/day). Six macaques were vaccinated twice
with ALVAC-SIV-gag-pol-env (gpe) (2 ? 108pfu), and five were vacci-
nated in a similar manner and treated with IL-2 (see Fig. 2).
Human rIL-2 was obtained from Amgen. The initial IL-2 doses were es-
timated from the maximum nontoxic doses defined for humans previously
as 250,000 Amgen U/m2body surface area, which amounts to ?6,000
U/kg body weight, or ?0.4 ?g/kg (specific activity ? 15,000 U/?g IL-2
protein) (35). Therefore, for a macaque of 10 kg, the initial doses used were
60,000 and 120,000 U (8 ?g). The Amgen IL-2 formulation is ?5-fold
more potent than the commercially available IL-2 preparation from Chiron,
so the equivalent dose of Chiron IL-2 is ?300,000–600,000 U (40–80
?g)/macaque. Plasma IL-2 concentrations were determined using a com-
mercially available ELISA kit from Endogen.
Quantification of plasma SIVmac RNA and CD3?, CD4?, and
CD8?T cell counts
SIVmac251 RNA in plasma was quantified by nucleic acid sequence-based
amplification (49). Briefly, RNA extracted from plasma was subjected to
isothermal amplification with primers specific for SIVmac251 and quanti-
fied by electrochemiluminescence chemistry using a coextracted internal
standard. The detection limit of this assay is 2 ? 103RNA copies/input
volume of 100 ?l of plasma.
CD4?and CD8?T cell counts were periodically determined on 100 ?l
of whole blood and by FACS analysis, according to the FACS/Lyse kit
(BD Biosciences) with minor modifications. Briefly, after incubating 10 ?l
of a mixture containing PerCP-CD4, CD8-allophycocyanin, CD45-PE, and
CD3-FITC Ab (BD Biosciences) for 30 min at room temperature, red cells
were lysed by adding 2 ml of FACS/Lyse solution for 15 min. Samples
were then centrifuged for 5 min at 1,200 rpm at room temperature, washed
(1% FCS, 0.05% NaAzide in PBS), resuspended in 500 ?l of wash buffer,
and stored at 4°C until acquired by a FACSCalibur flow cytometer (BD
Biosciences). The counts were expressed as percentage of CD3?CD4?or
CD3?CD8?lymphocytes per microliter or as absolute number of cells
with these phenotypes.
Table I. Prior history and clinical features of the animals studied
Levels Vaccination History
NYVAC-SIV-gpe ? DNA
NYVAC-SIV-gpe ? DNA
NYVAC-SIV-gpe ? DNA
ALVAC ? IL-2 16
NYVAC-SIV-gpe ? DNA
1914IL-2 TREATMENT AND VACCINATION OF SIV-INFECTED MACAQUES
T cell proliferation and tetramer staining
Ag-specific proliferation was measured in PBMCs from fresh blood sam-
ples. PBMCs were isolated by density gradient centrifugation on Ficoll,
resuspended in RPMI 1640 medium (Invitrogen Life Technologies) con-
taining 5% inactivated human A/B serum (Sigma-Aldrich), and cultured at
105cells/well for 3 days in the absence or presence of native purified
SIVmac p27 Gag, gp120, (Advanced BioScience Laboratories), or tetanus
toxoid and Con A. The cells were then pulsed overnight with 1 ?Ci of
[3H]thymidine before harvest. The relative rate of lymphoproliferation was
calculated as fold of thymidine incorporation into cellular DNA over con-
trol (stimulation index). The stimulation index was considered positive
Fresh PBMCs were stained with anti-human CD3 Ab (PerCP-labeled,
clone SP34; BD Pharmingen), anti-human CD8? Ab (FITC-labeled, BD
Biosciences), and Mamu-A*01 tetrameric complexes refolded in the pres-
ence of a specific peptide and conjugated to PE-labeled streptavidin (Mo-
lecular Probes). Gag181–189CM9 (CTPYDINQM) (Gag_CM9)-specific tet-
ramer was used. Samples were analyzed by FACSCalibur (BD
Biosciences), and the data are presented as percentage of tetramer-positive
cells of all CD3?CD8?lymphocytes.
Intracellular cytokine staining (ICS)
ICS was performed using the anti-TNF-? Ab with or without anti-IFN-?
Ab. A total of 106fresh PBMCs in 0.2 ml of complete medium was in-
cubated for 1 h at 37°C in the absence or presence of Gag181–189CM9 (10
?g/ml), Gag QI9 (10 ?g/ml), or a pool of Gag peptides (2 ?g/ml), and in
the presence of CD28 and CD49d (1 ?g/ml each) on a V-bottom-well plate.
After addition of 10 ?g/ml brefeldin A (Sigma-Aldrich), cells were incu-
bated for 5 h at 37°C and processed for surface and ICS. Briefly, cells were
washed with 1% FCS in PBS, surface-stained for 20 min with CD3?-FITC
and CD8-PerCP (4 ?l each) (BD Biosciences), washed again, and perme-
abilized with FACSPerm (BD Pharmingen) for 10 min at room temperature
in the dark. Following two further washes, cells were intracellularly stained
with PE-conjugated anti-TNF-? with or without PE-conjugated anti-IFN-?
(4 ?l/well each) (BD Pharmingen), with or without allophycocyanin-la-
beled CD69 (1.5 ?l/well each; BD Pharmingen), incubated for 20 min at
37°C, fixed with 180 ?l of 1% paraformaldehyde (Sigma-Aldrich) in PBS,
and analyzed by four-color flow cytometry (FACSCalibur-Multiwell Plate
Manager; BD Biosciences).
Cryopreserved and thawed PBMCs were maintained overnight in RPMI
1640 with 20% FCS and penicillin/streptomycin in 10 ml of complete R20
medium, washed in R10, and plated in triplicate on a 96-well round-bottom
plate. Mamu-A*01-positive herpes papio-transformed target cells were
stimulated 2 h with 10 ?g of Gag181–189CM9 or Gag QI9 peptides in 1 ml
of complete medium, labeled for 2 h with 100 ?Ci of51Cr, and washed in
Iscove’s medium plus 10% FCS and RPMI 1640 plus 10% FCS with 100
U penicillin/streptomycin, and 5 ? 103cells were added to each well.
Unstimulated target cells were used as a negative control. Cells were in-
cubated for 6 h at E:T ratios of 50:1, 25:1, 12.5:1, and 6.25:1, and the
percentage of released51Cr was calculated by dividing the difference be-
tween the mean cpm of experimental and spontaneous release by the dif-
ference between the mean cpm of total and spontaneous release ? 100.
CD25?T cell detection and depletion
Macaque PBMCs that were depleted or undepleted of CD25?T cells were
stainedwith CD3?-FITC, CD4-PerCP,
CD25-PE (BD Immunocytometry Systems) for 20 min in the dark at
4–8°C and washed with wash buffer (PBS supplemented with 1% FCS).
Following incubation, the cells were washed and resuspended in 200 ?l of
1% paraformaldehyde. Analysis was performed using the FACSCalibur
Macaque PBMCs were prepared according to standard isolation tech-
niques. The cells were passed through a prewetted 30-?m nylon mesh
(catalog no. 130-041-407; Miltenyi Biotec) to remove clumps. Cells were
washed, pelleted, resuspended in 100 ?l of selection buffer (PBS supple-
mented with 0.5% FCS and 2 mM EDTA), and stained with CD25 con-
jugated with r-PE Ab (catalog no. 341009; clone 2A3; BD Biosciences) for
10 min at 4–8°C in the dark. Following incubation, the cells were washed
twice and resuspended in 80 ?l. Twenty microliters per 107cells of anti-PE
microbeads (catalog no. 130-048-801; Miltenyi Biotec) was added and
incubated at 4–8°C for 15 min. The cells were washed, resuspended in 500
?l in selection buffer, and passed through magnetic MS columns for de-
pletion of CD25?T cells. Unlabeled effluent was collected and analyzed.
All differences were tested using repeated-measures ANOVA. Arc-sine or
logarithmic transformation was applied to ICS, tetramer staining, and pro-
liferative responses to normalize data distributions.
IL-2 pharmacokinetics in rhesus macaques
The bioavailability of IL-2 in serum of macaques was assessed
following IL-2 administration at 60,000 Amgen U (4 ?g) or
120,000 Amgen U (8 ?g) daily dose by the s.c. route to two ma-
caques. Plasma was collected at prescribed intervals following in-
oculation. The same animals were reinoculated after a few months
with the same dose of IL-2 to obtain duplicate results. Within 2 h
from s.c. inoculation of 120,000 Amgen U of the cytokine, IL-2
reached plasma peak levels (?50 pM) (Fig. 1A). By 6 h after
injection, the plasma level was near 10 pM, which is equivalent to
the equilibrium dissociation constant (Kd) or EC50of the high-
affinity heterotrimeric IL-2R (Fig. 1A). At the lower dose (60,000
rhesus macaques. A, Animals 291 and 294 were inoculated with a single dose
of IL-2 at 120,000 IU (left panel), whereas macaques 389 and 568 received
IL-2 at a dose of 60,000 IU (right panel). Plasma was collected at the time
(hours) indicated. Two independent inoculations of IL-2 at the same dose in
the same macaques were performed (see Materials and Methods). B, Study
design and animal numbers. ?, Designates Mamu-A*01-positive macaques. C,
Absolute number of CD3?, CD4?, and CD8?T cells in animals vaccinated
with (top panels) or without (bottom panels) IL-2.
Study design and pharmacokinetics of exogenous IL-2 in
1915The Journal of Immunology
Amgen U), the IL-2 plasma level also reached 50 pM within 2 h
from administration and was at or below 10 pM by 6 h (Fig. 1A).
Therefore, the IL-2 dose of 120,000 Amgen U was chosen for
further studies, because, in this case, the serum IL-2 concentration
for at least 6 h was equal to that necessary to half saturate the
high-affinity IL-2R (50).
functionality of Gag181–189CM9 CD8?T
cell response in vaccinated macaques with
and without IL-2. A, Frequency of tet-
ramer-positive cells in the blood of the
Mamu-A*01-positive macaques from
groups immunized with ALVAC-SIV
alone (top panel) or with ALVAC-SIV
plus IL-2 (bottom panel). B, Frequency of
Gag181–189CM9-specific CD8?T cells
producing TNF-? following stimulation
with the Gag peptide over time. C, Fre-
quency of CD8?T cells able to produce
TNF-? following stimulation with the en-
tire Gag peptide pool. The arrows in A–C
indicate the time of immunization. D–G,
Ex vivo cytolytic activity following stim-
ulation with the dominant Gag181–189
CM9 epitope (D and E) or the subdomi-
nant Gag QI9 epitope (66) (F and G).
Kinetics of induction and
1916 IL-2 TREATMENT AND VACCINATION OF SIV-INFECTED MACAQUES
IL-2 induces significant increase of vaccine-induced functional
CD8?T cell response
The effect of continuous low-dose IL-2 administration in combi-
nation with ALVAC-SIV-gpe was assessed in 11 macaques. Ten
of the 11 macaques were vaccinated before the current study with
either a combination of DNA and the NYVAC-SIV-gpe vaccine
candidate or the NYVAC-SIV-gpe vaccine candidate alone, as
summarized in Table I. All of these macaques had been chronically
infected with SIVmac251 before treatment with ART, but at the
time of this study, they had contained viral replication below a
detectable level (?2000 RNA copies/100 ?l input), and main-
tained normal levels (?700 CD4?T cells/mm3) over time (Table
I). Therefore, these animals were comparable to human long-term
nonprogressors and were considered to have been “primed” to SIV
Ags and to have protective immunity to SIV. Nevertheless, to min-
imize the possible contribution of subliminal viral replication to
the immune response induced by vaccination, all animals were
subjected to daily treatment with ART. Six of these macaques
were immunized 4 wk after initiation of ART with ALVAC-
SIV-gpe at 2 ? 108pfu by the i.m. route, and vaccination was
repeated in identical conditions 13 wk later (Fig. 1B). The re-
maining five macaques were immunized with the same regimen
except that IL-2 at 120,000 Amgen U was given daily by the s.c.
route up to wk 22 (Fig. 1B). During this time, CD4?and CD8?
T cell counts were analyzed every 2 wk, and at this IL-2 dose,
treatment did not result in a significant increase of CD4?or
CD8?T cell counts in the blood, which remained within the
normal ranges (Fig. 1C).
Mamu-A*01-positive macaques were included in both groups
(Table I) to quantify the dominant response to Gag181–189CM9 by
tetramer staining. ALVAC-SIV-gpe expanded the frequency of
Gag181–189CM9 tetramer-positive cells in all Mamu-A*01-posi-
tive macaques. Following the first immunization, no significant
difference was observed between the two groups (Fig. 2A). How-
ever, following the second immunization, the change in frequency
of the tetramer response relative to the response after the first im-
munization was significantly higher in the animals treated with
IL-2 (p ? 0.0011), indicating that IL-2 contributed to the expan-
sion and maintenance of this population of cells.
The ability of CD8?T cells with the same specificity to produce
TNF-? was assessed in parallel by ICS following in vitro stimu-
lation with the Gag181–189CM9 peptide. In agreement with the
tetramer data, significantly higher responses were observed in the
IL-2-treated group after the second immunization (p ? 0.041)
(Fig. 2B). Thus, both immunological assays demonstrated that IL-2
increased the frequency of functional immune response to this
dominant Gag181–189CM9 epitope.
We further analyzed the ability of CD8?T cells to produce
TNF-? using the entire Gag peptide array. As in the case of the
Gag181–189CM9 peptide, macaques treated with IL-2 developed
higher vaccine-induced Gag-specific CD8?T cell response fol-
lowing the second immunization (p ? 0.0009) (Fig. 2C), support-
ing the notion that the ability of IL-2 to increase vaccine-induced
CD8?T cell response is not limited to dominant epitopes.
Ex vivo CTL activity specific for the Gag181–189CM9 epitope
was measured in all macaques at wk 5, 13, 15, and 17, and was
found to be comparable in all of the Mamu-A*01-positive ma-
caques from both groups. As expected, no significant CTL activity
was measured in the Mamu-A*01-negative macaques 3067, 3065,
and 25 (Fig. 2, D and E). This result is not unexpected because
CTL activity measurement likely does not accurately reflect the
frequency of the Gag181–189CM9-specific CD8?T cell response.
Ex vivo CTL activity was also assessed against the subdominant
Gag QI9 epitope, and also in this case, no difference in the extent
of this response was observed between the two groups (Fig. 2, F
1917The Journal of Immunology
Vaccination-induced CD4?T cell responses are significantly
decreased by IL-2 treatment
In a previous study, we observed that IL-2 at the same dose used
here was associated with a decrease in the proliferative response to
Gag induced by vaccination with the NYVAC-SIV-gpe vaccine
candidate (40). However, in those experiments, a parallel system-
atic quantitative measurement of the Gag-specific proliferative re-
sponse and cytokine production by CD4?T cells was not per-
formed. Therefore, in this study, we assessed side-by-side the
CD4?T cell response to the entire Gag protein by ICS as well as
by p27 Gag-induced [3H]thymidine incorporation at six indepen-
dent time points in all 11 macaques.
Proliferative responses to Gag were significantly lower follow-
ing both the first and second immunizations in macaques treated
with IL-2 (p ? 0.0017 and p ? 0.0066, respectively, by ANOVA)
(Fig. 3A), thereby confirming and extending our previous
Interestingly, the number of CD4?T cells producing TNF-?
was also significantly lower in the IL-2-treated group following the
entire vaccination regimen (p ? 0.015), suggesting that IL-2 de-
creases the true frequency of vaccine-induced CD4?T cell re-
sponses rather than altering only their ability to produce IL-2 and
to proliferate (Fig. 3B).
IL-2 increases the number of CD4?CD25?T cells
In humans, treatment with a 10-fold higher dose of IL-2 (e.g.,
9–15 mU/day) is associated with an increase in the frequency of
circulating CD4?T cells expressing the IL-2R ?-chain (CD25)
(26, 51). Recently, T-reg cells with the CD4?CD25?phenotype
have been shown to be able to suppress IL-2 production and pro-
liferation by CD4?CD25?-responding T cells, and have been de-
scribed in both mice and humans (52). Therefore, we assessed
whether continuous low-dose IL-2 treatment of macaques in-
creased the frequency of this population in the blood. Flow cy-
tometry analysis of PBMCs from the IL-2-treated and untreated
vaccinated animals revealed an overall increase over time of the
percentage of CD4?CD25?T cells in IL-2-treated macaques (Fig.
4A) up to 6–7% at 6 wk of treatment, compared with ?1% without
IL-2 treatment (A). Surprisingly, this frequency subsequently de-
creased, despite continuous IL-2 treatment, and the decrease was
apparently unrelated to the induction of Abs to IL-2, because the
sera of these animals did not react in Western blot with the rIL-2
protein (data not shown). It is possible that either trafficking to
tissues or down-regulation of the CD25 molecule occurs after pro-
longed treatment with the cytokine. In either case, the level of
CD4?CD25?T cells remained higher in the IL-2-treated group
throughout the study.
The increase in CD4?CD25?T cells raised the possibility that
this population might have a suppressive effect on cytokine pro-
duction and proliferation, especially by CD4?T cells. Because we
observed a decrease in the CD4?T cell response, we sought to
investigate whether the depletion of the CD4?CD25?T cells in
vitro could restore the CD4?T cell response. To investigate this
hypothesis, total or CD25?T cell-depleted PBMCs were stimu-
lated with the entire Gag-overlapping peptide, and the ability of
CD4?and CD8?T cells to produce cytokines was measured in the
presence or absence of CD4?CD25?T cells. Examples of the
assay performed in the animals are presented for two animals in
Fig. 4, B and C, and summarized in a histogram for the other four
animals in D. Although depletion of the CD25?T cell population
was highly effective, it did not consistently result in an increase of
virus-specific CD4?or CD8?T cell response, and overall did not
differ significantly in animals vaccinated in the presence or ab-
sence of IL-2.
In this study, we have demonstrated in macaques able to naturally
contain SIVmac251 replication (long-term nonprogressors) that
low-dose IL-2 given together with a T cell vaccine and maintained
throughout the immunization regimen had opposite effects on vac-
cine-induced Ag-specific CD4?and CD8?T cells. IL-2 signifi-
cantly increased the frequency and function of a dominant CD8?
T cell response, but significantly decreased the frequency of virus-
specific CD4?T cells. These data may be reconciled to the known
biological effects of IL-2. IL-2 induces T cell proliferation but also
limits T cell expansion by down-regulating the ?c-chain and Bcl2
on cycling T cells and by causing apoptosis (53). IL-2 also causes
activation-induced cell death (54) through up-regulation of Fas
(CD95), Fas ligand (CD178), Fas-associating protein with death
domain transcription, and down-regulation of FLICE/FLIP (55–
57). The importance of the negative effect of IL-2 is supported by
the finding that IL-2 and IL-2R knockout mice have lymphoid
hyperplasia and autoimmune syndromes (58–60).
Thus, the effect of IL-2 likely depends on the timing and dose of
administration. In fact, a recent study demonstrated that the effect
of IL-2 varies greatly according to its presence at different times
during the development of an adaptive immune response (61). In
mice that received low doses of IL-2 during the expansion (8
days), contraction (8 to 15 days), or memory (60 days) phase of the
immune response following lymphocytic choriomeningitis virus
infection (61), the timing of IL-2 administration determined the
extent of virus-specific CD4?and CD8?effector memory T cell
responses. During the expansion phase, IL-2 decreased the number
of CD4?T cells and had no significant effect on virus-specific
CD8?T cell response. In contrast, IL-2 administration during the
contraction phase resulted in the persistence of high virus-specific
CD8?and even higher virus-specific CD4?T cell response up to
6 mo following infection. However, by 6 mo, the level of both
CD4?and CD8?memory T cell responses to lymphocytic cho-
riomeningitis virus did not differ in IL-2-treated and untreated an-
imals, suggesting that administration of IL-2 did not increase the
Similarly, in our study in macaques chronically infected with
SIVmac251, IL-2 augmented the expansion of CD8?tetramer-
positive cells after vaccination but did not influence the contraction
to p27 Gag. A, The proliferative response to p27 Gag over time is presented
for each animal in the two left panels. B, Both right panels show the
virus-specific CD8?(CD4?) response measured with the ICS assay for
TNF-? following stimulation with the entire Gag peptide pool.
Kinetics of vaccine-induced virus-specific helper response
1918 IL-2 TREATMENT AND VACCINATION OF SIV-INFECTED MACAQUES
of this response. Indeed, in the long term, the frequency of tetramer-
positive cells did not differ in the animals vaccinated in the presence
or the absence of IL-2. The decrease in CD4?T cells observed here
may have resulted from trafficking of Ag-specific CD4?T cells to
tissues; activation-induced cell death, as IL-2 was maintained at
pharmacological levels during the entire immunization regimen; or
increased frequency in the number of CD4?CD25?T cells with
suppressive activity in tissues but not in blood (62).
IL-2 administration augmented the frequency over time of circu-
lating CD4?CD25?T cells in the macaques studied here, as observed
in HIV-1-infected individuals (63). However, this increase was tran-
sient and completely reversible once the cytokine administration was
frequency of CD4?CD25?T cells.
A, Quantitation of CD4?CD25?T
cells in blood of four animals before
treatment with IL-2 and vaccine
(?1) and after. B and C, Raw FACS
analysis data from two animals in
the study. Data on two animals are
presented. Animal 22 was vacci-
nated and treated with IL-2 (B),
whereas animal 3076 received vac-
cine only (C). ?2 is 2 wk before
treatment, and 4 and 7 wk are after
vaccination with or without IL-2. In
B and C, the top two panels demon-
strate the efficiency of depletion of
the CD4?and CD8?T cell popula-
tions, whereas the two bottom panels
demonstrate the change in the ability
of Gag-specific CD4?CD69?
CD8?CD69?T cells to produce
TNF-? in the presence (nonde-
pleted) or absence (depleted) of
CD4?CD25?T cells. Data for all
animals tested using the same ap-
proach are summarized in C.
IL-2 augments the
1919 The Journal of Immunology
discontinued. Initially, this finding appeared to provide a possible ex-
and human CD4?T cells that also express CD25 exert suppressive
activity in lymphocyte proliferative assays (42, 64). However, when
tested directly, the removal of the CD4?CD25?T cells in vitro did
not result consistently in restoration of higher frequency CD4?T cell
Thus, the mechanism underlying the IL-2-associated decreased SIV-
specific response remains unclear.
Importantly, however, our data complement findings of others in
IL-2-treated HIV-1-infected individuals on ART whereby a de-
crease in HIV-1-specific CD4?effector responses was also ob-
served together with an increase of CD4?CD25?T cells (63).
Decreasing the frequency of virus-specific CD4?T cells while
maintaining functional CD8?T cells may not necessarily be an
undesirable result in HIV-1 infection because Ag-specific CD4?T
cells are preferentially infected by the virus (65). Hopefully, an-
swers to this question may stem from ongoing human trials
whereby IL-2 has been associated with vaccination.
We thank Jim Tartaglia of Aventis Pasteur for providing ALVAC and
Steven Snodgrass for editorial assistance.
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