Dysfunction of Simian Immunodeficiency Virus/Simian Human
Immunodeficiency Virus-Induced IL-2 Expression by Central
Memory CD4?T Lymphocytes1
Yue Sun,* Jo ¨rn E. Schmitz,* Paula M. Acierno,* Sampa Santra,* Ramu A. Subbramanian,*
Dan H. Barouch,* Darci A. Gorgone,* Michelle A. Lifton,* Kristin R. Beaudry,*
Kelledy Manson,†Valerie Philippon,†Ling Xu,‡Holden T. Maecker,§John R. Mascola,‡
Dennis Panicali,†Gary J. Nabel,‡and Norman L. Letvin2*‡
Production of IL-2 and IFN-? by CD4?T lymphocytes is important for the maintenance of a functional immune system in infected
individuals. In the present study, we assessed the cytokine production profiles of functionally distinct subsets of CD4?T lym-
phocytes in rhesus monkeys infected with pathogenic or attenuated SIV/simian human immunodeficiency virus (SHIV) isolates,
and these responses were compared with those in vaccinated monkeys that were protected from immunodeficiency following
pathogenic SHIV challenge. We observed that preserved central memory CD4?T lymphocyte production of SIV/SHIV-induced
IL-2 was associated with disease protection following primate lentivirus infection. Persisting clinical protection in vaccinated and
challenged monkeys is thus correlated with a preserved capacity of the peripheral blood central memory CD4?T cells to express
this important immunomodulatory cytokine. The Journal of Immunology, 2005, 174: 4753–4760.
sponses and by maintaining effective CTL (1, 2). Recent studies
suggest that functional CD4?T lymphocytes are also required at
the time of immune priming for the development of memory
CD8?T lymphocytes (3–5).
CD4?T lymphocytes of HIV-infected individuals display func-
tional defects, including reduced proliferative responses to both
Ags and mitogens (6). These functional CD4?T lymphocyte de-
fects are associated with reduced production of IL-2, which can be
partially corrected in vitro by the addition of exogenous IL-2 (7,
8). The integrity of CD4?T lymphocytes that secrete IL-2, or a
subset of CD4?T lymphocytes that secrete both IFN-? and IL-2,
is associated with good clinical outcome in HIV-infected individ-
uals (9–11). Thus, production of IL-2 by CD4?T lymphocytes is
important for the maintenance of a functional immune system in
CD4?T lymphocytes have been divided into central memory
and effector memory cell subsets based on their homing capacity
and effector function (12, 13). Central memory T lymphocytes
irus-specific CD4?T lymphocytes play a central role in
the immune containment of HIV. They contribute to
HIV clearance both by providing help for B cell re-
home to lymphoid organs, have little or no effector function, pro-
duce predominantly IL-2, and have a high capacity to proliferate.
In contrast, effector memory T lymphocytes migrate to peripheral
tissues, display effector function, produce primarily IFN-?, and
lack significant proliferative capacity. Precisely how the defective
cellular production of cytokines is associated with the develop-
ment of memory CD4?T lymphocytes in HIV-infected individu-
als remains to be elucidated.
In the present study, the cytokine production of CD4?T lym-
phocyte subsets was analyzed in rhesus monkeys infected with
pathogenic viruses and attenuated viruses, and in vaccinated mon-
keys that were protected from developing disease following patho-
genic simian human immunodeficiency virus (SHIV)3challenge.
The results of this study suggest that the ability of SIV/SHIV-
stimulated central memory CD4?T lymphocytes to synthesize
IL-2 is an immune correlate of disease protection following pri-
mate immunodeficiency virus infection.
Materials and Methods
Animals and viruses
Heparinized blood samples were obtained from rhesus monkeys (Macaca
mulatta). All animals were maintained in accordance with the guidelines of
the Committee on Animals for the Harvard Medical School and the Guide
for the Care and Use of Laboratory Animals (14). Viruses used in this study
were the nonpathogenic SHIV-89.6, the pathogenic SHIV-89.6P, the
pathogenic SIVmac251, the attenuated J5 strain of SIVmac251, and the
CD4?T lymphocyte counts and plasma viral RNA levels
Peripheral blood CD4?T lymphocyte counts were calculated by multiply-
ing the total lymphocyte count by the percentage of CD3?CD4?T cells
determined by mAb staining and flow cytometric analysis. Plasma viral
RNA levels were measured by an ultrasensitive branched DNA amplifica-
tion assay with a detection limit of 125 copies per ml (Bayer Diagnostics).
*Division of Viral Pathogenesis, Department of Medicine, Beth Israel Deaconess
Medical Center, Harvard Medical School, Boston, MA 02115;†Therion Biologics,
Cambridge, MA 02142;‡Vaccine Research Center, National Institute of Allergy and
Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; and§BD
Biosciences, San Jose, CA 95131
Received for publication September 7, 2004. Accepted for publication February
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by National Institutes of Health Grants AI20729, AI30033,
and AI48394, and by Dana-Farber Cancer Institute/Beth Israel Deaconess Medical
Center/Children’s Hospital Center for AIDS Research Grant AI28691.
2Address correspondence and reprint requests to Dr. Norman L. Letvin, Division of
Viral Pathogenesis, Department of Medicine, Beth Israel Deaconess Medical Center,
Harvard Medical School, Research East Room 113, 330 Brookline Avenue, Boston,
MA 02215. E-mail address: email@example.com
3Abbreviations used in this paper: SHIV, simian human immunodeficiency virus;
rAd5, recombinant adenovirus serotype 5; MVA, modified vaccinia virus Ankara;
SEB, staphylococcal enterotoxin B.
The Journal of Immunology
Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00
first group of monkeys (vaccinated/SHIV-89.6P challenged) was immunized
three times by the i.m. route with 10 mg of plasmid DNA containing
SIVmac239 Gag/Pol/Nef, with or without HIV-1 89.6P Env. Following an
of recombinant adenovirus serotype 5 (rAd5) expressing the same SIV/HIV-1
genes by the i.m. route. Animals were challenged at wk 38 with 100 50%
monkey infectious doses (MID50) of SHIV-89.6P by the i.v. route. The
second group of monkeys (vaccinated only) was immunized two to four
times by the i.m. route with plasmid DNA containing gag, pol, nef, and
env genes encoding for CTL epitopes (15), and boosted by i.m. injection
with recombinant modified vaccinia virus Ankara (MVA), vaccinia
virus, or rAd5 constructs. The third group of monkeys (vaccinated only)
either was immunized three times by i.m. route with 10 mg of plasmid
DNA containing SIVmac239 Gag/Pol/Nef and HIV-1 89.6P Env, or
received no plasmid DNA immunization. Following an 18-wk rest, all
DNA-primed monkeys, as well as a group of naive monkeys, received
2 ? 1012PFU of rAd5 expressing the same SIV/HIV-1 genes. Monkeys
received a second inoculation of rAd5 constructs 133 wk later.
The Abs used in this study were directly coupled to FITC, PE, PE-Texas
Red (ECD), allophycocyanin, PerCP-Cy5.5, Alexa Fluor 700, or PE-Cy7.
Table I. Vaccination/challenge history of study animals
90–98, 95–98, 135–97, 128–97,
Plasmid DNAs expressing
genes encoding gag, pol, nef, and
env CTL epitopes
Pox virus or rAd5 expressing
SIVmac239 Gag/Pol, HIV-1 89.6P
Plasmid DNA i.m.
MVA, vaccinia, or rAd5 i.m.
AW2P, AW13, AW28, AV83
Plasmid DNAs and/or rAd5
SIVmac239 Gag/Pol/Nef, HIV-1
Plasmid DNA i.m.—wk 0, 4, 8
rAd5 i.m.—wk 26, 159No
PKB, AV8T, AK67, AK85,
AW2V, PWE, AV2E, AV67,
Plasmid DNAs and rAd5 expressing
SIVmac239 Gag/Pol/Nef, HIV-1
Plasmid DNA i.m.—wk 0, 4, 8
rAd5 i.m.—wk 26
SHIV-89.6P i.v.—wk 38
JBA, TKF, AV65, TCJ, AK6X,
Plasmid DNAs and rAd5 expressing
SIVmac239 Gag/Pol/Nef Plasmid DNA i.m.—wk 0, 4, 8
rAd5 i.m.—wk 26
SHIV-89.6P i.v.—wk 38
Table II. Clinical data on rhesus monkeys infected with pathogenic or attenuated viruses
Plasma Viral RNA
Monkeys with no detected
plasma viral RNAa
Monkeys with detected
plasma viral RNAb
aReferred to in the figures as nonpathogenic virus.
bReferred to in the figures as pathogenic virus.
4754DETERMINANTS OF DISEASE PROGRESSION IN SIV/SHIV INFECTION
The following mAbs were used: anti-CD28-FITC (28.2; BD Biosciences),
anti-Ki67-FITC (B56; BD Biosciences), anti-CD28-PE (28.2; BD Bio-
sciences), anti-CD95-PE (DX2; BD Biosciences), anti-CCR5-PE (3A9;
BD Biosciences), anti-CCR7-PE (150503; R&D System), anti-CD11a-PE
(HI111; BD Biosciences), anti-CD45RA-PE (2H4; Beckman Coulter), anti-
CD62L-PE (SK11; BD Biosciences), anti-CD25-PE (M-A215; BD Bio-
sciences), anti-Bcl-2-PE (Bcl-2/100; BD Biosciences), anti-Granzyme B-PE
(CLB-GB11; PeliCluster), anti-CD4-ECD (19thy5d7; Beckman Coulter), anti-
CD8?-ECD (7PT; Beckman Coulter), anti-CD95-allophycocyanin (DX2; BD
Biosciences), anti-IFN-?-allophycocyanin (B27; BD Biosciences), anti-TNF-
?-allophycocyanin (MAb11; BD Biosciences), anti-IL-2-allophycocyanin
(MQ1-17H12; BD Biosciences), anti-CD4-PerCP-Cy5.5 (L200; BD Bio-sci-
ences), anti-CD3-Alexa Fluor 700 (SP34-2; BD Biosciences), and anti-IFN-
?-PE-Cy7 (B27; BD Biosciences).
PBMC stimulation and intracellular cytokine staining
PBMC were separated from whole blood by Ficoll density gradient cen-
trifugation. PBMC (106) were incubated at 37°C in a 5% CO2environment
for 6 h in the presence of RPMI 1640/10% FCS medium alone (unstimu-
lated), a pool of 15-mer Gag peptides (5 ?g/ml each peptide), or staphy-
lococcal enterotoxin B (SEB) (5 ?g/ml, Sigma-Aldrich) as a positive con-
trol. All cultures contained Monensin (GolgiStop; BD Biosciences) as well
as 1 ?g/ml anti-CD49d (BD Biosciences). The cultured cells were stained
with mAbs specific for cell surface molecules before fixation. The PBMC
were washed twice with PBS-2% FCS and then fixed and permeabilized
with Cytofix/Cytoperm solution (BD Biosciences) in accordance with the
manufacturer’s protocol. Cells were washed twice with 1? Perm/Wash
buffer (BD Biosciences) and then stained with anti-cytokine mAb. Anti-
cytokine mAb titers for optimal staining were determined in preliminary
experiments. Cells were washed twice with 1? Perm/Wash buffer and then
fixed in 1.5% formaldehyde-PBS. The following mAb combinations were
used: CD28-FITC/CD95-PE/CD4-ECD/cytokine-allophycocyanin (IFN-?,
IL-2, and TNF-?) and CD28-FITC/CD95-PE/CD8?-ECD/IL-2-allophyco-
cyanin/CD4-PerCP-Cy5.5/CD3-Alexa700/IFN-?-PE-Cy7. Samples were
collected either on a FACSCalibur instrument (BD Biosciences) and ana-
lyzed with CellQuest software (BD Biosciences) or on a LSR II instru-
ment (BD Biosciences) and analyzed using FlowJo software (Tree
Star). Approximately 100,000 to 300,000 events in the lymphocyte gate
were acquired. The background level of cytokine staining varied from
sample to sample, but was typically ?0.04% of the CD4?T lympho-
cytes. The only samples considered positive were those in which the
percentage of cytokine-staining cells was at least twice that of the back-
ground or in which there was a distinct population of cytokine brightly
A comparison of values was first performed using the Kruskal-Wallis test.
If any results were statistically significant (p ? 0.05), the data pairs were
compared by the Mann-Whitney U test. The Holm’s method was used to
account for multiple comparisons, and the p values were adjusted accord-
ingly; only significant values (p ? 0.05) after applying the Holm’s method
were indicated in the figures. A Spearman correlation test was performed
to analyze the association between the cytokine responses and plasma viral
RNA level. A value of p ? 0.05 was considered significant and was high-
lighted in the figures with an asterisk (?), and p values that approached 0.05
were also shown.
IL-2, IFN-?, and TNF-? production by CD4?T lymphocytes in
naive, vaccinated, and SIV/SHIV-infected rhesus monkeys
To determine the functional capacity of CD4?T lymphocytes
from naive, vaccinated, and SIV/SHIV-infected rhesus monkeys,
the expression of selected cytokines by SEB-stimulated lympho-
cytes was first characterized. One of the cohorts of vaccinated
animals that was evaluated received plasmid DNA prime and re-
combinant poxvirus boost immunizations 6 mo before evaluation
lymphocytes of normal, vaccinated, and SIV/SHIV-in-
fected rhesus monkeys. The evaluated animals included
healthy, uninfected monkeys; monkeys primed with
plasmid DNA and boosted with a recombinant poxvirus
vaccine or recombinant adenovirus vaccine; monkeys
infected with attenuated SIV/SHIV; and monkeys in-
fected with pathogenic SIV isolates. CD4?T lympho-
cyte responses to SEB (A) or pooled Gag peptides (B)
were measured. IFN-?, IL-2, and TNF-? expression are
shown as percent positive CD4?T lymphocytes. Values
for each monkey are depicted as separate points, and the
bars represent the median value for each group. Nonre-
sponders (zero values) are shown on the base of each log
plot at the frequency of 0.01. The Kruskal-Wallis test
was used to evaluate significant differences between
groups (bolded p values), and the Mann-Whitney U test
was used to investigate the statistical significance of the
individual data pairs. To account for multiple compari-
sons, the p values were adjusted by the Holm’s method.
Statistically significant differences using this test are
highlighted in the figure by an asterisk (?).
Cytokine expression profiles of CD4?T
IL-2?only, and IFN-??IL-2?double-positive cells following Gag stimu-
lation in PBMC of monkeys infected with attenuated SIV/SHIV and mon-
keys infected with pathogenic SIV isolates. Nonresponders (zero values)
are shown on the base of each log plot at the frequency of 0.01. Significant
differences are shown as determined by the Mann-Whitney U test.
Comparison of CD4?T cell frequencies of IFN-??only,
4755The Journal of Immunology
(Table I). The other cohort of vaccinated monkeys that was studied
was given plasmid DNA prime and recombinant adenovirus boost
immunizations 5 wk before analysis (Table I). Because the mon-
keys vaccinated by either of the regimens developed comparable
immune responses, the data from both cohorts were combined for
purpose of analysis. Monkeys infected with nonpathogenic SHIV-
89.6, attenuated SIVmac251(J5), or highly pathogenic SIVmac251
and SIVsmE660 isolates were characterized to determine plasma
viral RNA levels and CD4?T lymphocyte counts (Table II).
No statistically significant differences were noted in the frequen-
cies of IFN-?-, IL-2-, and TNF-?-producing CD4?T lymphocytes
in response to SEB from normal, vaccinated, and infected mon-
keys (Kruskal-Wallis test) (Fig. 1A). However, a trend toward
lower frequencies of IFN-?- and IL-2-producing CD4?lympho-
cytes was seen in monkeys infected with the pathogenic SIV iso-
lates. We also noticed markedly decreased in vitro viability of
PBMC from monkeys with advanced disease following stimula-
tion with SEB, with the remaining CD4?T cells having no cyto-
kine-producing capacity after stimulation (data not shown). Of
note, monkeys infected with pathogenic SIV isolates had signifi-
cantly lower frequencies of Gag-specific IL-2-producing CD4?T
cells than those infected with nonpathogenic virus (p ? 0.027,
Holm’s test) (Fig. 1B). Thus, the capacity of Gag-specific CD4?T
lymphocytes to express IL-2 was associated with the clinical status
of the infected monkeys.
Gag-specific IFN-??alone, IL-2?alone, and
IFN-??IL-2?CD4?T cell frequencies in monkeys infected
with attenuated SIV/SHIV or pathogenic SIV isolates
Recent studies have shown that the maintenance of a subset of
HIV-specific CD4?T cells that produce both IFN-? and IL-2 is
associated with good clinical outcome in HIV-infected individuals
(11). We therefore sought to determine whether the loss of IL-2-
producing cells in the monkeys infected with pathogenic virus re-
flected a loss of cells producing IL-2 alone or cells producing IL-2
and IFN-?. To this end, we quantitated Gag-specific CD4?T cells
that were IFN-??alone, IL-2?alone, and IFN-??IL-2?in PBMC
of monkeys infected with attenuated SIV/SHIV and of monkeys
infected with pathogenic SIV isolates (Fig. 2). PBMC were stained
simultaneously for both IFN-? and IL-2, and the frequency of
CD4?T cells that produced IFN-? or IL-2, as well as cells that
produced both IFN-? and IL-2 was measured in a seven-color flow
cytometric assay. The percentage of Gag-specific CD4?T cells
producing IFN-? alone was similar in PBMC of both groups of
monkeys. However, the median frequency of Gag-specific IFN-
??IL-2?CD4?T cell responses was significantly higher in mon-
keys infected with the attenuated SIV/SHIV than in monkeys in-
fected with pathogenic SIV isolates (p ? 0.034). Moreover,
although very few Gag-specific CD4?T cells produced IL-2 alone
in the evaluated monkeys, a trend toward lower frequencies of
these cells was seen in monkeys infected with pathogenic SIV
with maturation and function on CD4?T lymphocytes
subsets defined by CD28 and CD95. Cell surface ex-
pression of molecules associated with maturation and
function on CD28?CD95?(naive), CD28?CD95?
(central memory), and CD28?CD95?(effector mem-
ory) CD4?T cell subsets was evaluated. Lymphocytes
from five healthy, uninfected monkeys were studied.
The symbols ??, ?, and ? reflect the relative staining
intensities of the anti-CD28 mAb.
The expression of molecules associated
memory subsets of CD4?T lymphocytes from normal, vaccinated, and
SIV/SHIV-infected rhesus monkeys. A, The expression of CD28 and CD95
on peripheral blood CD4?T lymphocytes of healthy uninfected monkeys
(n ? 5), monkeys primed with plasmid DNA and boosted with a recom-
binant poxvirus vaccine (n ? 6), monkeys infected with attenuated SIV/
SHIV (n ? 5), and monkeys infected with pathogenic SIV isolates (n ?
10). B, Comparison of the expression of molecules associated with matu-
ration and function on CD4?T lymphocyte subsets defined by CD28 and
CD95 expression in each of these four groups of monkeys. The interquar-
tile range is indicated for each bar.
Maturation and function-associated molecules on naive and
4756 DETERMINANTS OF DISEASE PROGRESSION IN SIV/SHIV INFECTION
isolates than in monkeys infected with attenuated virus (p ?
0.054). These results suggest that the presence of Ag-specific
CD4?T cells able to produce IL-2 is associated with effective
immune control of virus.
Cell surface characterization of CD4?T lymphocytes:
maturation and functional markers on lymphocyte subsets
defined on the basis of CD28 and CD95 expression
The expression of CD28 and CD95 has been used to define distinct
subsets of CD4?T lymphocytes (16). Naive CD4?T cells can be
identified by their intermediate expression of CD28 and lack of
CD95 expression. Memory CD4?T cells acquire CD95 expres-
sion and can be divided into central memory and effector memory
subsets based on CD28 expression.
To examine the expression of other putative naive and memory
cell-associated surface molecules on rhesus monkey CD4?T lym-
phocytes, we performed phenotypic and functional analyses of
PBMC defined by CD28 and CD95 expression in five normal an-
imals. Naive CD4?T cells (CD28?CD95?) were homogenous
small lymphocytes that expressed moderate-to-high levels of lym-
phoid tissue-homing molecules (CCR7?, CD62L?), low levels of
adhesion molecules (CD11alow), and lacked expression of CCR5,
a molecule associated with homing to effector sites (Fig. 3). These
cells showed minimal proliferation (Ki67?, Bcl-2high) and no ef-
fector function (no cytokine production capacity, Granzyme B?).
In contrast, effector memory CD4?T cells (CD28?CD95?) ex-
pressed cell surface molecules that facilitate homing to effector
sites (CCR7?, CD62L?, CD11ahigh, CCR5?/?). They had pheno-
typic characteristics of effector cells (Granzyme B?, and were ca-
pable of producing IFN-? and TNF-?). Central memory CD4?T
cells (CD28?CD95?) had a different phenotypic profile. Two-
thirds of the central memory CD4?T cells were CCR7?. The
central memory CD4?T cells had the capacity to produce IL-2 and
proliferate, but lacked cytotoxic effector function (Granzyme B?).
Some naive and central memory CD4?T cells were CD25?and
may therefore have regulatory function. These observations con-
firmed that the rhesus monkey CD4?T lymphocyte subsets de-
fined by CD28 and CD95 expression exhibited physiologic char-
acteristics of naive, central memory, and effector memory CD4?T
cells as previously described (12, 16).
Maturation and function of CD4?T lymphocytes in naive,
vaccinated, and SIV/SHIV-infected rhesus monkeys
Having demonstrated a loss of IL-2 production by Gag-specific
CD4?T cells in monkeys with progressive disease (Fig. 1B), we
sought to determine whether this reflected changes in the relative
representation of naive and memory CD4?T cells in the peripheral
blood of these animals or resulted from the loss of cytokine pro-
duction capacity by a particular subset of CD4?T cells. We there-
fore evaluated naive and memory CD4?T cells defined by CD28
and CD95 expression in the naive, vaccinated, and infected cohorts
monkey CD4?T lymphocyte subsets. Intracellular cy-
tokine expression was measured in SEB-stimulated (A)
or Gag pooled peptide-stimulated (B) PBMC from
healthy uninfected monkeys, monkeys primed with plas-
mid DNA and boosted with a recombinant poxvirus vac-
cine or a recombinant adenovirus vaccine, and monkeys
infected with pathogenic SIV isolates. IFN-?, IL-2, and
TNF-? expression are shown as percent positive
CD28?CD95?effector memory (EM) CD4?T lympho-
cytes. Values for each monkey are depicted as separate
points, and the bars represent the median value for each
group. Nonresponders (zero values) are shown on the
base of each log plot at the frequency of 0.01. Significant
differences are shown as determined by the Mann-Whit-
ney U test. To account for multiple comparisons, the p
values were adjusted by the Holm’s method.
Cytokine expression profiles of rhesus
blood and peripheral lymph node of a SHIV-89.6P-infected rhesus mon-
key. CD4?T cells from PBMC and from a peripheral lymph node (LN)
were examined for their expression of CD28 and CD95 (percent positive
noted in each dot plot for the designated cell subsets). Cytokine expression
profiles of rhesus monkey CD4?T lymphocyte from PBMC and LN were
compared. A study of cells from the representative monkey 196-88 infected
with pathogenic SHIV-89.6P is shown.
Naive and memory CD4?T lymphocytes are present in
4757The Journal of Immunology
of monkeys (Fig. 4A). Although the numbers of CD4?T cells de-
creased in the monkeys infected with pathogenic viruses, no signifi-
cant difference in the percentage of total CD4?T cells with a naive
(CD28?CD95?) (p ? 0.264), central memory (CD28?CD95?)
(p ? 0.296), or effector memory (CD28?CD95?) (p ? 0.117) phe-
notype was noted among the various groups of monkeys. A more
detailed phenotypic analysis showed that monkeys infected with
pathogenic virus had a relatively higher turnover of naive and mem-
ory CD4?T cells (increased expression of Ki67, p ? 0.06; decreased
expression of Bcl-2, p ? 0.06), and a significantly lower percentage
of CCR5?CD4?T cells compared with the other groups (p ? 0.005)
(Fig. 4B). Taken together, these data suggest that the loss of IL-2
production by Gag-specific CD4?T cells in monkeys with progres-
subsets. However, as expected, there was a decrease in cells that
might serve as targets for SIV infection (CD4?CCR5?).
Cytokine expression by CD4?T cell subsets in naive,
vaccinated, and pathogenic SIV-infected monkeys
To evaluate further the functional activity of CD4?T cell subsets
in normal, vaccinated, and SIV-infected monkeys, cytokine pro-
duction by central memory and effector memory cells was ana-
lyzed (Fig. 5). In normal monkeys, SEB-induced IFN-? production
by CD4?T lymphocytes was found predominantly in the effector
memory cell population (median, 19.2 vs 4.97%). Conversely,
IL-2 production by SEB-stimulated CD4?T lymphocytes origi-
nated predominantly from central memory cells (median, 8.98 vs
2.77%). TNF-? expression was comparable in SEB-stimulated
central memory and effector memory CD4?T lymphocytes (me-
dian, 15.34 vs 20.47%). No significant differences in SEB-induced
cytokine expression patterns were observed between the CD4?T
lymphocyte populations of vaccinated and normal monkeys. How-
ever, there was a significant decrease of IL-2 expression by central
memory CD4?T cells of monkeys infected with pathogenic virus
(Fig. 5A). A trend toward a statistical difference between the nor-
mal monkeys and the monkeys infected with pathogenic virus in
the SEB-stimulated IL-2-producing CD4?T cells was also seen.
In contrast, Ag stimulation by a Gag peptide pool induced very
different cytokine expression patterns in subpopulations of CD4?
T lymphocytes of vaccinated or SIV-infected monkeys (Fig. 5B).
There were low but measurable levels of IFN-?, IL-2, or TNF-?
expression by Gag-specific CD28?CD95?effector memory cells.
Cytokine production by Gag-specific CD4?T lymphocytes was
found predominantly in cells with a central memory phenotype
(CD28?CD95?). A selective decrease in IL-2 expression was seen
in central memory CD4?T cells of monkeys with progressive
clinical disease, sometimes in the absence of changes in the ex-
pression of IFN-? or TNF-?. These data suggest that loss of IL-2
production by Gag-specific CD4?T cells in monkeys with pro-
gressive disease does not reflect changes in the relative represen-
tation of naive and memory CD4?T cells in the peripheral blood
of these animals. Rather, it results from the loss of IL-2 production
capacity by central memory CD4?T cells.
Naive and memory CD4?T lymphocytes are present in blood
and peripheral lymph nodes of infected rhesus monkeys
Because it has been shown that central memory CD4?T cells can
preferentially be found in lymphoid tissues, we sought to deter-
mine whether the frequency of central memory CD4?T cells was
high enough in the peripheral blood to reflect the biology of this
cell subpopulation. We therefore compared the relative frequencies
of naive and memory phenotype CD4?T cells in the peripheral
blood and the secondary lymphoid tissues in infected monkeys.
Data from a representative monkey with a normal CD4?T cell
count and undetectable viral load are shown in Fig. 6. Central
memory CD4?T cell frequencies in peripheral blood tended to be
a little lower than frequencies in the lymph node, and as expected,
effector memory CD4?T cells were absent from the lymph node.
More importantly, Gag-specific cytokine-producing CD4?T cells
Table III. Clinical data on vaccinated/SHIV-89.6P-infected rhesus
Plasma Viral RNA
aAll monkeys were infected for 15 mo.
disease is associated with preserved central memory CD4?T lymphocyte
functional capacity. Rhesus monkeys were vaccinated with immunogens
expressing Gag/Pol/Nef with or without Env. Gag-specific IL-2 expression
was evaluated in CD4?T lymphocytes of previously vaccinated rhesus
monkeys 15 mo after SHIV-89.6P challenge. A comparison of IL-2-pro-
ducing CD28?CD95?central memory CD4?T cells (A) and IL-2-produc-
ing CD28?CD95?effector memory CD4?T cells (B) from vaccinated/
challenged monkeys and unvaccinated monkeys infected with pathogenic
virus demonstrated that, similar to monkeys infected with attenuated SIV/
SHIV, central memory CD4?T cells of the vaccinated/SHIV-89.6P-chal-
lenged monkeys have a preserved capacity to produce IL-2. Significant
differences are shown as determined by the Mann-Whitney U test. The p
values were adjusted by the Holm’s method.
Vaccine protection against SHIV-89.6P-induced clinical
4758DETERMINANTS OF DISEASE PROGRESSION IN SIV/SHIV INFECTION
were present at twice the frequency in the peripheral blood than in
the lymph node. Thus, central memory CD4?T lymphocytes in
the peripheral blood should reflect those seen in lymph nodes.
Vaccine protection against SHIV-induced clinical disease is
associated with preserved central memory CD4?T lymphocyte
We next assessed cytokine expression profiles of Gag-specific
CD4?T cells from a cohort of monkeys that was vaccinated and
subsequently challenged with SHIV-89.6P (Tables I and III). In
this cohort, monkeys were vaccinated first with plasmid DNA and
then with recombinant adenovirus constructs expressing SIV Gag/
Pol/Nef with or without HIV-1 Env. Following challenge with
SHIV-89.6P, all vaccinated monkeys maintained low plasma viral
RNA levels and normal peripheral blood CD4?T lymphocyte
counts. Three monkeys that received Gag/Pol/Nef but no Env-
containing vaccines had relatively high plasma viral RNA levels
and reduced CD4?T lymphocyte counts. Gag peptide-stimulated
CD4?T lymphocyte cytokine expression profiles were assessed in
PBMC of these monkeys 15 mo postchallenge.
Similar to monkeys infected with attenuated SIV/SHIV, IL-2
production by central memory CD4?T cells of the vaccinated/
challenged monkeys was better preserved than IL-2 production by
central memory CD4?T cells in monkeys infected with patho-
genic virus in the absence of prior vaccination (p ? 0.014, Holm’s
test) (Fig. 7A). The only vaccinated and then challenged monkeys
that did not demonstrate preserved SIV Gag-stimulated IL-2 pro-
duction by central memory CD4?T lymphocytes were those with
high plasma viral RNA levels. These results demonstrate that vac-
cine protection against SHIV-induced clinical disease is associated
with preserved central memory CD4?T lymphocyte function.
Correlation between the frequency of IL-2-producing central
memory CD4?T cells and plasma viral RNA levels
The observed association between high viral loads and loss of
Gag-specific IL-2-producing central memory CD4?T cells in this
group of vaccinated/challenged monkeys suggested that such an
association be evaluated in a larger cohort of animals. Therefore,
the frequencies of Gag-specific IL-2-producing central memory
CD4?T cells were assessed for an association with plasma viral
RNA levels for all evaluated monkeys, including monkeys infected
with attenuated SIV/SHIV, monkeys infected with pathogenic vi-
rus, and monkeys vaccinated and challenged with SHIV-89.6P. A
significant inverse correlation was observed between these param-
eters (r ? ?0.70, p ? 0.0001) (Fig. 8). However, no significant
correlation was noted between IFN-? or TNF-? production by cen-
tral memory CD4?T cells and the plasma viral RNA levels in
these animals (data not shown). Therefore, the maintenance of IL-
2-producing central memory CD4?T cells is ultimately associated
with virologic control in the monkeys.
Although IFN-? production assays are commonly used to measure
T lymphocyte immune responses to HIV Ags, these analyses do
not reflect the complete profile of the functional capacity of HIV-
specific T cells. In fact, considerable recent work suggests that the
production of other cytokines by CD4?T lymphocytes plays an
important functional role in disease pathogenesis. IL-2 is essential
to the expansion and survival of T lymphocytes, and accruing data
suggest that IL-2-secreting Ag-specific CD4?T lymphocytes rep-
resent a key component of an effective immune response (7, 11,
17). In the present study, CD4?T lymphocytes of infected mon-
keys with uncontrolled viremia and reduced CD4?T lymphocyte
counts preserved their capacity to produce IFN-? and TNF-?,
whereas their ability to synthesize IL-2 was impaired. Therefore,
the preservation of Gag-specific IL-2-producing CD4?T lympho-
cytes is associated with effective immune control of virus. IL-2
production by CD4?T lymphocytes may be lost during an early
stage of disease in these monkeys, with a loss of IFN-? and TNF-?
production as disease progresses. This possibility is consistent with
the recent finding that lymphocytic choriomeningitis virus-specific
T cells lose their ability to produce IL-2 before their ability to
produce TNF-? and IFN-? as lymphocytic choriomeningitis virus-
induced disease progresses in mice (18).
A number of studies have shown that Ag-specific CD8?T cells
can be separated into three functional subsets: early-, intermedi-
ate-, and late-differentiated phenotypes. HIV-specific CD8?T
lymphocytes can exist in an intermediate differentiated state in the
peripheral blood of infected individuals (19, 20). A similar matu-
ration phenotype has also been observed for HIV-specific CD4?T
lymphocytes in the peripheral blood of these individuals (21, 22).
The phenotypic profiles of the SIV Gag-specific CD4?T cells of
the infected monkeys evaluated in the present study are consistent
with this intermediate differentiated state. Cytokine production by
Gag-specific CD4?T lymphocytes was found predominantly in
cells with a central memory phenotype (CD28?CD95?). The rea-
son for this atypical state of T cell maturation is likely multifac-
torial. First, because compartmentalization of T lymphocyte sub-
populations may occur, the population of cells identified in the
peripheral blood of infected individuals might reflect changes in T
cell trafficking and are not necessarily representative of the entire
virus-specific T cell population. Second, intermediate T cell dif-
ferentiation may reflect the normal immune response to an immu-
nodeficiency virus during chronic infection. HIV-specific CD4?T
cells that have a central memory/effector memory phenotype may
be targeted to lymph nodes where HIV replication is ongoing. A
third possible explanation for this atypical T cell maturation is that
replicating HIV may inhibit or redirect T cell differentiation and, in
so doing, affect the clinical course of disease progression. Con-
flicting data have been reported bearing on this last possibility.
Some investigators have reported that, although HIV-specific
CD4?T cells are less mature than CMV-specific CD4?T cells,
ing CD28?CD95?central memory CD4?T cells and plasma viral RNA
levels. Data on all evaluated animals (monkeys infected with attenuated
SIV/SHIV, monkeys infected with pathogenic virus, and monkeys vacci-
nated and challenged with SHIV-89.6P) are indicated by individual filled
diamonds (?). The relationship between the percentage of IL-2-producing
central memory CD4?T cells and plasma viral RNA levels was evaluated
using the Spearman correlation test (r ? ?0.7; R2? 0.49; p ? 0.0001).
Inverse correlation between the frequencies of IL-2-produc-
4759The Journal of Immunology
there is no correlation between HIV-specific T cell maturation and
disease progression (22). Other investigators have reported that
HIV-1 replication skews Gag-specific CD4?T cells away from an
IL-2-producing central memory phenotype and toward a poorly
proliferating effector memory phenotype, and this change may
limit the effectiveness of the HIV-specific immune response (23).
These conflicting results may reflect the different approaches
used by these investigators to define naive, central memory, and
effector memory T lymphocyte subsets. CD4?T lymphocytes
have been divided into three major functional subsets on the basis
of their expression of pairs of surface molecules (12, 20, 24). The
pairing molecules used for such studies include CCR7 or CD62L
with CD45RA, CD28 with CD27, and CD28 or CD27 with
CD45RA. However, it is very likely that a more precise definition
of functional subsets of cells can be accomplished using mAbs
that bind to additional surface molecules. For example,
CD28?CD45RA?central memory T cells might be further di-
vided into CD28?CD45RA?CCR7?and CD28?CD45RA?
CCR7?T cell subpopulations. Therefore, a more precise definition
of functional T lymphocyte subpopulations may facilitate an elu-
cidation of the role of these cells in disease pathogenesis.
In the present study, surface expression of CD28 and CD95 has
been used to define CD4?T lymphocyte subsets. We showed that
cell subpopulations defined in this way have physiologic charac-
teristics of naive, central memory, and effector memory CD4?T
cells as has been previously described (16, 25). Most of the cyto-
kine production by Gag-specific CD4?T lymphocytes was con-
tributed by cells with a CD28?CD95?central memory phenotype.
This was true in healthy vaccinated monkeys, and in monkeys
infected with pathogenic virus. The consistency of these findings
suggests that the function of SIV-specific CD4?T cells with a
central memory phenotype is correlated with disease progression.
Finally, it is also possible that late effector cells that acquire CCR5
expression become targets for HIV infection, and are therefore
depleted. This would result in cells of intermediate-differentiated
phenotype being detected predominantly in infected individuals
with progressive disease.
We and others have previously shown that monkeys vaccinated
and challenged with a pathogenic immunodeficiency virus have
low viral loads, preserved CD4?counts, and no evidence of clin-
ical disease (26–28). However, the mechanism accounting for this
persistent clinical protection is poorly understood. In the present
study, we show that the central memory CD4?T lymphocytes
from such vaccinated and then infected monkeys had a preserved
capacity to produce IL-2, comparable with that seen in animals that
were infected with nonpathogenic virus. This observation suggests
that persistent clinical protection in vaccinated and challenged
monkeys is correlated with a preserved capacity of the peripheral
blood central memory CD4?T cells to express virus-induced im-
munomodulatory cytokine IL-2.
We are grateful to Mark Cayabyab, Gail Mazzara, Michael Wyand,
Linda Gritz, Alicia Gomez-Yafal, Rebecca S. Gelman, Vi Dang,
Srinivas Rao, and Michael H. Newberg for generous advice and providing
The authors have no financial conflict of interest.
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4760DETERMINANTS OF DISEASE PROGRESSION IN SIV/SHIV INFECTION