Published in Volume
113, Issue 6
(March 15, 2004)J Clin Invest.
Copyright © 2004, American Society for Clinical
Blockade of T cell costimulation reveals interrelated actions of
CD4+ and CD8+ T cells in
control of SIV replication
1Emory Vaccine Center,
2Department of Medicine, and
3Yerkes National Primate
Research Center, Emory University, Atlanta, Georgia, USA.
of Surgery, Duke University Medical Center, Durham, North Carolina, USA.
5Department of Surgery and
6Department of Microbiology
& Immunology, Emory University, Atlanta, Georgia, USA.
Address correspondence to: Mark Feinberg, Emory Vaccine Center, 954 Gatewood
Road, Atlanta, Georgia 30329, USA. Phone: (404) 727-4374; Fax: (404)
727-8199; E-mail: firstname.lastname@example.org.
Published March 15, 2004
Received for publication July 9,
2003, and accepted in revised form December 16,
In vivo blockade of CD28 and CD40 T cell costimulation pathways during acute
simian immunodeficiency virus (SIV) infection of rhesus macaques was performed
to assess the relative contributions of CD4+ T cells,
CD8+ T cells, and Ab responses in modulating SIV
replication and disease progression. Transient administration of CTLA4-Ig and
anti–CD40L mAb to SIV-infected rhesus macaques resulted in dramatic
inhibition of the generation of both SIV-specific cellular and humoral immune
responses. Acute levels of proliferating CD8+ T cells
were associated with early control of SIV viremia but did not predict ensuing
set point viremia or survival. The level of in vivo CD4+
T cell proliferation during acute SIV infection correlated with concomitant peak
levels of SIV plasma viremia, whereas measures of in vivo
CD4+ T cell proliferation that extended into chronic
infection correlated with lower SIV viral load and increased survival. These
results suggest that proliferating CD4+ T cells function
both as sources of virus production and as antiviral effectors and that
increased levels of CD4+ T cell proliferation during SIV
infections reflect antigen-driven antiviral responses rather than a compensatory
homeostatic response. These results highlight the interrelated actions of
CD4+ and CD8+ T cell responses
in vivo that modulate SIV replication and pathogenesis.
The mechanisms by which host immune responses may control immunodeficiency virus
replication and why they almost invariably fail to prevent progression to AIDS
remain incompletely understood. Acute HIV infection of humans and simian
immunodeficiency virus (SIV) infection of rhesus macaques are characterized by a
transient peak in viremia, the partial resolution of which correlates temporally
with the emergence of virus-specific CD8+ T cell and Ab
responses (1–4). The resulting set point level of viremia is a robust
predictor of the ensuing rate of progression to AIDS in both HIV-infected humans
(5) and SIV-infected macaques (6–8). Observations of several host-virus interactions during primary HIV (or
SIV) infection suggest that acute virus-specific CD8+ T cell
responses can regulate (at least partially) the extent of virus replication. These
interactions include temporal correlations between CD8+ T
cell responses and initial decline in acute viremia (1–4) and the emergence
of CTL-escape mutants during acute infection (9–11). In addition,
Ab-mediated depletion of CD8α+ cells during both
primary and chronic SIV infection of rhesus macaques results in significant
increases in SIV replication and has been interpreted as evidence for antiviral
regulation of SIV replication by SIV-specific CD8+ T cells
(12–14). The observed increases in SIV viremia reported in
these CD8α+ depletion studies, however,
potentially derive from factors in addition to the loss of SIV-specific
CD8+ T cell–mediated antiviral control. These
factors include global depletion of CD8α+ T cell
subsets with ensuing disruption of overall lymphocyte homeostasis, activation of
CD4+ T cells in response to bolus injections of xenogenic
and antigenic CD8α-depleting Ab’s, depletion of
CD8α+ NK cells (a cell population that plays
an important role in innate immune responses and that proliferates in response to
acute SIV infection) (15), and potential
reactivation of latent infectious agents (e.g., CMV) that result in increased levels
of CD4+ T cell activation. Thus, the quantitative
contribution of virus-specific CD8+ T cell responses toward
control of acute and chronic immunodeficiency virus replication remains largely
unknown. Furthermore, in contrast to models of CD8+ T cell
regulation of acute immunodeficiency virus replication, it has been theorized that
the kinetics of acute HIV replication can be wholly explained by exhaustion of the
supply of CD4+ target cells that produce virus, independent
of antiviral immune responses (16).
We sought to experimentally induce SIV antigen-specific tolerance in vivo, without
disruption of lymphocyte homeostasis or unintentional depletion of other cell types,
in order to assess the relative contributions of CD4+ target
cells, antigen-specific CD4+ Th and
CD8+ T cells, and virus-specific Ab’s to the
dynamics of acute SIV replication. Toward this end, we have used in vivo
administration of CTLA4-Ig and anti-CD40L mAb during acute SIV infection of rhesus
macaques to achieve costimulation (CS) blockade of SIV antigen-specific T and B cell
responses. CTLA4-Ig (17) and anti-CD40L mAb
block CD28-CD80/86 and CD40-CD40L lymphocyte-signaling pathways, respectively, that
are required for the generation of primary T and T-dependent B lymphocyte responses
(18–21). CTLA4-Ig is a soluble form of the extracellular
domain of human CTLA4, which binds CD80/86 on APCs with high avidity and suppresses
T cell–dependent Ab responses against foreign antigens in mice (22, 23).
Anti-CD40L mAb prevents CD40-CD40L interactions between CD4+
T cells and B cells. These interactions are essential for development of
antigen-specific humoral immune responses, including activation and differentiation
of B cells, immunoglobulin class switching, formation of B cell germinal centers in
lymph nodes, and development of B cell memory (24). In addition, CD40L-CD40 interactions between
CD4+ Th cells and DCs (21) or CD8+ T cells (25) also augment the generation of memory CD8+
CTLs following virus (e.g., lymphocytic choriomeningitis virus
[LCMV]) infection of mice (26–30). Simultaneous
in vivo blockade of the CD28-CD80/86 and CD40-CD40L pathways, by coadministration of
CTLA4Ig and anti-CD40L mAb, results in potent antigen-specific immunosuppression as
evidenced by long-term acceptance of skin and cardiac allografts in mice (31) and renal allografts in rhesus macaques
In the current study, two groups of Mamu
A*01+ rhesus macaques (treatment or
control, n = 4 per group) were infected with pathogenic
SIVmac239. The treatment group received infusions of CTLA4-Ig and anti-CD40L mAb,
which began 1 day prior to infection and continued for 27 days following SIV
infection, according to a dose and schedule previously used to induce tolerance to
MHC-mismatched renal allografts in rhesus macaques (see Methods). The use of
Mamu A*01+ macaques allowed
us to track the development of SIV-specific CD8+ T cell
responses by flow-cytometric detection of CD8+ T cells that
bound tetrameric Mamu A*01 MHC complexes (tetramers) (34) that present known immunodominant
Gag181–189 (p11C, CTPYDINQM) and
Tat28–35 (SL8, STPESANL) SIV epitopes (35–37). To our knowledge this study represents the first use of
CTLA4-Ig/anti-CD40L mAb-mediated CS blockade in primates wherein antigen-specific
immune responses could be tracked longitudinally.
Animals and virus. Rhesus macaques (Macacca mulatta) used in this study harbored
the Mamu A*01 allele as determined through typing
MHC class I alleles by PCR (38)
(Supplemental Table 1; supplemental data available at [http://www.jci.org/cgi/content/full/113/6/836/DC1]). Male, 2.5- to
4.5-year-old, Mamu A*01+
macaques were randomly assigned to treatment (n =
4) and control groups (n = 4). Recombinant human
CTLA4-Ig and anti-CD40L (anti-gp39) mAb, which cross-reacts with macaque CD28
and CD40L (C. Larsen, unpublished results), were obtained from Bristol-Myers
Squibb Co. (New York, New York, USA). Macaques in the CS blockade group received
intravenous infusions of CTLA4-Ig (20 mg/kg) and anti-CD40L mAb (20 mg/kg) in
sterile 0.9% sodium chloride solution (Baxter Healthcare Corp.,
Deerfield, Illinois, USA) 1 day prior to SIV infection and again on days 3, 6,
8, 10, 13, 20, and 27 following infection with SIV. Macaques in both CS blockade
and untreated control groups were inoculated intravenously on day 0 with
SIVmac239 (1.8 ng p27 per macaque; titered stock of SIVmac239 kindly provided by
R. Desrosiers, Harvard Medical School, Boston, Massachusetts, USA) (39). Infected animals were maintained in
accordance with institutionally and federally approved animal welfare protocols
Flow cytometry. Peripheral blood (heparinized) or lymph node cellular suspensions were stained
for surface markers by incubation with titrated combinations of
fluorochrome-conjugated Ab’s obtained from BD Biosciences
Pharmingen, San Diego, California, USA): FITC-,
phycoerythrin–CD3ε (clone SP34), peridinin chlorophyll
protein–CD8 (clone SK1), APC-CD4 (clone SK3), or APC-conjugated Mamu
A*01 tetramers (Gag181–189 and
Tat28–35). Contaminating red blood cells were lysed
by incubation of samples with FACS Lysing Buffer (BD Biosciences Immunocytometry
Systems, San Jose, California, USA). Intracellular staining with FITC-anti-Ki67
(clone B56; BD Biosciences Pharmingen) was performed following permeabilization
of cells (FACS Permeabilizing Solution; BD Biosciences Immunocytometry Systems).
Data were acquired (≥50,000 lymphocytes) on a FACSCalibur flow
cytometer (BD Biosciences Immunocytometry Systems) and analyzed with FlowJo
software (Treestar Inc., Ashland, Oregon, USA). For analyses, peripheral blood
cells were gated on lymphocytes as determined by forward and side-scatter
profiles. T cells were defined by positive staining with anti-CD3 Ab. For
tetramer analyses, CD8+ cells (medium and bright
staining) that were identified within lymphocyte/CD3+
gates were gated for binding to tetramer reagents and calculated as a percentage
of CD8+ T cells.
Enzyme-linked immunosorbent spot analysis of IFN-γ production. Multiscreen 96-well filter plates (Millipore, Billerica, Massachusetts, USA) were
coated with anti–human-IFN-γ mAb (D1K; Biosource
International, Camarillo, California, USA) in 0.1 M NaHCO3 buffer (pH
9.5). Duplicate PBMC samples (500,000 per well) were stimulated in vitro with 2
μg/ml Gag181–189 (p11C) peptide or negative
control peptide (LCMV NP396–404) in RPMI for
16–24 hours at 37°C, 5% CO2.
Bound IFN-γ was visualized following incubation of plates with
biotinylated anti–human-IFN-γ mAb (7-B6-1; Mabtech AB,
Nacka Strand, Sweden) and colorimetric detection by Vectastain ABC peroxidase
kit (Vector Laboratories, Burlingame, California, USA) and stable
3,3′-diaminobenzidine (Research Genetics, Carlsbad, California,
SIV viral load assay. Quantitative real-time RT-PCR assay to determine SIV viral load (40) was performed as described (41).
SIV sequence determination. Twenty thousand copies of viral RNA, determined by real-time RT-PCR
quantification, were primed with random hexamers and reverse transcribed
(Multiscribe RT) to produce cDNA. PCR amplification (40 cycles) of
tat exon 1 was performed on 10,000 copy equivalents of cDNA per
animal per time point with the following exceptions: day 42, Rvy5 =
8,000; day 42, Ryt5 = 7,250; day 42, RBm6 = 188 copy
equivalents cDNA. PCR primers were as follows: SIV6511F (forward),
5′-AGTTAGCAAGCGAGGATCA-3′; SIV6900R (reverse),
5′-AGCAAGATGGCGATAAGCAG-3′ (9). PCR reaction products were gel purified and
cloned into the pCR-BluntII-TOPO vector (Invitrogen Corp., Carlsbad, California,
USA). Plasmid DNA was prepared using QIAprep Spin Minipreps (QIAGEN Inc.,
Valencia, California, USA) and used as the template for sequencing of insert DNA
with M13 forward and M13 reverse primers. Automated DNA sequencing reactions
were carried out by the Emory DNA Core Facility using fluorescent dye terminator
cycle sequencing methodology. Sequence data were analyzed with Lasergene
software suite (DNASTAR Inc., Madison, Wisconsin, USA).
Ab titers. Anti-SIV Ab titers were determined by Genetic Systems HIV-2 EIA (Bio-Rad
Laboratories Virus Division, Redmond, Washington, USA) and expressed in
arbitrary units that were derived from fitting A450 nm readings to
the linear range of a standard curve that was generated from positive control
(HIV-1) patient sera. Neutralizing Ab titers against SIVmac239 were measured as
the reciprocal plasma dilution at which p27 production was reduced
80% in human PBMCs relative to the amount of p27 synthesized in the
absence of a plasma sample (42).
Neutralizing Ab titers against SIVmac251 were determined by CEMx174 cell-killing
assay and are reported as the reciprocal plasma dilution at which
50% of cells were protected from virus-induced killing as measured
by neutral red uptake (42).
Statistics. Repeated measures analyses (43) for each
outcome were performed using a means model (SAS PROC MIXED) with SAS/STAT (SAS
Institute, Cary, North Carolina, USA), providing separate estimates of the means
by days after infection and treatment group. A compound symmetry
variance-covariance form among the repeated measurements was assumed for each
outcome, and robust estimates of the standard errors were used to perform tests
and construct 95% confidence intervals. Fisher’s exact,
Mann-Whitney, Spearman correlation, and t tests were calculated
with the StatView software package (SAS Institute).
Effects of CS blockade on SIV replication. We first determined the effects of transient T cell CS blockade on SIVmac239
replication by measuring levels of SIV in plasma by a quantitative real-time
RT-PCR assay (Figure 1). Macaques in both
treatment and control groups exhibited similar rates of initial increase in SIV
viremia (P = 0.82, comparison of regression slopes
between groups, days 3–10 after infection; Figure 1A). Peak SIV viremia in the control group (day 10,
log10 geometric mean viral RNA (vRNA) = 8.26)
occurred earlier (at day 10 after infection in three of four macaques) than in
the treatment group (at day 13 after infection in four of four macaques) and was
significantly greater (fourfold) than the corresponding level of viremia in the
treatment group (day 10 log10 vRNA = 7.62;
P = 0.009, repeated measures analysis of means;
Figure 1B, shaded region). Comparison of
postpeak declines in SIV viremia (through day 27 after infection, the last day
of treatment) revealed that untreated control macaques exhibited an average 1.86
log reduction in viral load (range = 1.08–2.88), whereas
treated animals exhibited a 1 log reduction in postpeak SIV viremia (range
= 0.46–1.74). Control macaques (three of four) exhibited
continued declines in SIV viremia for an additional 1–2 weeks.
Following cessation of CTLA4-Ig and anti-CD40L mAb infusions, treated macaques
displayed a transiently (approximately 4 weeks) increased geometric mean level
of SIV viremia that was significantly greater (21- to 30-fold) than that
observed in control animals (Figure 1B,
shaded region). During the same period of time, the control group exhibited a
gradual progressive increase (threefold) in geometric mean SIV viral load
(Figure 1B). By 14 weeks after infection,
both groups reached similar set point levels of SIV plasma viremia at
approximately 107 SIV RNA copies per milliliter (Figure 1B). Thus, CS blockade during acute SIV
infection had a moderate, but significant effect on the level and timing of peak
SIV viremia and reduced control (in both magnitude and duration) of postpeak
viral load, but did not affect ultimate set point levels of SIV viremia.
Effects of CS blockade on SIV replication. (A) SIV viral load
(log10 SIV RNA copies per milliliter plasma) from macaques in the
control group (left panel) or CS blockade treatment group (right panel).
Indicated are times at which individual macaques were sacrificed because of
clinical deterioration due to simian AIDS (circled x). Control macaques:
ROz5 (black), RBm6 (blue), RVy5 (red), RYt5 (green). CS blockade macaques:
REp6 (black), REo6 (blue), RIc6 (red), RCr5 (green). The gray bar denotes
the treatment period. (B) Geometric mean SIV viral load (log10
SIV RNA copies per milliliter plasma) for CS blockade group (red) and
control group (black). The yellow shading indicates times at which
significant differences were determined between groups (P
≤ 0.05, repeated measures analysis of means). The gray bar
denotes the treatment period.
CS blockade attenuates SIV-specific CD8+ T cell
responses. Macaques undergoing CS blockade generated substantially reduced levels of SIV
Tat28–35-specific CD8+ T cells
compared with untreated control animals during acute SIV infection (Figure 2A). In control macaques,
CD8+ T cells directed against the
Gag181–189 epitope were detected in peripheral blood
as early as 10 days following SIV infection (Figure 2A) and exhibited a mean peak level of
2.6% (range = 2.3–3.2%) at day
13 after infection. In contrast, macaques in the treatment group exhibited a
significantly lower (eightfold) mean level of
Gag181–189-specific CD8+ T cells
(0.34%, range = 0.21–0.43%;
P < 0.0001, t test). In
treated macaques, the mean peak level of
Tat28–35-specific CD8+ T cells was
reduced and occurred earlier than in control macaques (CS peak day 16, mean
= 1.1%, range =
0.7–2.0%; control peak, day 20, mean =
3.2%, range = 1.3–8.2%;
P = 0.04 Mann-Whitney U test)
CS blockade attenuates the generation and in vivo CTL activity of
SIV-specific CD8+ T cell responses. (A) Levels of
CD8+ T cells (red trace),
CD8+ T cells (yellow trace), or the sum of
Tat28–35-specific CD8+ T cells
(blue trace) as percentages of CD8+ T cells in
peripheral blood (left axes) and the level of the sum of
Tat28–35-specific CD8+ T cells
as a percentage of CD8+Ki67+ T
cells (black trace) in peripheral blood (right axes). Left column, control
macaques; right column, CS blockade macaques. Gray bars denote the treatment
period. (B) Levels (%) of peripheral blood
CD8+ T cells that exhibit the Ki67 antigen, a marker
of cellular proliferation, by flow cytometry. Control macaques: ROz5
(black), RBm6 (blue), RVy5 (red), RYt5 (green). CS blockade macaques: REp6
(black), REo6 (blue), RIc6 (red), RCr5 (green). The gray bar denotes the
treatment period. (C) Nucleotide sequences of cDNA clones encoding SIV
tat exon 1 were determined at the indicated times after
infection and were compared to the WT parental SIVmac239 sequence.
Frequencies of cDNA clones that exhibited one or more nonsynonymous
mutations within the region encoding the Tat28–35
(STPESANL) epitope are shown for individual macaques in the control or CS
blockade groups. (D) Correlation of the level of
CD8+ T cells and the frequency of mutation within
the Tat28–35 coding region at 20 days following SIV
infection. The P value was determined by the Spearman rank
correlation test. Control macaques (circles): ROz5 (black), RBm6 (blue),
RVy5 (red), RYt5 (green); CS blockade macaques (triangles): REp6 (black),
REo6 (blue), RIc6 (red), RCr5 (green).
Measurement of CD8+ T cells directed against only two Mamu
A*01–restricted SIV epitopes
(Gag181–189 and Tat28–35)
assesses only a subset of the total potential CD8+ T cell
responses mounted by individual outbred macaques against SIV (36). We reasoned that measurement of in vivo
CD8+ T cell proliferation during acute SIV infection
may predominantly reflect antigen-specific expansion of SIV-specific precursors,
rather than nonspecific CD8+ T cell activation or
homeostatic lymphocyte proliferation. This is because initial increases in the
levels of proliferating CD8+ T cells coincides temporally
with the emergence of SIV-specific CD8+ T cell responses
of known epitope specificities (Figure 2, A
and B) and because the acute CD8+ T cell proliferation
that occurs in other experimental viral infections predominantly reflects viral
antigen-specific responses (44). We
measured acute CD8+ T cell proliferation in SIV-infected
macaques by flow-cytometric detection of the Ki67 (45), a nuclear antigen that identifies cells
progressing through the cell cycle (i.e., not in G0)
(46, 47). Untreated macaques exhibited a significant increase (fourfold)
over baseline in the mean level of proliferating CD8+ T
cells (day 13 after infection, P = 0.02,
t test) that coincided temporally with the peak
Gag181–189-specific CD8+ T
cell response. In contrast, this acute CD8+ T cell
proliferative response was blunted in macaques undergoing CS blockade (twofold
increase over baseline at day 13 after infection) (Figure 2B). The fraction of proliferating
CD8+ T cells that were specific for
Gag181–189 or Tat28–35 was
calculated to assess the focus of each macaque’s anti-SIV
CD8+ T cell response to these previously defined
Gag181–189 and Tat28–35 Mamu
A*01–restricted epitopes (Figure 2A, black traces). Because this parameter is
calculated from independent measurements of
CD8+ tetramer-positive lymphocytes, it potentially
reflects a partial contribution from changes in CD8+ T
cell homeostatic proliferation or aberrant cell cycle arrest. At the times of
peak responses, Gag181–189- and
Tat28–35-specific CD8+ T cells
accounted for 20–100% of the CD8+
T cell proliferative response in control animals and
15–100% in treated macaques. The relatively narrow
antigenic focus of CD8+ T cell responses exhibited during
acute SIV infection of some macaques may be due to homozygosity of the
Mamu A*01 allele (e.g., ROz5 typed positive
only for the Mamu A*01 MHC allele out of eight
different MHC-I alleles assayed; see Supplemental Table 1) or to exceptional
immunodominance of Mamu A*01–restricted
Gag181–189 and Tat28–35
epitopes over others potentially restricted by additional (but unidentified) MHC
class I alleles.
CS blockade attenuates in vivo SIV-specific CTL function. To assess CD8+ CTL effectiveness in vivo, frequencies of
SIV Tat28–35 CTL escape mutants (9) were determined at 13, 20, and 42 days following
SIV infection of macaques (Figure 2C; see
also Supplemental Table 2). Frequencies of CTL-escape mutants were determined as
the number of SIV cDNA clones that harbored one or more nonsynonymous mutation
within the region encoding the Tat28–35 epitope per total
cDNA clones analyzed. Nonsynonymous mutations in tat exon 1,
when present, occurred almost exclusively within the region encoding the
Tat28–35 epitope (representative alignments shown in
Supplemental Figure 1).
Treated macaques, which exhibited lower levels and shorter duration of
Tat28–35-specific CD8+ T cells
than untreated controls (Figure 2A), also
harbored significantly lower frequencies of Tat28–35
CTL-escape mutants (Figure 2C). At day 13
after infection, mutation within the region encoding the
Tat28–35 epitope was observed only in cDNA clones
from control macaques (≤5%) (Figure 2C). At 20 days after infection, the time point that
corresponded with the peak
Tat28–35-CD8+ T cell response in
this group, three of four control macaques exhibited mutation frequencies
greater than 77% (Figure 2C).
The fourth control animal, Ryt5, exhibited a lower frequency of mutation at day
20 after infection (17% mutant clones) and also exhibited the lowest
peak level of Tat28–35-specific
CD8+ T cells among animals in the control group (Figure
2, A and C). Treated animals exhibited
significantly lower frequencies of mutation within the
Tat28–35 epitope at days 20 and 42 following infection as
compared with controls (CS block versus control: day 20, P
< 0.0001; day 42, P < 0.0001;
Fisher’s exact test). Identical results were obtained when these
analyses were performed using a restricted definition of an escape mutant as one
that had previously been shown to exhibit impaired binding to the Mamu
A*01 MHC-I molecule (Table 1 in ref. 9). Overall, a significant positive correlation (P
= 0.028, ρ = 0.83, Spearman rank
correlation) was observed between the peak level of
CD8+ T cells (observed during the first 20 days
following SIV infection) and the frequency of SIV sequence clones (at 20 days
after infection) that harbored one or more nonsynonymous mutations within the
region encoding the Tat28–35 epitope (Figure 2D). These results provide strong evidence
for in vivo CD8+ T cell–mediated selection
against the WT Tat28–35 (STPESANL) epitope and ensuing
virus escape from Tat28–35-specific
CD8+ T cells by mutation (9, 48). Furthermore, because
Tat28–35-specific CD8+ T cells
became undetectable following acute infection of treated macaques (Figure 2A), even though WT SIV still predominated
(Figure 2C), these results demonstrate that
emerging antiviral CD8+ T cell function is abrogated in
the setting of CS blockade.
Acute SIV viral load is inversely related to acute
CD8+ T cell proliferative responses. The correlation of acute Gag181–189- and
Tat28–35-specific CD8+ T cell
responses with postpeak reduction in SIV viral load revealed only weak trends
that did not achieve statistical significance (Figure 3, A and B). The SIV-specific
CD8+ T cell responses mounted by our cohort of outbred
animals, as measured by the available MHC class I tetramers, may not reflect
total SIV-specific CD8+ T cell responses, as suggested by
the observation that they represent only a fraction of
CD8+Ki67+ T cells. Therefore,
we also considered acute (through day 27 after infection) levels of total
CD8+ T cell proliferation
(CD8+Ki67+ T cells) as a
surrogate measure of SIV-specific CD8+ T cell responses
directed against all existing and newly emerging SIV epitopes (44). Using this measure, it was found that in vivo
CD8+ T cell proliferation during acute SIV infection
was significantly correlated with the magnitude of postpeak decline in SIV
viremia (P = 0.03, Figure 3C). Similarly, a more sensitive mathematical modeling
analysis of our data that takes into account all available information on target
cells and SIV-specific CD8+ T cells during primary
infection shows that SIV-specific CD8+ T cell responses
play a crucial role in the control of early SIV replication in the untreated
macaques, whereas SIV replication kinetics in macaques undergoing CS blockade
are the result of target cell exhaustion (49).
Reduction of acute SIV viral load is directly related to
CD8+ T cell proliferative responses. Correlation
of the magnitude of postpeak decline in SIV viremia and
Tat28–35-specific (B), and
Ki67+ (C) CD8+ T cells. The
magnitude of the postpeak decline in SIV viremia is reported as the
logarithm of the fold reduction of SIV viremia between peak and day 27 after
infection (last day of treatment). All CD8+ T cell
parameters were calculated as AUCs from baseline through day 27 after
infection. Control macaques (circles): ROz5 (black), RBm6 (blue), RVy5
(red), RYt5 (green); CS blockade macaques (triangles): REp6 (black), REo6
(blue), RIc6 (red), RCr5 (green). P and ρ
values were determined by the Spearman rank correlation test. The solid
lines are regression lines.
Chronic SIV-specific CD8+ T cell responses are
impaired following acute CS blockade. In the absence of effective CS, T cells exposed to antigen can become anergic or
undergo apoptosis (50). Similarly, in the
absence of effective CD4+ T cell help during chronic
viral infection, viral antigen-specific CD8+ T cells may
also become anergic or undergo deletion (51). The relatively shorter duration and lower levels, of
Tat28–35-specific CD8+ T cells
observed in treated macaques is consistent with this model (Figure 2A). Interestingly,
Gag181–189-specific CD8+ T
cells were detected by tetramer staining in treated animals beginning at 18
weeks after infection (14 weeks following cessation of the CS blockade, data not
shown). The Gag181–189-specific
CD8+ T cells that emerged during chronic infection of
treated macaques may represent new thymic emigrants that are (partially)
responsive to high levels of SIV antigen. Alternatively, these cells may have
arisen from CD8+
Gag181–189-precursors within the naive repertoire, which
were present at initial SIV infection, but whose expansion was blocked during
the period of effective CS blockade. We performed enzyme-linked immunosorbent
spot (ELISPOT) assays to assess the functional capacity of SIV-specific
CD8+ T cells. The
Gag181–189-specific CD8+ T cells
that emerged late (20 weeks after infection) in (two of four) treated animals
(Figure 4A) exhibited attenuated production
of IFN-γ in response to in vitro stimulation with cognate
Gag181–189 (CTPYDINQM) peptide (Figure 4B). This impairment of SIV-specific
CD8+ T cells that arose during chronic SIV infection
is consistent with functional exhaustion or impaired induction of
CD8+ T cell responses that are generated in the
absence of effective CD4+ T cell help (51–53).
Chronic SIV-specific CD8+ T cell responses are
impaired following acute CS blockade. Levels of
CD8+ T cells determined by tetramer staining of
peripheral blood at 20 weeks following SIV infection (A) and corresponding
ELISPOT quantification of IFN-γ–producing cells
following in vitro stimulation of PBMCs with
Gag181–189 (CTPYDINQM) peptide (B).
Acute CS blockade delays SIV seroconversion. We characterized the development of humoral immune responses against SIV in
treated versus control macaques and determined their relationship to SIV
replication. Development of B cell germinal centers (GCs) in lymph nodes,
assessed by histological analysis of proliferating
(Ki67+) lymphocytes, was evident at 10 days following SIV
infection of control macaques but was profoundly inhibited in treated animals
(Figure 5A). Similar inhibition of GC
development by CS blockade was also observed in lymph nodes from treated animals
at 6 weeks after infection (not shown) and is temporally consistent with the
delayed seroconversion observed in treated animals (Figure 5B). These effects on GC development and
seroconversion are consistent with the inhibitory effects on humoral immunity
following in vivo disruption of CD40L-CD40 lymphocyte signaling in other animal
Effects of acute CS blockade on humoral immune responses. (A) B cell germinal
centers (arrows) in representative lymph node sections from macaques in
control or treatment (CS blockade) groups at 10 days following SIV
infection. Ki67+ lymphocytes are brown; magnification
×200. (B and C) SIV-specific Ab titers were determined by ELISA
(B) or SIVmac239-neutralization assay (C) on plasma samples from control
macaques (circles): ROz5 (black), RBm6 (blue), Rvy5 (red), RYt5 (green); or
CS blockade macaques (triangles): REp6 (black), REo6 (blue), RIc6 (red),
RCr5 (green). Gray bars denote the treatment period; note different scales.
(D) Correlation of SIV-specific Ab titer (by ELISA) and SIV plasma viral
load around the time of seroconversion for macaques in the control group
(days 16–42 after infection) or CS
blockade–treatment group (days 50–98 after
infection). Ab titers are plotted as natural log transforms of the Ab titers
While animals in both control and treatment groups exhibited seroconversion
against SIV, albeit with different kinetics, in neither group did macaques
produce Ab’s that were able to neutralize SIVmac239 infection ex
vivo (Figure 5C). Interestingly,
SIV-specific Ab’s that were elicited during SIVmac239 infection of
both treated and control macaques, while not capable of neutralizing SIVmac239,
were capable of ex vivo neutralization of a related tissue
culture–adapted SIV isolate (SIVmac251) (Supplemental Figure 2). The delayed emergence of
SIVmac251-specific neutralizing Ab’s in treated versus control
macaques (Supplemental Figure 2) indicates
that generation of neutralizing Ab responses against SIV is prevented by the CS
blockade. These Ab responses therefore must require effective
CD4+ T cell help and do not arise in a T
cell–independent manner, as previously suggested (42, 54).
SIV viral load is inversely related to SIV-specific Ab titers. In both groups, significant inverse correlations were observed between the levels
of SIV plasma viremia and SIV-specific Ab titers around their respective times
of seroconversion (Figure 5D; control
group, P < 0.001; CS blockade group,
P < 0.001: comparison of slopes of regression lines
against zero; that is, null hypothesis indicates no association). This inverse
correlation was greater for the control group than for the treatment group
(Figure 5D; P
= 0.03, comparison of slopes for control versus treatment group). In
addition, inverse rank order correlations between SIV plasma viremia and
SIV-specific Ab titers were observed in individual animals at numerous time
points following seroconversion that extend into the period of steady-state SIV
viremia (compare Figure 1A and Figure 5B).
Peak SIV viremia is predicted by early levels of proliferating
CD4+ T cells. Upon infection with a CD4+-tropic lentivirus, activated
virus-specific CD4+ T cells can act as both targets for
productive viral infection and as effector Th cells that augment antiviral
humoral and cellular immune responses. Because SIV replication is augmented in
activated/proliferating CD4+ T cells (versus resting
cells) both in vitro (55) and in vivo
(56), we reasoned that levels of in
vivo proliferating target cells that precede the development of antiviral
effectors should predict the ensuing peak of SIV viremia. At 10 days following
SIV infection, the level of proliferating CD4+ T cells
(CD4+Ki67+) in lymph nodes, an
anatomic site that supports high levels of SIV replication (57), exhibited a significant direct correlation with
peak SIV plasma viremia (Figure 6A;
P = 0.03, Spearman’s ρ
=0.81). Similarly, a significant direct correlation was also
observed between the level of proliferating CD4+ T cells
(CD4+Ki67+) in peripheral
blood (day 10 after infection) and peak SIV viremia (Figure 6A; P = 0.02,
Spearman’s ρ =0.90). These data provide
direct evidence that higher levels of activated CD4+ T
cells in vivo during the initial phase of SIV infection contribute to increased
levels of SIV viremia. The fact that control macaques exhibited higher levels of
CD4+ T cell proliferation in lymph nodes and blood
(Figure 6A; lymph nodes, P
= 0.04; blood, P = 0.04; Mann-Whitney
U test) than did macaques undergoing CS blockade indicates
that this increased CD4+Ki67+
response in control animals reflects antigen-driven proliferation of
SIV-specific CD4+ T cells.
In vivo CD4+ T cell proliferative responses predict
the peak level and subsequent control of SIV viremia, titers of SIV-specific
Ab’s, and survival of infected macaques. (A) Correlation between
the level of CD4+Ki67+ T cells
(day 10 after infection) in lymph node or peripheral blood and peak SIV
plasma viremia. (B) Levels of
CD4+Ki67+ T cells in
peripheral blood of macaques in the control group and treatment group (CS
blockade). The yellow shading indicates times at which significant
differences were determined between groups (P =
0.05, repeated measures analysis of means). The gray bar denotes the
treatment period. (C and D) Correlation between the level of
CD4+Ki67+ T cells in
peripheral blood (AUC: baseline to 16 weeks after infection) and level of
anti-SIV Ab’s (AUC: day 6–18 weeks after infection)
(C) or the level of SIV plasma viremia (AUC: day 3–16 weeks
after infection) (D). (E) Survival of macaques in the control (black) or CS
blockade treatment group (red). (F) Correlation between
CD4+Ki67+ T cells (AUC:
baseline to 16 weeks after infection) and period of survival. (A, C, D, and
F) Treated macaques, triangles; control macaques, circles.
P values were determined by the Spearman rank correlation
test. The solid lines represent regression lines. (A, B, C, D, and F)
Control macaques: ROz5 (black), RBm6 (blue), RVy5 (red), RYt5 (green); CS
blockade macaques: REp6 (black), REo6 (blue), RIc6 (red), RCr5 (green). PB,
SIV viral load is inversely related to CD4+ T cell
proliferative responses. SIV-infected CD4+ T cells, including those specific for
SIV as well as other antigens, serve as a source of SIV production during
infection. Additionally, SIV-specific CD4+ T cells also
potentially function as antiviral effectors through direct and/or indirect
mechanisms. To examine the relationship between CD4+ T
cell responses to SIV infection and viral load, we measured in vivo
CD4+ T cell proliferation during the first four
months of SIV infection (Figure 6B) and
compared this with SIV-specific Ab responses (Figure 6C) and viral loads (Figure 6D). Acute CS blockade treatment of SIV-infected
macaques resulted in a significant reduction in levels of proliferating
(Ki67+) CD4+ T cells in
peripheral blood as compared with untreated control animals (Figure 6B). The mean level of
CD4+Ki67+ T cells in treated
macaques, which decreased threefold below baseline level during the first week
of treatment, did not increase during acute SIV infection as was observed for
untreated controls, and remained suppressed through at least 7 weeks after
infection (Figure 6B). This observation is
most readily explained by destruction of highly permissive
(Ki67+) CD4+ T cells by SIV
infection and the simultaneous prevention of expansion of SIV-specific
CD4+ T cells by effective CS blockade.
Following cessation of the CS blockade, delayed recovery of
CD4+ T cell proliferation was observed in two of four
treated macaques, and this CD4+ T cell response
correlated temporally with SIV seroconversion (Figure 5B and Figure 6B). For treated and control macaques, the duration and magnitude of
CD4+Ki67+ T cell responses
measured in peripheral blood during the initial 4 months of SIV infection,
measured as area under the curve (AUC) (Figure 6B) correlated significantly with the cumulative SIV-specific Ab
response (AUC, Figure 5B)
(P = 0.05, Spearman rank correlation; Figure 6C). Moreover, comparison of AUCs for SIV
viremia and CD4+Ki67+ T cell
levels, as measures of their magnitude and duration, also revealed a significant
correlation between in vivo CD4+ T cell proliferation and
lower SIV plasma viral load (Figure 6D;
P = 0.05, Spearman rank correlation). In
contrast, similar analyses did not reveal any significant associations between
CD8+ T cell proliferation and SIV viral load
(Supplemental Figure 3).
In vivo CD4+ T cell proliferative responses predict
survival. Because in vivo levels of CD4+Ki67+
T cells correlated with increased anti-SIV humoral immunity and decreased SIV
viremia, we reasoned that the in vivo CD4+ T cell
proliferative response to infection, if protective, should also correlate with
increased periods of survival of infected macaques. Within the treatment group,
three of four macaques progressed rapidly to simian AIDS (≤25 weeks
post-infection), whereas only one of four control macaques exhibited accelerated
disease progression (Figure 6E). The
magnitude and duration of CD4+ T cell proliferation
(measured as AUC; Figure 6B) exhibited a
significant direct correlation with increased periods of survival for macaques
in both the treatment and control groups (Figure 6F; P = 0.01, Spearman’s
ρ = 0.952), whereas absolute levels of peripheral blood
CD4+ T cells (which were similar between treatment
and control groups) were not associated with survival (Supplemental Figure 4).
The relative roles played by multiple host immune responses in regulating HIV
replication and the mechanisms underlying their ultimate failure to successfully
control virus infection and prevent immunodeficiency disease progression remain
incompletely understood. For a better understanding of the relative contributions of
antiviral CD8+ T cells, CD4+ target
cells, antiviral CD4+ Th cells, and Ab responses in
modulating CD4+-tropic lentivirus replication and
pathogenesis, we have used CTLA4-Ig/anti-CD40L mAb-mediated blockade of in vivo T
cell CS pathways during acute SIV infection of rhesus macaques. We show here that
transient in vivo CS blockade during acute SIV infection of rhesus macaques results
in attenuation of SIV-specific cellular and humoral immune responses and a reduced
capacity to control acute SIV replication. We conclude that control of postpeak
viremia during acute (untreated) SIV infection is largely mediated by SIV-specific
CD8+ T cells, which is in agreement with previous studies
predicated on mAb-mediated depletion of CD8α-expressing cells (12–14). This conclusion is supported by correlational analyses presented
here (Figure 3), as well as by our results from
mathematical modeling of this data set to discriminate between target cell
limitation versus immune control models of acute SIV replication (49).
We found evidence for CD8+ T cell–mediated control
of SIV replication at early, but not late, times following infection. Interestingly,
the magnitude of decline in postpeak SIV viremia correlates more strongly with the
acute CD8+ T cell proliferative response to SIV infection, a
surrogate measure of total SIV-specific CD8+ T cell
responses, than with either of two specific immunodominant
CD8+ T cell responses (Gag181–189 and
Tat28–35) and suggests that the predominance of these
Mamu-A*01–restricted CD8+ T cell
responses does not necessarily imply effective control of SIV replication. While our
studies do not indicate the precise mechanism through which
Gag181–189-specific CD8+ T cells
fail to contain SIV replication, it is conceivable that multiple mechanisms,
including SIV escape from CTL recognition by epitope mutation (9, 10), as shown
for the Tat28–35 epitope STPESANL (Figure 2D), improper differentiation in the setting of chronic
antigen exposure (58, 59), dysregulation of CTL effector mechanisms (58, 60–62), and
inefficient killing of infected targets by CTL (63), may function simultaneously to render particular SIV-specific
CD8+ T cell responses ineffective at controlling SIV
In addition to the role of CD8+ T cells in regulation of acute
SIV replication, our results highlight the dual role of SIV-specific
CD4+ T cells as both targets for productive virus
infection and antiviral Th cells. At the earliest stages of SIV infection,
activated/proliferating CD4+ T cells (of all antigenic
specificities) that become infected appear to contribute to net increases in SIV
viremia. Subsequent expansion of virus-specific CD4+ T cells,
which, by analogy to HIV infection, may be preferentially infected with SIV (64), in turn may lead to either further
increases in SIV viremia by providing an expanded pool of permissive target cells or
lead to a decline in SIV viremia through exertion of antiviral effects. Our finding
that duration and levels of proliferating CD4+ T cells in
circulation are directly correlated with titers of SIV-specific Ab’s and
indirectly correlated with SIV viremia provides strong, albeit indirect, evidence
that the observed in vivo CD4+ T cell proliferative response
is composed largely of SIV-specific CD4+ T cells that are
responding to SIV as an antigen and may, to various degrees, exert a net antiviral
effect. The alternative scenario, as envisioned in “tap and
drain” models of HIV pathogenesis (65), is that CD4+ T cell proliferation occurs as a
compensatory response to an imbalance in lymphocyte homeostasis caused by increased
destruction of CD4+ T cell targets. This model, which
predicts a direct correlation between levels of CD4+ T cell
proliferation and SIV viremia, does not appear likely in light of our data.
Our observation, in both treated and untreated macaques, that increased titers of
nonneutralizing SIV-specific Ab’s are associated with lower SIV viral
loads at their respective times of seroconversion suggests that production of these
Ab’s constitutes one potential mechanism of antiviral regulation, but
does not exclude additional potential mechanisms, such as production of antiviral
cytokines and chemokines by underlying SIV-specific CD4+ Th
cell responses. Non-neutralizing SIV-specific Ab’s potentially function
to lower levels of SIV replication through Ab-dependent cellular
cytoxicity–mediated clearance of infected cells (66) and by clearance, via the mononuclear phagocytic
system, of virions in complement-containing immune complexes (67). Because SIVmac239 is less susceptible to Ab-mediated
neutralization than are other SIV strains (e.g., SIVmac251), our use of SIVmac239 in
this study likely underestimates the potential antiviral effects of neutralizing
Ab’s (68). Moreover, the
observation that SIV seroconversion and concomitant reduction in SIV viral load
occurred prior to the emergence of SIV-specific
(Gag181–189-specific) CD8+ T cells in
treated macaques further suggests that SIV-specific Ab’s (or another
aspect of the underlying SIV-specific CD4+ T cell response)
mediate reduction of SIV viremia, independent of CD4+ Th
effects on CD8+ CTLs. Regardless, the observations that, over
time, an increased CD4+ T cell proliferative response to SIV
infection predicts increased titers of SIV-specific Ab’s, lower viral
load, and increased time of survival provide evidence that this response is composed
of SIV-specific CD4+ T cells that exert an antiviral effect.
Progressive loss of peripheral CD4+ T cells in association
with progressive susceptibility to opportunistic infections, a hallmark of HIV
infection that occurs by accelerated destruction and impaired regeneration of this
cell population (reviewed in ref. 69), was
also observed in SIV-infected macaques with increasing duration of SIV infection.
The end-stage levels of peripheral blood CD4+ T cells,
however, did not correlate with time of AIDS-free survival (Supplemental Figure
4). In light of these results, progression
to simian AIDS that follows infection with highly virulent isolates of SIV (such as
SIVmac239), as evidenced by severe weight loss and development of multiple
opportunistic infections, likely reflects a pronounced impairment of
CD4+ T cell functionality in addition to actual depletion
of peripheral blood CD4+ T cells. In this regard, the use of
such virulent SIV isolates may mask subtleties relevant to understanding HIV
pathogenesis in humans. During HIV infection of humans, reduced numbers of
peripheral blood CD4+ T cells have been shown to correspond
with an increased fraction of proliferating cells within this compartment (70). Because the extent of depletion of
peripheral blood CD4+ T cells in humans with progressive HIV
infection appears to be greater than that occurring in SIV-infected macaques (71), coincident increases in fractions of
proliferating CD4+ T cells in humans may exhibit a positive
association with disease progression, whereas the magnitude and duration of the in
vivo CD4+ T cell proliferative response during SIV infection
of macaques is predictive of increased periods of AIDS-free survival.
In this regard, it is becoming increasingly appreciated that the deleterious
“indirect” consequences of chronic, generalized immune
activation, in addition to direct virus-mediated killing of
CD4+ T cells, play prominent roles in the pathogenesis of
AIDS (72–75). Important evidence supporting the view that
generalized immune activation is a primary determinant of disease progression
following CD4-tropic lentivirus infection of humans and nonhuman primates that are
not natural hosts of these viruses (such as rhesus macaques) is the observation that
apathogenic SIV infection of natural host species, such as sooty mangabey monkeys,
is characterized by limited bystander immunopathology despite chronic high-level SIV
viremia (74). Given that experimental SIV
infection of rhesus macaques leads to high levels of immune activation and
progression to AIDS, it is conceivable that CS blockade in SIV-infected rhesus
macaques might have resulted in attenuation, rather than aggravation, of disease
progression through reduction of the pathogenic role of immune activation. This
outcome, however, was clearly not observed, potentially the result of a number of
factors. First, transient administration of CS blockade reagents in rhesus macaques
did not induce either complete or durable tolerance against SIV antigens, as
revealed through longitudinal assessments of SIV antigen-specific immune responses
(Figure 4A and Figure 5B). This impermanent attenuation of immune responses by
CS blockade in macaques is in contrast to the more pronounced and durable effects of
transient CS blockade in induction of antigen-specific immune tolerance in mice
(31). Second, the control of
immunopathology in SIV-infected sooty mangabey monkeys might be the result of active
anti-inflammatory processes (e.g., induction of beneficial T regulatory responses)
that would not be elicited or perhaps would even be blocked in the setting of CS
blockade, as well as attenuated inflammatory responses. Finally, CS blockade affects
only adaptive immune responses, whereas the interface between innate immunity and
SIV may be a key difference both in determining and maintaining the balance between
beneficial and detrimental immune responses to SIV infection in natural and
Nevertheless, the experimental paradigm developed in this study will likely also be
relevant to the analysis of other targeted immune interventions designed to probe
the beneficial and detrimental determinants of AIDS virus infections in nonhuman
primates. It is anticipated that such studies will enable a greater understanding of
the mechanisms of AIDS pathogenesis and lead to the identification of immune
interventions that successfully block deleterious effects of chronic immune
activation without compromising beneficial host antiviral responses. The ultimate
success of any such strategy will depend on the nature and specificity of the
immunomodulatory intervention selected and the extent to which the SIV infection
model chosen recapitulates the biology of HIV disease in humans.
View Supplemental data
This research was supported by NIH grants R01-AI49155-02 (to M.B. Feinberg),
T32-AI07470 and T32-AI07442 (to D.A. Garber), P51 RR00165-42 (to Yerkes Primate
Research Center), and P30 AI50409-04A1 (to Emory/Atlanta Center for AIDS Research).
D.A. Garber is an Elizabeth Glaser Scholar of the Pediatric AIDS Foundation. The
authors wish to acknowledge the contributions of the following people: Chris Ibegbu
and Ashley Carter for assistance with flow cytometry, Lily Wang for providing
tetramer reagents, Kirk Easley for assistance with statistical analyses, Diane
Hollenbaugh (Bristol-Myers Squibb) for providing us with CTLA4-Ig and anti-CD40L
mAb, David Watkins, Nancy Wilson and David Lorentzen (HLA/Molecular Diagnostics
Laboratory, University of Wisconsin) for providing macaque MHC genotyping services,
Stephanie Ehnert and Andrew Adams for providing expert animal handling and care, Dan
Anderson for conducting macaque necropsies, Jeff Safrit and Robert Mittler for
providing helpful discussions, and Kate Garber for critical reading of the
See the related Commentary beginning on page 808.
Nonstandard abbreviations used: area under the curve (AUC);
costimulation (CS); enzyme-linked immunosorbent spot (ELISPOT); germinal center
(GC); simian immunodeficiency virus (SIV).
Conflict of interest: The authors have declared that no conflict of
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